Dll3 binding moieties and uses thereof

Abstract

The present application provides single-domain antibodies that bind to DLL3 and chimeric antigen receptors comprising the same. The present application further provides engineered immune cells (e.g., T cells) comprising the chimeric antigen receptors provided herein. The present application also provides pharmaceutical compositions, kits, and methods of treating a disease or disorder.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480060736.X (22) Application Date 2024.09.26 (66) Domestic Priority Data PCT / CN2023 / 121699 2023.09.26 CN (85) PCT International Application Entering National Phase Date 2026.03.23 (86) PCT International Application Application Data PCT / CN2024 / 121443 2024.09.26 (87) PCT International Application Publication Data WO2025 / 067338 EN 2025.04.03 (71) Applicant Nanjing Legend Biotechnology Co., Ltd. Address 211100 No. 568 Longmian Avenue, Jiangning District, Nanjing City, Jiangsu Province, Building 6, Nanjing Life Science Town (72) Inventors: Tang Fengyuan, Yang Lei, Zhang Wangzhuang, Qiu Chuan, Yang Xiaocheng, Fang Yun, Gao Rui, Liu Qianqian, Tu Xiaojie, Wu Yunxiang, Hu Zhongsheng (74) Patent Agency: China Council for the Promotion of International Trade Patent & Trademark Agency Co., Ltd., 11038 Patent Attorney: Cheng Yingying (51) Int.Cl. C07K 16 / 28 (2006.01) C07K 16 / 30 (2006.01) C07K 19 / 00 (2006.01) C12N 15 / 62 (2006.01) C12N 15 / 63 (2006.01) C12N 5 / 0783 (2006.01) A61P 35 / 00 (2006.01) (54) Title of Invention: DLL3 Binding Part and Its Use (57) Abstract This application provides a single-domain antibody that binds to DLL3 and a chimeric antigen receptor comprising thereto. This application further provides engineered immune cells (e.g., T cells) comprising the chimeric antigen receptor provided herein. This application also provides pharmaceutical compositions, kits, and methods for treating diseases or conditions. Claims 4 pages, Description 117 pages, Sequence Listing (electronic publication), Drawings 16 pages, CN 122003436 A 2026.05.08 CN 1 22 00 34 36 A 1. An anti-DLL3 (δ-like ligand 3) single-domain antibody (sdAb), comprising: (1) CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO: 1; (2) CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO: 2; (3) CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO: 3; (4) having amino acid sequences as shown in SEQ ID NO: 1;(5) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 4; (6) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 5; (7) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 6; (8) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 7; (9) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 9. and CDR3; or (10) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 10. 2. The anti-DLL3 sdAb according to claim 1, wherein said 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. 3. The anti-DLL3 sdAb according to claim 1 or claim 2, wherein the anti-DLL3 sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 30; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 32; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33; CDR1 containing the amino acid sequence of SEQ ID NO: 24, CDR2 containing the amino acid sequence of SEQ ID NO: 15, and CDR2 containing the amino acid sequence of SEQ ID NO: 24.(6) CDR1 containing the amino acid sequence of SEQ ID NO: 16, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO: 35; (7) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 26, and CDR3 containing the amino acid sequence of SEQ ID NO: 36; (8) CDR1 containing the amino acid sequence of SEQ ID NO: 18, CDR2 containing the amino acid sequence of SEQ ID NO: 27, and CDR3 containing the amino acid sequence of SEQ ID NO: 37; (9) CDR1 containing the amino acid sequence of SEQ ID NO: 19, CDR2 containing the amino acid sequence of SEQ ID NO: 28, and CDR3 containing the amino acid sequence of SEQ ID NO: 38; or (10) 4. The anti-DLL3 sdAb comprising the amino acid sequence of SEQ ID NO: 20, the CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and the CDR3 comprising the amino acid sequence of SEQ ID NO: 39. 5. The anti-DLL3 sdAb according to any one of claims 1-3, wherein the anti-DLL3 sdAb further comprises one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in any one of SEQ ID NO: 1-10 and 40-73. 6. The anti-DLL3 sdAb according to any one of claims 1-4, wherein the anti-DLL3 sdAb is a camel antibody, a chimeric antibody, a human antibody, or a humanized antibody. 7. The anti-DLL3 sdAb according to any one of claims 1-5, wherein the anti-DLL3 sdAb comprises a VHH structure. 7. The anti-DLL3 sdAb according to any one of claims 1-6, wherein the anti-DLL3 sdAb comprises the amino acid sequence of any one of SEQ ID NO: 1-10 and 40-73, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it. 8. The anti-DLL3 sdAb according to any one of claims 1-7, wherein the anti-DLL3 sdAb is genetically fused or chemically conjugated with an agent. 9. The anti-DLL3 sdAb according to any one of claims 1-8, wherein the anti-DLL3 sdAb further comprises the immunoglobulin heavy chain constant region or a fragment thereof;Preferably, the immunoglobulin is IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, or IgA. 10. A multispecific antibody comprising at least one anti-DLL3 sdAb according to any one of claims 1-9, and / or another antibody or an antigen-binding fragment thereof. 11. The multispecific antibody according to claim 10, wherein the multispecific antibody comprises two anti-DLL3 sdAbs according to any one of claims 1-9. 12. The multispecific antibody according to claim 10 or 11, wherein the multispecific antibody comprises the amino acid sequence of any one of SEQ ID NO: 74-75, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with it. 13. An isolated nucleic acid encoding an anti-DLL3 sdAb according to any one of claims 1-9, or a multispecific antibody according to any one of claims 10-12. 14. A vector comprising the isolated nucleic acid according to claim 13. 15. A host cell comprising the isolated nucleic acid according to claim 13 or the vector according to claim 14. 16. A chimeric antigen receptor (CAR) comprising a polypeptide, said polypeptide comprising: (a) an extracellular antigen-binding domain comprising one or more anti-DLL3 sdAbs according to any one of claims 1-9; (b) a transmembrane domain; and (c) an intracellular signal transduction domain. 17. The CAR of claim 16, wherein the extracellular antigen-binding domain comprises two anti-DLL3 sdAbs. Claims 2 / 4 Page 3 CN 122003436 A 18. The CAR according to claim 16 or 17, wherein the two anti-DLL3 sdAbs comprise a first anti-DLL3 sdAb and a second anti-DLL3 sdAb, and wherein: (1) the first anti-DLL3 sdAb comprises CDR1, CDR2 and CDR3 as shown in the anti-DLL3 sdAb comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 64; and (2) the second anti-DLL3 sdAb comprises CDR1, CDR2 and CDR3 as shown in the anti-DLL3 sdAb comprising the amino acid sequence of SEQ ID NO: 68. 19. The CAR according to any one of claims 16-18, wherein the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains. 20. The CAR according to claim 19, wherein the one or more additional antigen-binding domains are selected from...The CAR binds to one or more antigens from the group consisting of: CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. 21. The CAR according to any one of claims 16-20, wherein the transmembrane domain is derived from a molecule selected from the group consisting of: CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD-1; preferably, the transmembrane domain is derived from CD8α. 22. The CAR according to any one of claims 16-21, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. 23. The CAR according to claim 22, wherein the primary intracellular signaling domain is derived from CD3-ζ. 24. The CAR of any one of claims 16-23, wherein the intracellular signal transduction domain further comprises a co-stimulatory signal transduction domain. 25. The CAR of claim 24, wherein the co-stimulatory signal transduction domain is derived from a co-stimulatory molecule selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand binding to CD83, and combinations thereof. 26. The CAR of claim 25, wherein the co-stimulatory signal transduction domain is derived from CD137. 27. The CAR of any one of claims 16-26, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. 28. The CAR of claim 27, wherein the hinge domain is derived from CD8α. 29. The CAR of any one of claims 16-28, further comprising a signal peptide located at the N-terminus of the polypeptide. 30. The CAR of claim 29, wherein the signal peptide is derived from CD8α. 31. The CAR of any one of claims 16-30, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 77-86, 88-123, and 130-175. 32. An isolated nucleic acid comprising a nucleic acid sequence encoding the CAR of any one of claims 16-31. 33. A vector comprising the isolated nucleic acid of claim 32. 34. An engineered immune cell comprising the CAR of any one of claims 16-31, and according to the claims...32. The isolated nucleic acid as described in claim 32, or the vector as described in claim 33. 35. The engineered immune cell as described in claim 34, wherein the immune cell is a T cell, NK cell, peripheral blood mononuclear cell (PBMC), hematopoietic stem cell, pluripotent stem cell, embryonic stem cell, or any combination thereof. 36. A method for producing engineered immune cells, the method comprising introducing the vector as described in claim 33 into cells. Claims 3 / 4 pages 4 CN 122003436 A 37. A pharmaceutical composition comprising an anti-DLL3 sdAb as described in any one of claims 1-9, a multispecific antibody as described in any one of claims 10-12, a CAR as described in any one of claims 16-31, the isolated nucleic acid as described in claim 13 or 32, the vector as described in claim 14 or 33, or the engineered immune cell as described in claim 34 or 35, and a pharmaceutically acceptable excipient. 38. A method of treating a subject for a disease or condition, the method comprising administering to the subject an effective amount of an anti-DLL3 sdAb according to any one of claims 1-9, a multispecific antibody according to any one of claims 10-12, engineered immune cells according to claim 34 or 35, or a pharmaceutical composition according to claim 37. 39. The method of claim 38, wherein the disease or condition is a DLL3-related disease or condition. 40. The method of claim 38 or 39, wherein the disease or condition is a tumor or cancer. 41. The method according to any one of claims 38-40, wherein the disease or condition is selected from the group consisting of: neuroendocrine tumors (NETs) or neuroendocrine carcinomas (NECs); preferably, the disease or condition is selected from the group consisting of: lung cancer (e.g., small cell lung cancer (SCLC)), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumors (GI-NEC), bladder cancer (e.g., small cell bladder cancer (SCBC)), neuroendocrine prostate cancer (NEPC), melanoma, glioma (e.g., low-grade glioma), glioblastoma, medullary thyroid carcinoma, neuroendocrine bladder cancer (NEBC), testicular cancer, and pancreatic neuroendocrine tumors (PNETs). 42. The anti-DLL3 sdAb according to any one of claims 1-9, the multispecific antibody according to any one of claims 10-12, the CAR according to any one of claims 16-31, the isolated nucleic acid according to claim 13 or 32, the vector according to claim 14 or 33, and the engineered immunosorbent assay (IRISA) according to claim 34 or 35.Use of the pharmaceutical composition according to claim 37 in the manufacture of a medicament for use in treating a disease or condition (e.g., cancer) in a subject. Claims 4 / 4 Page 5 CN 122003436 A DLL3 binding portion and its use

[0001] Cross Reference

[0002] This application claims priority to International Patent Application No. PCT / CN2023 / 121699, filed on September 26, 2023, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application incorporates by reference a sequence listing filed with this application, which is an XML file entitled "IEC240393PCT-seql.xml", created on September 22, 2024, and is 222,695 bytes in size. 1. Technical Field

[0005] This disclosure relates to anti-DLL3 single-domain antibodies, chimeric antigen receptors, engineered immune cells, and methods of using the same. This disclosure further relates to the activation and expansion of cells for therapeutic purposes, particularly to T-cell immunotherapy based on chimeric antigen receptors. 2. Background Art

[0006] DLL3 (δ-like ligand 3) is a non-canonical Notch ligand that acts in a cell-autonomous manner to inhibit Notch signaling, thereby blocking intercellular interactions and the internalization of Notch in target cells. DLL3 is a neuroendocrine lineage marker that is highly expressed in SCLC and other neuroendocrine tumors, but expressed at minimal levels in normal tissues. Notch signaling is downregulated during the growth of neuroendocrine tumors and is inhibited by DLL3 expression. Other indications involving DLL3 include melanoma, low-grade glioma, glioblastoma, medullary thyroid carcinoma, diffuse neuroendocrine tumors of the pancreas, bladder, and prostate, testicular cancer, and lung adenocarcinoma with neuroendocrine features. 3. Summary of the Invention

[0007] In a first aspect, this invention provides an anti-DLL3 (δ-like ligand 3) single-domain antibody (sdAb), the anti-DLL3 sdAb comprising:

[0008] (1) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1;

[0009] (2) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 2;

[0010] (3) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 3;

[0011] (4) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 4.CDR2 and CDR3;

[0012] (5) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 5;

[0013] (6) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 6;

[0014] (7) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 7;

[0015] (8) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 8;

[0016] (9) having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 8; CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 10; or

[0017] (10) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 10.

[0018] In some embodiments, 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.

[0019] In some embodiments, the anti-DLL3 sdAb comprises:

[0020] (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 30;

[0021] (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31;

[0022] (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 32;

[0023] (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 14. CDR3 of amino acid sequence 33;

[0024] (5) Containing SEQ ID NO:CDR1 containing the amino acid sequence of SEQ ID NO: 15, CDR2 containing the amino acid sequence of SEQ ID NO: 24, and CDR3 containing the amino acid sequence of SEQ ID NO: 34;

[0025] (6) CDR1 containing the amino acid sequence of SEQ ID NO: 16, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO: 35;

[0026] (7) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 26, and CDR3 containing the amino acid sequence of SEQ ID NO: 36;

[0027] (8) CDR1 containing the amino acid sequence of SEQ ID NO: 18, CDR2 containing the amino acid sequence of SEQ ID NO: 27, and CDR3 containing the amino acid sequence of SEQ ID NO: 37;

[0028] (9) Containing SEQ ID NO: CDR1 containing the amino acid sequence of SEQ ID NO: 29, CDR2 containing the amino acid sequence of SEQ ID NO: 28, and CDR3 containing the amino acid sequence of SEQ ID NO: 38; or

[0029] (10) CDR1 containing the amino acid sequence of SEQ ID NO: 20, CDR2 containing the amino acid sequence of SEQ ID NO: 29, and CDR3 containing the amino acid sequence of SEQ ID NO: 39.

[0030] In some embodiments, the anti-DLL3 sdAb further comprises one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in any one of SEQ ID NO: 1-10 and 40-73.

[0031] In some embodiments, the anti-DLL3 sdAb is a camel antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0032] In some embodiments, the anti-DLL3 sdAb comprises a VHH structure.

[0033] In some embodiments, the anti-DLL3 sdAb comprises the amino acid sequence of any one of SEQ ID NO: 1-10 and 40-73, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

[0034] In some embodiments, the anti-DLL3 sdAb is genetically fused or chemically conjugated with a drug.

[0035] In some embodiments, the anti-DLL3 sdAb further comprises the immunoglobulin heavy chain constant region or a fragment thereof.

[0036] In some embodiments, the immunoglobulin is IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, or IgA.

[0037] In a second aspect, this document provides a multispecific antibody comprising at least one of the anti-DLL3 sdAbs disclosed above, and / or another antibody or an antigen-binding fragment thereof.

[0038] In some embodiments, the multispecific antibody comprises two anti-DLL3 sdAbs disclosed above.

[0039] In some embodiments, the multispecific antibody comprises the amino acid sequence of any one of SEQ ID NO: 74-75, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with it.

[0040] In a third aspect, this document provides an isolated nucleic acid encoding an anti-DLL3 sdAb disclosed above, or a multispecific antibody disclosed above.

[0041] In a fourth aspect, this document provides a vector comprising the isolated nucleic acid disclosed above.

[0042] In a fifth aspect, this document provides a host cell comprising the isolated nucleic acid or the vector disclosed above.

[0043] In a sixth aspect, this document provides a chimeric antigen receptor (CAR) comprising a polypeptide comprising:

[0044] (a) an extracellular antigen-binding domain comprising one or more anti-DLL3 sdAbs disclosed above;

[0045] (b) a transmembrane domain; and

[0046] (c) an intracellular signal transduction domain.

[0047] In some embodiments, the extracellular antigen-binding domain comprises two anti-DLL3 sdAbs.

[0048] In some embodiments, the two anti-DLL3 sdAbs comprise a first anti-DLL3 sdAb and a second anti-DLL3 sdAb, wherein:

[0049] (1) the first anti-DLL3 sdAb comprises CDR1, CDR2, and CDR3 as shown in the anti-DLL3 sdAb comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 64; and

[0050] (2) the second anti-DLL3 sdAb comprises CDR1, CDR2, and CDR3 as shown in the anti-DLL3 sdAb comprising the amino acid sequence of SEQ ID NO: 68.

[0051] In some embodiments, the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains.

[0052] In some embodiments, one or more additional antigen-binding domains are selected from the group consisting of...One or more antigens bind: CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77.

[0053] In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of: CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD-1.

[0054] In some embodiments, the transmembrane domain is derived from CD8α.

[0055] In some embodiments, the intracellular signal transduction domain comprises a primary intracellular signal transduction domain of an immune effector cell.

[0056] In some embodiments, the primary intracellular signal transduction domain is derived from CD3ζ.

[0057] In some embodiments, the intracellular signal transduction domain further comprises a co-stimulatory signal transduction domain.

[0058] In some embodiments, the co-stimulatory signal transduction domain is derived from a co-stimulatory molecule selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands binding to CD83, and combinations thereof. Specification 3 / 117 page 8 CN 122003436 A

[0059] In some embodiments, the co-stimulatory signal transduction domain is derived from CD137.

[0060] In some embodiments, the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0061] In some embodiments, the hinge domain is derived from CD8α.

[0062] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide.

[0063] In some embodiments, the signal peptide is derived from CD8α.

[0064] In some embodiments, the CAR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 77-86, 88-123, and 130-175.

[0065] In a seventh aspect, this document provides an isolated nucleic acid comprising a nucleic acid sequence encoding the CAR disclosed above.

[0066] In an eighth aspect, this document provides a vector comprising the isolated nucleic acid disclosed in the seventh aspect.

[0067] In a ninth aspect, this document provides an engineered immune cell comprising the CAR disclosed above, the isolated nucleic acid disclosed in the seventh aspect, or the vector disclosed in the eighth aspect.

[0068] In some embodiments, the immune cell is a T cell, NK cell, peripheral blood mononuclear cell (PBMC), hematopoietic stem cell, etc.Cells, pluripotent stem cells, embryonic stem cells, or any combination thereof.

[0069] In a tenth aspect, this document provides a method for producing engineered immune cells, the method comprising introducing a vector disclosed in the eighth aspect into cells.

[0070] In an eleventh aspect, this document provides a pharmaceutical composition comprising the anti-DLL3 sdAb disclosed above, the multispecific antibody disclosed above, the CAR disclosed above, the isolated nucleic acid disclosed in the third or seventh aspect, the vector disclosed in the fourth or eighth aspect, or the engineered immune cells disclosed above, and a pharmaceutically acceptable excipient.

[0071] In a twelfth aspect, this document provides a method for treating a subject for a disease or condition, the method comprising administering to the subject an effective amount of the anti-DLL3 sdAb disclosed above, the multispecific antibody, the engineered immune cells, or the pharmaceutical composition.

[0072] In some embodiments, the disease or condition is a DLL3-related disease or condition.

[0073] In some embodiments, the disease or condition is a tumor or cancer.

[0074] In some embodiments, the disease or condition is selected from the group consisting of: neuroendocrine tumor (NET) or neuroendocrine carcinoma (NEC).

[0075] In some embodiments, the disease or condition is selected from the group consisting of: lung cancer (e.g., small cell lung cancer (SCLC)), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), bladder cancer (e.g., small cell bladder cancer (SCBC)), neuroendocrine prostate cancer (NEPC), or melanoma, glioma (e.g., low-grade glioma), glioblastoma, medullary thyroid carcinoma, neuroendocrine bladder cancer (NEBC), testicular cancer, and pancreatic neuroendocrine tumor (PNET).

[0076] In a thirteenth aspect, this document provides the use of the anti-DLL3 sdAb disclosed above, the multispecific antibody disclosed above, the CAR disclosed above, the isolated nucleic acid disclosed in the third or seventh aspect, the vector disclosed in the fourth or eighth aspect, the engineered immune cells disclosed above, or the pharmaceutical composition disclosed above in the manufacture of a medicament for use in treating a disease or condition (e.g., cancer) in a subject. Specification 4 / 117 pages 9 CN 122003436 A 4. Description of Drawings

[0077] Figures 1A-1D show the cytolytic activity of camel-based anti-DLL3 VHH CAR-T cells against the tumor cell line NCI-H82, with an E:T ratio of 0.5:1.

[0078] Figures 2A-2P respectively show the effects of the camel-based anti-DLL3 VHH CAR-T cells provided in this disclosure on different cell lines (e.g., CHO-K1, CHO-K1 hDLL1, CHO-K1 hDLL3, and hCHO-K1).The cell lysis specificity of hDLL4 was observed, with an E:T ratio of 2:1.

[0079] Figures 3A-3D show the cytokine release of the tumor cell line NCI-H82 by CAR-T cells based on camel anti-DLL3 VHH, with an E:T ratio of 0.5:1, and ▲ indicates the lower limit of concentration.

[0080] Figures 4A-4D show the in vivo efficacy of CAR-T cells based on camel anti-DLL3 VHH in the NCI-H82 subcutaneous xenograft model.

[0081] Figures 5A-5D show the percentage of CAR-T cells based on camel anti-DLL3 VHH in the peripheral blood of NCG mice.

[0082] Figures 6A-6J show the cytolytic activity of humanized anti-DLL3 VHH CAR-T cells against the NCI-H82 tumor cell line compared to camel-based anti-DLL3 VHH CAR-T cells and UnT cells, with an E:T ratio of 2:1.

[0083] Figures 7A-7J show the in vitro persistence of humanized anti-DLL3 VHH CAR-T cells under repeated TAA stimulation compared to camel-based anti-DLL3 VHH CAR-T cells and UnT cells.

[0084] Figures 8A-8J show the in vitro expansion of humanized anti-DLL3 VHH CAR-T cells under repeated TAA stimulation compared to camel-based anti-DLL3 VHH CAR-T cells.

[0085] Figures 9A-9C show the in vivo efficacy of humanized anti-DLL3 VHH-based CAR-T cells in the NCI-H82 subcutaneous xenograft model compared to camel-based anti-DLL3 VHH CAR-T cells and UnT cells.

[0086] Figures 10A-10C show the percentage of humanized anti-DLL3 VHH-based CAR-T cells in the peripheral blood of NCG mice compared to camel-based anti-DLL3 VHH CAR-T cells and UnT cells.

[0087] Figures 11A-11D show the in vitro efficacy (e.g., persistence and expansion) of bispecific / bivalent humanized anti-DLL3 VHH-based CAR-T cells under repeated TAA stimulation compared to monospecific humanized anti-DLL3 VHH-based CAR-T cells and / or UnT cells.

[0088] Figures 12A-12B show the in vivo efficacy (e.g., antitumor activity and percentage) of CAR-T cells based on bispecific / bivalent humanized anti-DLL3 VHH under repeated TAA stimulation compared to CAR-T cells and UnT cells based on monospecific humanized anti-DLL3 VHH. 5. Detailed Description

[0089] This disclosure is based in part on novel single-domain antibodies (e.g., VHH domain) that bind to DLL3, chimeric antigen receptors or engineered cells containing them, and their improved properties.

[0090] 5.1. Definitions

[0091] The techniques and procedures described or referenced herein include those well understood and / or commonly employed by those skilled in the art using conventional methods, such as the widely used methods described below: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition 2001); Current Protocols in Molecular Biology (edited by Ausubel et al., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009); Monoclonal Antibodies: Methods and Protocols (edited by Albitar, 2010); and Antibody Engineering, Volumes 1 and 2 (edited by Kontermann and Dübel, 2nd edition 2010). Unless otherwise defined herein, the technical and scientific terms used in this specification shall have the meanings commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of the terms shall apply, and where appropriate, terms used in the singular shall also include the plural, and vice versa. In the event of any conflict between any description of a stated term and any document incorporated herein by reference, the description of the term stated below shall prevail.

[0092] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably herein and in the broadest sense, and particularly cover, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having multi-epitope or single-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), single-chain antibodies and fragments thereof (e.g., domain antibodies), as described below. Antibodies can be human antibodies, humanized antibodies, chimeric antibodies, and / or affinity-matured antibodies, as well as antibodies derived from other species (e.g., mice, rabbits, llamas, etc.). The term "antibody" is intended to include B-cell polypeptide products of immunoglobulin-like peptides that are capable of binding to specific molecular antigens and consist of two pairs of identical polypeptide chains, each pair having a heavy chain (approximately 50–70 kDa) and a light chain (approximately 25 kDa).Each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain includes a constant region. See, for example, Antibody Engineering (edited by Borrebaeck, 2nd edition, 1995); and Kuby, Immunology (3rd edition, 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinant antibodies, including single-domain antibodies from camel species (e.g., llamas or alpacas) or their humanized variants, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments of any of the above (e.g., antigen-binding fragments) that are part of the antibody heavy or light chain polypeptide and retain some or all of the binding activity of the antibody from which the fragment is derived. 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, biantibodies, triantibodies, tetraantibodies, and miniantibodies. In particular, the antibodies provided herein comprise immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, such as antigen-binding domains or molecules containing antigen-binding sites (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 (edited by Myers, 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2nd edition, 1990). The antibodies described herein can be 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). Antibodies can be agonist or antagonist. Antibodies can be neither agonist nor antagonist.

[0093] An “antigen” is a structure that an antibody can selectively bind to. Target antigens can be peptides, carbohydrates, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds. Target antigens can be peptides. Antigens can be cell-related, for example, present on or within cells.

[0094] An “intact” antibody is an antibody that contains an antigen-binding site as well as a CL and at least heavy chain constant regions CH1, CH2, and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof. An intact antibody may have one or more effector functions.

[0095] A “single-chain Fv” (also abbreviated as “sFv” or “scFv”) is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. For example, the sFv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding, as described in the specification 6 / 117 pages 11 CN 122003436 A. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0096] 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 4-chain antibodies. Cameloideas (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0097] As used herein, “single-domain antibody” or “sdAb” refers to a single monomeric variable antibody domain that is capable of binding an antigen (e.g., a single-domain antibody that binds to DLL3). Single-domain antibodies include the VHH domain as described herein. Examples of single-domain antibodies include, but are not limited to, antibodies that are naturally lacking in the light chain, such as antibodies from cameloid species (e.g., llamas), single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. For example, as described herein, single-domain antibodies can be derived from antibodies produced in cameloid species such as camels, llamas, dromedary camels, alpacas, and guanacos. Other species besides camelids can produce naturally occurring heavy-chain antibodies lacking the light chain; VHHs derived from such other species are within the scope of this disclosure. The single-domain antibodies (e.g., VHHs) provided herein can have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single-domain antibodies can be genetically fused or chemically conjugated to another molecule (e.g., a pharmaceutical agent) as described herein. Single-domain antibodies may be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0098] The term “binds” refers to interactions between molecules, including, for example, the formation of a complex. Interactions can be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions.Function. Complexes can also comprise the binding of two or more molecules linked together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interaction 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 for that epitope. The ratio (koff / kon) of the dissociation rate (koff) of a binding molecule (e.g., an antibody) to the association rate (kon) of a monovalent antigen is the dissociation constant KD, which is inversely proportional to affinity. The lower the KD value, the higher the affinity of the antibody. KD values ​​vary from antibody to antigen complex and depend on kon and koff. The dissociation constant KD of antibodies provided herein can be determined using any of the methods provided herein or any other method known to those skilled in the art. Affinity at a 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) comes into contact with an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site increases the likelihood of a reaction occurring at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called affinity.

[0099] Regarding the binding molecules described herein, terms such as “binding,” “specific binding,” and similar terms may be 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 immunoassay, Octet®, Biacore®, or other techniques known to those skilled in the art. A binding molecule or antigen-binding domain can bind to an antigen or specifically bind to an antigen when it binds to an antigen with a higher affinity than when it binds to any cross-reactive antigen, as determined by experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, the specific or selective response will be at least twice the background signal or noise, and can be more than 10 times the background. See, for example, Fundamental Immunology 332-36 (edited by Paul, 2nd edition 1989) for a discussion on binding specificity. The degree to which a binding molecule or antigen-binding domain binds to a "non-target" protein can be approximately 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, for example, as determined by fluorescence-activated cell sorting (FACS) analysis or RIA. Binding molecules or antigen-binding domains that bind to antigens include those capable of binding antigens with sufficient affinity so that the binding molecule can be used as, for example, a therapeutic agent and / or diagnostic agent targeting the antigen. (See page 7 / 117, 12 CN 122003436 A for details.)Antigen-binding domain. The binding molecule or antigen-binding domain that binds to the antigen may have a dissociation constant (KD) less than or equal to 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. The binding molecule or antigen-binding domain can bind to conserved antigenic epitopes from antigens from different species.

[0100] 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 in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and in fragments of such antibodies, provided they exhibit the desired biological activity (see U.S. Patent No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). The chimeric sequence may include a humanized sequence.

[0101] The binding molecule or antigen-binding domain may comprise portions of a “humanized” form of a non-human antibody (e.g., a camel, mouse, or non-human primate antibody) comprising a sequence derived from a human immunoglobulin (e.g., a receptor antibody), wherein native CDR residues are replaced by residues from a corresponding CDR derived from a non-human species (e.g., a donor antibody) having the desired specificity, affinity, and capability, such as a camel, mouse, rat, rabbit, or non-human primate. One or more FR region residues of the human immunoglobulin sequence may be replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the receptor or donor antibody. These modifications are made to further improve antibody performance. The heavy or light chain of the humanized antibody may comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to the CDRs of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. The humanized antibody may comprise at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a 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); US Patent Nos.: 6,800,738, 6,719,971, 6,639,055, 6,407,213 and 6,054,297.

[0102] The binding molecule or antigen-binding domain may comprise a portion of a “fully human antibody” or a “human antibody,” wherein these terms are used interchangeably herein and refer to an antibody comprising a human variable region and, for example, a human constant region. The binding molecule may comprise a single-domain antibody sequence. These terms may refer to an antibody comprising a human-derived variable region and a constant region. A “fully human” antibody may also encompass an antibody that binds a polypeptide and is encoded by 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 immunoglobulin sequences, as described by Kabat et al. (see Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is an antibody having an amino acid sequence corresponding to that of antibodies produced by humans and / or having been produced using any technology used to manufacture human antibodies. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques 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)). Methods for preparing human monoclonal antibodies are also described in the following literature: 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). Human antibodies can be produced by applying antigens to a modified substrate in response to antigen attack, as per the specification page 8 / 117, 13 CN 122003436 A.Such antibodies are prepared from transgenic animals (e.g., mice) whose endogenous loci are inactive (see, for example, Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Brüggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Patent Nos. 6,075,181 and 6,150,584 concerning the XENOMOUSE™ technology). For human antibodies generated via human B-cell hybridoma technology, see also, for example, Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006).

[0103] The binding molecule or antigen-binding domain may include the phrase “recombinant human antibody,” wherein the phrase includes human antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combined human antibody library, antibodies isolated from transgenic and / or transchromosomally transgenic animals (e.g., mice or cattle) for the human immunoglobulin gene (see, for example, Taylor, L.D. et al., Nucl. Acids Res. 20:6287–6295 (1992)), or antibodies prepared, expressed, generated, or isolated by any other means involving splicing a human immunoglobulin gene sequence into another DNA sequence. 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, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242). However, such recombinant human antibodies can be mutagenized in vitro (or, when using transgenic animals with human Ig sequences, in vivo somatic cell mutagenesis), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and associated with human germline VH and VL sequences, may not be naturally present in human antibody germline libraries in vivo.

[0104] The binding molecule or antigen-binding domain may comprise a portion of a “monoclonal antibody,” where the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, for example, except for possibly naturally occurring mutations or well-known post-translational modifications (such as amino acid isomerization or deamidation, methionine oxidation, or aspartate amino acid oxidation) that may be present in small amounts.Apart from asparagine or glutamine deamidation, the individual antibodies comprising this population are identical, and each monoclonal antibody will typically recognize a single epitope on the antigen. As used herein, a “monoclonal antibody” can be an antibody produced from a single hybridoma or other cell. The term “monoclonal” is not limited to any particular method used to prepare the antibody. For example, monoclonal antibodies that can be used in this disclosure can be prepared by a hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be prepared in bacterial or eukaryotic or plant cells using a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). “Monoclonal antibodies” can also be isolated from phage antibody libraries using techniques described, for example, those 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 therefrom are well known in the art. See, for example, Short Protocols in Molecular Biology (edited by Ausubel et al., 5th edition, 2002).

[0105] A typical 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is connected to the H chain by a covalent disulfide bond, while the two H chains are connected to each other by one or more disulfide bonds depending on the H chain isoform. Each H chain and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isoforms. Each L chain has a variable domain (VL) at its N-terminus and a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form an interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (edited by Stites et al., 8th edition 1994); and Immunobiology (edited by Janeway et al., 5th edition 2001). Specification 9 / 117 pages 14 CN 122003436 A

[0106] The term “Fab” or “Fab region” refers to the antibody region that binds to the antigen. Conventional IgG typically contains two Fab regions.Each Fab region is located on one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of a variable region and a constant region, one for the heavy chain and one for the light chain. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions in the Fab region can be arranged in various ways to confer antigen-binding ability according to this disclosure. For example, the VH and CH1 regions can be on a single polypeptide, and the VL and CL regions can be on separate polypeptides, similar to the Fab region of conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the following sections.

[0107] The terms “variable region,” “variable domain,” “V region,” or “V domain” refer to a portion of the light or heavy chain of an antibody, typically located at the amino terminus of the light or heavy chain, and having a length of approximately 120 to 130 amino acids in the heavy chain and approximately 100 to 110 amino acids in the light chain, and are used for the binding and specificity of each specific antibody to its specific antigen. The variable region of the heavy chain may be referred to as “VH.” The variable region of the light chain may be referred to as “VL.” The term “variable” refers to the fact that certain segments of the variable region in an antibody are significantly different in sequence. The V region mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, the variability is not uniformly distributed within the 110 amino acid range of the variable region. Instead, the V region consists of segments of approximately 15–30 amino acids called frame regions (FRs) with less variation (e.g., relatively constant), which are separated by shorter regions of approximately 9–12 amino acids each called “hypervariates” with greater variation (e.g., extreme variation). The variable regions of both the heavy and light chains each contain four FRs that predominantly adopt a β-sheet configuration. These FRs are linked by three hypervariable regions that form a loop and, in some cases, form part of a β-sheet structure. The hypervariable regions in each chain are tightly bound together by the FRs and, together with hypervariable regions from the other chain, 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)). Constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions differ significantly in sequence between different antibodies. Variable regions can be human variable regions.

[0108] When used to refer to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50–70 kDa, wherein the amino-terminal portion containsThe heavy chain comprises a variable region of about 120 to 130 or more amino acids, and a carboxyl-terminal portion comprising a constant region. Based on the amino acid sequence of the constant region of the heavy chain, the constant region can be one of five different types (e.g., isotypes) known as alpha (α), delta (δ), epsilon (ε), gamma (γ), and muon (μ). The different heavy chains are of different sizes: α, δ, and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with light chains, these different types of heavy chains produce antibodies of five well-known classes (e.g., isotypes), IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

[0109] When used to refer to antibodies, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion comprises a variable region of about 100 to about 110 or more amino acids, and the carboxyl-terminal portion comprises a constant region. The light chain is approximately 211 to 217 amino acids in length. Based on the amino acid sequence of constant domains, there are two different types, called Kabat (κ) or Lambda (λ).

[0110] As used herein, the terms “hypervariant 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 the VH β-sheet frame of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VL β-sheet frame of an antibody. Thus, a CDR is a variable region sequence scattered within a frame region sequence.

[0111] CDR regions are well known to those skilled in the art and are defined by well-known numbering schemes. For example, the Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (see, for example, Kabat et al., ibid.). Chothia refers to the location of the structural loop on page 10 / 117 of the specification, CN 122003436 A (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When 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 the insertion at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software (see, for example, Antibody).Engineering, Volume 2 (edited by Kontermann and Dübel, 2nd edition 2010). The “contact” hypervariable region is based on the analysis of available complex crystal structures. Another universal numbering scheme that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1):55–77 (2003)). IMGT is a comprehensive information scheme specifically for the study of immunoglobulins (IG), T cell receptors (TCR), and major histocompatibility complex (MHC) in humans and other vertebrates. In this paper, CDR refers to the amino acid sequence and position within the light or heavy chain. Because the “position” of CDR within the structure of the variable domain of immunoglobulins is conserved across species and exists in structures called loops, CDRs and framework residues are easily identified by using a numbering scheme that compares the variable domain sequence based on structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into receptor frameworks typically derived from human antibodies. Honegger and Plückthun, J. Mol. Biol. 309: 657-70 (2001) developed an additional numbering scheme (AHon). Correspondences between numbering schemes, including, for example, the Kabat numbering and the IMGT unique numbering scheme, are well known to those skilled in the art (see, for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.). Residues from each of these hypervariable regions or CDRs are illustrated in Table 1 below.

[0112] Table 1. Exemplary CDRs according to various numbering schemes

[0113]

[0114] The boundaries of a given CDR may vary depending on the scheme used for identification. Therefore, unless otherwise stated, the terms “CDR” and “complementarity-determining region” for a given antibody or its regions (such as variable regions) and the individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody or its regions should be understood to encompass the complementarity-determining region as defined by any known scheme described above. In some cases, a scheme is specified for identifying one or more specific CDRs, such as those defined by IMGT, Kabat, AbM, Chothia, or Contact methods. In some cases, one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number. For exemplary numbering of the VHH domain according to Kabat, see, for example, Deschacht et al., 2010. J Immunol 184: Specification.Page 11 / 117, 16 CN 122003436 A 5696-704. In other cases, a specific amino acid sequence of the CDR is given. It should be noted that the CDR region can also be defined by a combination of various numbering schemes (e.g., a combination of the Kabat and Chothia numbering systems, a combination of the Kabat and AbM numbering systems, or a combination of the Kabat and IMGT numbering systems). Therefore, the term "CDR1 as shown in a specific VH or VHH" includes, but is not limited to, any CDR1 defined by the exemplary CDR numbering schemes described above. Once a variable region (e.g., VHH, VH, or VL) is given, those skilled in the art will understand that the CDR within that region can be defined by different numbering systems or combinations thereof.

[0115] Hypervariable regions 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.

[0116] The term “constant region” or “constant domain” refers to the carboxyl-terminal portion of the light and heavy chains that does not directly participate in antibody-antigen binding but exhibits various effector functions, such as interaction with Fc receptors. The term also refers to a portion of an immunoglobulin molecule that, relative to another portion of the immunoglobulin, is a variable region, having a more conserved amino acid sequence containing antigen-binding sites. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0117] The term “frame” or “FR” refers to those variable region residues located on either side of the CDR. FR residues are present in, for example, chimeric antibodies, humanized antibodies, human antibodies, domain antibodies (e.g., single-domain antibodies), biantibodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues outside of the hypervariable region residues or CDR residues.

[0118] The term “Fc region” as used herein is used to define the C-terminal region of the immunoglobulin heavy chain, including, for example, the native sequence Fc region, the recombinant Fc region, and the variant Fc region. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at the Cys226 or Pro230 position 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 recombinantly engineering the nucleic acid encoding the antibody heavy chain. Therefore, a complete antibody composition can comprise antibody groups with all K447 residues removed, antibody groups without K447 residues removed, and antibody groups consisting of mixtures containing and without K447 residues.The "functional Fc region" possesses the "effective function" of the native sequence Fc region. Exemplary "effective functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions typically require combining the Fc region with a binding region or binding domain (e.g., antibody variable region or domain) and can be evaluated using various assays known to those skilled in the art. The "variant Fc region" comprises an amino acid sequence that differs from the native sequence Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion). The variant Fc region may have at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent peptide, for example, about one to about ten amino acid substitutions, or about one to about five amino acid substitutions in the native sequence Fc region or the Fc region of the parent peptide. The variant Fc region described herein may have at least about 80% homology with the native sequence Fc region and / or the Fc region of the parent peptide, or at least about 90% homology with it, for example, at least about 95% homology with it.

[0119] As used herein, “epitope” is a term in the art and refers to a local region of an antigen that a binding molecule (e.g., an antibody comprising a single-domain antibody sequence) can specifically bind to. An epitope can be a linear epitope or a conformational, nonlinear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be a continuous amino acid of the polypeptide (“linear” epitope), or an epitope can comprise amino acids from two or more discontinuous regions of the polypeptide (“conformational,” “nonlinear,” or “discontinuous” epitope). Those skilled in the art will understand that, in general, a linear epitope can depend on or not depend on secondary, tertiary, or quaternary structures. For example, a binding molecule can bind to a set of amino acids, regardless of whether these amino acids are folded in the native three-dimensional protein structure. A binding molecule may require the amino acid residues constituting the epitope to exhibit a specific conformation (e.g., bending, twisting, turning, or folding) to recognize and bind to the epitope. Specification 12 / 117 pages 17 CN 122003436 A

[0120] “Blocking” antibodies or “antagonist” antibodies are antibodies that inhibit or reduce the biological activity of the antigens they bind to. Blocking antibodies or antagonist antibodies can substantially or completely inhibit the biological activity of the antigen.

[0121] “Agonist” or activating antibodies are antibodies that can enhance or initiate signal transduction of the antigens they bind to. Agonist antibodies can induce or activate signal transduction in the absence of a natural ligand.

[0122] The “percentage (%) amino acid sequence identity” and “homology” of peptide, polypeptide, or antibody sequences are defined as the amino acid residues in the candidate sequence that are identical to the amino acid residues in a specific peptide or polypeptide sequence after alignment of the sequence and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity.The percentage of bases. Alignment can be performed using publicly available calculator software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software, in various ways within the skill of the art, to determine the percentage of amino acid sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full-length sequences being compared.

[0123] The term “specificity” refers to the selective recognition of a specific epitope of an antigen by an antigen-binding protein (e.g., CAR or sdAb). For example, natural antibodies are monospecific. As used herein, the term “multispecific” means that an antigen-binding protein (e.g., CAR or sdAb) has two or more antigen-binding sites, at least two of which bind different antigens (or epitopes). As used herein, “bispecific” means that an antigen-binding protein (e.g., CAR or sdAb) has two different antigen-binding specificities. As used herein, the term “monospecific” means that an antigen-binding protein (e.g., CAR or sdAb) has one or more binding sites, each binding the same antigen (or epitope).

[0124] As used herein, the term “valence” indicates the presence of a specified number of binding sites in an antigen-binding protein (e.g., a CAR or sdAb). For example, a natural antibody or full-length antibody has two binding sites and is divalent. Thus, the terms “trivalent,” “tetravalent,” “pentavalent,” and “hexavalent” indicate the presence of two, three, four, five, and six binding sites in an antigen-binding protein (e.g., a CAR or sdAb), respectively.

[0125] As used herein, “chimeric antigen receptor” or “CAR” refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells such as T cells. Some CARs are also referred to as “artificial T cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” A CAR may contain an extracellular antigen-binding domain specific to one or more antigens (e.g., tumor antigens), a transmembrane domain, and an intracellular signaling domain for T cells and / or other receptors. “CAR-T cell” refers to a T cell expressing a CAR.

[0126] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid groups. These terms also cover amino acid polymers that have been naturally modified or modified by intervention; for example, by forming disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. This definition also includes, for example, polypeptides containing one or more amino acid analogs, including but not limited to non-natural amino acids, and other modifications known in the art.As the polypeptides disclosed herein may be based on antibodies or other members of the immunoglobulin superfamily, a “polypeptide” may exist as a single chain or as two or more related chains.

[0127] The terms “polynucleotide” or “nucleic acid” as used interchangeably herein refer to a polymer of nucleotides of any length and include DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that may be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and analogs thereof. As used herein, an “oligonucleotide” refers to a short, usually single-chain, synthetic polynucleotide whose length is typically (but not necessarily) less than about 200 nucleotides. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells that produce the binding molecules disclosed herein may include parental hybridoma cells, as well as bacterial and eukaryotic host cells in which nucleic acids encoding antibodies have been introduced. Unless otherwise stated, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of a nascent RNA transcript is referred to as the transcription direction; the sequence region on a DNA strand having the same sequence as the RNA transcript as the RNA transcript as the RNA transcript 5' to 5' end is referred to as the "upstream sequence"; the sequence region on a DNA strand having the same sequence as the RNA transcript as the RNA transcript 3' to 3' end is referred to as the "downstream sequence".

[0128] "Separated nucleic acid" is a nucleic acid (e.g., RNA, DNA, or a mixture of nucleic acids) that is substantially separated from other genomic DNA sequences naturally associated with the native sequence, as well as proteins or complexes such as ribosomes and polymerases. "Separated" nucleic acid molecules are nucleic acid molecules separated from other nucleic acid molecules present in the natural source of nucleic acid molecules. Furthermore, “isolated” nucleic acid molecules, such as cDNA molecules, when produced via recombinant technology, may be substantially free of other cellular material or culture medium, or when chemically synthesized, substantially free of chemical precursors or other chemicals. One or more nucleic acid molecules encoding single-domain antibodies or antibodies as described herein can be isolated or purified. This term includes nucleic acid sequences that have been removed from their natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized from heterologous systems. A substantially pure molecule may include the isolated form of the molecule. In particular, the “isolated” nucleic acid molecules encoding CARs or sdAbs described herein are nucleic acid molecules identified and isolated from at least one contaminant nucleic acid molecule, which…The molecule typically associates with at least one contaminant nucleic acid molecule in the environment in which it is produced.

[0129] The term “control sequence” refers to a DNA sequence necessary for the expression of an operablely linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0130] As used herein, the term “operably linked” and similar phrases (e.g., gene fusion), when used to refer to nucleic acids or amino acids, refer to the operable linking of nucleic acid sequences or amino acid sequences, respectively, that are functionally related to each other. For example, an operablely linked promoter, enhancer element, open reading frame, 5' and 3' UTR, and terminator sequence result in the accurate production of a nucleic acid molecule (e.g., RNA). Operablely linked nucleic acid elements can lead to transcription of the open reading frame and ultimately to the production of a polypeptide (i.e., expression of the open reading frame). As an example, an operablely linked peptide is a peptide in which functional domains are placed at appropriate distances from each other to confer the intended function of each domain.

[0131] The term "vector" refers to a substance used to carry or include a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, to introduce the nucleic acid sequence into a host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selective sequences or markers that can be stably integrated into the host cell chromosome. Additionally, a vector may include one or more selective marker genes and appropriate expression control sequences. Selective marker genes that may be included, for example, provide resistance to antibiotics or toxins, supplement nutritional deficiencies, or supply key nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., antibody heavy and light chains or antibody VH and VL), two nucleic acid molecules may be inserted, for example, into a single expression vector or a separate expression vector. For single vector expression, the encoding nucleic acid may be operatively ligated to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. These methods include, for example, nucleic acid analysis, such as Northern blotting or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for gene product expression, or other suitable analytical methods for testing the introduced nucleic acid sequence or the expression of its corresponding gene product. Those skilled in the art will understand that nucleic acid molecules are expressed in amounts sufficient to produce the desired product, and will further understand that methods well known in the art can be used. (See page 14 / 117, 19 CN 122003436 A)To optimize expression levels to achieve adequate expression.

[0132] As used herein, the term “host” refers to an animal, such as a mammal (e.g., a human).

[0133] As used herein, the term “host cell” refers to a specific target cell that can be transfected with a nucleic acid molecule, as well as the offspring or potential offspring of such cells. The offspring of such cells may differ from the parent cells transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or the integration of the nucleic acid molecule into the host cell genome.

[0134] As used herein, the term “autologous” is intended to refer to any material derived from the same individual, wherein the material is subsequently reintroduced into that individual.

[0135] “allogeneic” refers to a graft derived from a different individual of the same species.

[0136] As used herein, the terms “transfected” or “transformed” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into a host cell. “Transfected” or “transformed” or “transduced” cells are cells that have been transfected, transformed, or transduced using exogenous nucleic acids. This includes primary target cells and their progeny.

[0137] As used herein, the term “pharmaceuticalally acceptable” means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeia for use in animals, and more particularly for use in humans.

[0138] “Excipient” means a pharmaceutically acceptable material, composition, or medium, 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, extenders, fillers, flavorings, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, 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 medium.

[0139] Excipients may be pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 amino acid residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are found at Remington and Gennaro, Remington's PharmaceuticalAs described in Sciences (18th edition, 1990).

[0140] A component may be “pharmaceutically acceptable” in the sense of compatibility with other components of a pharmaceutical preparation and is suitable for contact with human or animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity or other problems or complications, in proportion to a reasonable benefit / risk ratio. See, for example, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th edition; edited by Rowe et al.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd edition; edited by Ash and Ash; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd edition; edited by Gibson; CRC Press LLC: Boca Raton, FL, 2009. Pharmaceutically acceptable excipients may be non-toxic to cells or mammals exposed to them at the doses and concentrations used. Pharmaceutically acceptable excipients may be aqueous pH buffer solutions.

[0141] Excipients may be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary excipient when the composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions, glucose solutions, and glycerol solutions may also be used as liquid excipients, particularly for injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. The composition may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions, including formulations, may include standard excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0142] Compositions including pharmaceutical compounds may contain, for example, conjugated molecules (e.g., antibodies) in isolated or purified form and appropriate amounts of excipients.

[0143] As used herein, the term “effective amount” or “therapeutic effective amount” means an amount of single-domain antibody or therapeutic molecule comprising a pharmaceutical agent and the single-domain antibody or pharmaceutical composition provided herein sufficient to produce the desired results.

[0144] The terms “subject” and “patient” are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate or primate (e.g., a human). A subject can be a human. A subject can be a mammal diagnosed with a disease or condition, such as a human. A subject can be a mammal at risk of developing a disease or condition, such as a human.

[0145] “Administration” means the act of injecting or otherwise physically delivering a substance present outside the body into a patient, such as via mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or known in the art.

[0146] As used herein, the terms “treat”, “treatment”, and “treating” mean reducing or improving the progression, severity, and / or duration of a disease or condition caused by the administration of one or more therapies. Treatment can be determined by assessing whether one or more symptoms associated with the underlying condition have been reduced, alleviated, and / or alleviated, resulting in observed improvement in the patient, although the patient may still have the underlying condition. The term “treatment” includes controlling and improving the disease. The term “control (manage, managing, and management)” refers to a beneficial effect obtained by the subject from a treatment that does not necessarily lead to a cure for the disease.

[0147] The term “prevent (preventing, and prevention)” refers to reducing the likelihood of the onset (or recurrence) of a disease, condition, ailment, or related symptoms (e.g., diabetes or cancer).

[0148] As used herein, “delaying” the development of cancer means postponing, hindering, slowing, decelerating, stabilizing, and / or delaying the development of the disease. This delay can have different durations depending on the history of the disease and / or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay can effectively cover prevention, since the individual has not developed the disease. The method of “delaying” the development of cancer is a method of reducing the likelihood of disease development and / or reducing the severity of the disease within a given time frame, compared to not using this method. Such comparisons are typically based on clinical studies using statistically significant numbers of individuals. Standard methods can be used to detect cancer development, including but not limited to computed tomography (CAT) scans, magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Development can also refer to cancer progression that may initially be undetectable, and includes occurrence, recurrence, and onset.

[0149] As used herein, “DLL3-related disease or condition” refers to a disease or condition comprising cells or tissues expressing DLL3. DLL3-related disease or condition may include cells with abnormal DLL3 expression (e.g., higher DLL3 expression compared to normal or healthy cells).

[0150] The terms “about” and “approximately” mean within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a given value or range.

[0151] As used in this disclosure and claims, the singular forms “a / an” and “the” include the plural forms unless the context clearly indicates otherwise. Specification 16 / 117 pages 21 CN 122003436 A

[0152] It should be understood that while embodiments are described anywhere herein using the term “comprising”, similar embodiments described as “consisting of” and / or “substantially consisting of” are also provided. It should also be understood that wherever embodiments are described with the phrase “substantially consisting of…”, similar embodiments described with “consisting of…” are also provided.

[0153] The term “between…” as used in the phrases “between A and B” or “between A and B” refers to a range including A and B.

[0154] The term “and / or” as used herein in phrases such as “A and / or B” is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to cover 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).

[0155] 5.2 Single-domain antibodies

[0156] 5.2.1 Single-domain antibodies binding to DLL3

[0157] In one aspect, this document provides single-domain antibodies (e.g., VHH domains) capable of binding to DLL3.

[0158] The single-domain antibodies (e.g., VHH domains) provided herein can bind to human DLL3. Human DLL3 (δ-like ligand 3) is a single transmembrane protein and a member of the Notch ligand family. The human DLL3 protein consists of approximately 618 amino acids, and its complete structure includes a DSL domain, an intracellular domain, and six epidermal growth factor-like (EGF-like) domains. Steinbuck, et al. Frontiers in immunology 9 (2018): 1230.

[0159] The anti-DLL3 single-domain antibodies provided herein can modulate one or more DLL3 activities. The anti-DLL3 single-domain antibodies provided herein can be antagonist antibodies or agonist antibodies.

[0160] The anti-DLL3 single-domain antibody provided herein can be used at < 1 μM, <A dissociation constant (KD) of 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10⁻⁷ M or less, e.g., 10⁻⁷ M to 10⁻¹³ M) binds to DLL3 (e.g., human DLL3). Various methods for measuring binding affinity are known in the art, any of which may be used for the purposes of this disclosure.

[0161] In some embodiments, the anti-DLL3 single-domain antibody provided herein is a VHH domain. The exemplary VHH domains provided herein are generated as described in Part 6 below, and these VHH domains are referred to as AS297272, AS300695, AS300724, AS301940, AS297693, AS300115, AS301207, AS302102, AS298907, AS301180, AS297272H4, AS297272H5, AS297272H6, AS297272H7, AS297272H8, AS297693H5, AS297693H6, AS297693H7, AS298907H7-3, AS298907H10-2, AS298907H10-3, AS298907H13, and AS300115H4. AS300115H5, AS300115H6, AS300115H7, AS300695H6, AS300695H8, AS300724H6, AS300724H7, AS301940H4, AS301940H5, AS301940H6, AS301940H7, AS301940H8, AS301940H9, AS301940H10, AS302102H7, AS302102H8, AS302102H9, AS301207H5, AS301207H6, AS301207H7 and AS301180H9 are also shown in Table 2 below.

[0162] Therefore, the single-domain antibodies provided herein may include AS297272, AS300695, AS300724, AS301940, AS297693, AS300115, AS301207, AS302102, AS298907, AS301180, AS297272H4, AS297272H5, AS297272H6, AS297272H7, AS297272H8, AS297693H5, AS297693H6, AS297693H7, AS298907H7-3. AS298907H10‑2, AS298907H10‑3, AS298907H13, AS300115H4, AS300115H5, AS300115H6, AS300115H7, AS300695H6, AS300695H8, AS300724H6, AS300724H7, AS301940H4, AS301940H5, AS301940H6, AS301940H7, AS301940H8, AS301940H9, AS301940H10, AS302102H7, One or more CDR sequences of any one of AS302102H8, AS302102H9, AS301207H5, AS301207H6, AS301207H7 and AS301180H9. The single-domain antibody for binding to DLL3 provided herein may contain the following structures: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequence is selected from the following: AS297272, AS300695, AS300724, AS301940, AS297693, AS300115, AS301207, AS302102, AS298907, AS301180, AS297272H4, AS297272H5, AS297272H6, AS297272H7, AS297272H8, AS297693H5, AS297693H6, AS297693H7. AS298907H7‑3, AS298907H10‑2, AS298907H10‑3, AS298907H13, AS300115H4, AS300115H5, AS300115H6, AS300115H7, AS300695H6, AS300695H8, AS300724H6, AS300724H7, AS301940H4, AS301940H5, AS301940H6, AS301940H7, AS301940H8, AS301940H9, AS301940H10, AS302102H7, AS302102H8, AS302102H9, AS301207H5, AS301207H6, AS301207H7 and / or AS301180H9.

[0163] Table 2. Exemplary single-domain antibody specification 18 / 117 pages 23 CN 122003436 A

[0164]

[0165] The anti-DLL3 single-domain antibody provided herein may comprise: (i) comprising as SEQ ID NO:(ii) CDR1 comprising the amino acid sequence of CDR1 shown in any one of SEQ ID NO: 1-10 and 40-73; and / or (iii) CDR3 comprising the amino acid sequence of CDR2 shown in any one of SEQ ID NO: 1-10 and 40-73; and / or (iii) CDR3 comprising the amino acid sequence of CDR3 shown in any one of SEQ ID NO: 1-10 and 40-73.

[0166] The anti-DLL3 single-domain antibody provided herein may comprise: (i) CDR1 comprising the amino acid sequence of CDR1 as shown in any one of SEQ ID NO: 1-10 and 40-73; (ii) CDR2 comprising the amino acid sequence of CDR2 as shown in any one of SEQ ID NO: 1-10 and 40-73; and (iii) CDR3 comprising the amino acid sequence of CDR3 as shown in any one of SEQ ID NO: 1-10 and 40-73.

[0167] The anti-DLL3 single-domain antibody provided herein may comprise:

[0168] (i) CDR1, comprising:

[0169] (a) an amino acid sequence of any one of SEQ ID NO: 11-20;

[0170] (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of SEQ ID NO: 11-20; or

[0171] (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to any one of SEQ ID NO: 11-20;

[0172] (ii) CDR2, comprising:

[0173] (a) SEQ ID NO: The amino acid sequence of any one of SEQ ID NO: 21-29;

[0174] (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of SEQ ID NO: 21-29; or

[0175] (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to any one of SEQ ID NO: 21-29;

[0176] and / or

[0177] (iii) CDR3, comprising:

[0178] (a) SEQ ID NO: 21-29

[0179] (b) An amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any of SEQ ID NO: 30-39; or

[0180] (c) An amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to any of SEQ ID NO: 30-39.

[0181] The anti-DLL3 single-domain antibody provided herein may comprise: (i) a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 11-20; (ii) a CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 21-29; and (iii) a CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 30-39.

[0182] The anti-DLL3 single-domain antibody provided herein may comprise one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 1. The anti-DLL3 single-domain antibody provided herein may comprise one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 2. The anti-DLL3 single-domain antibody provided herein may comprise one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 3. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 4. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 5. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 6. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 7. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 8. The anti-DLL3 single-domain antibody provided herein...The antibody may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 9. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 10. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 40. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 41. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 42. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 43. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 44. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 45. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 46. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 47. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 48. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 49. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 50. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 51.All three CDRs (CDR1, CDR2, and / or CDR3). The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 52. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 53. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 54. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 55. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 56. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 57. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 58. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 59. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 60. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 61. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 62. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 63. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 64. The anti-DLL3 single-domain antibody provided herein may contain SEQ ID NO:The amino acid sequence of SEQ ID NO: 65 may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3). The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 66. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 67. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 68. The anti-DLL3 single-domain antibody provided herein may contain one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 69. The anti-DLL3 single-domain antibody provided herein may comprise one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 70. The anti-DLL3 single-domain antibody provided herein may comprise one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 71. The anti-DLL3 single-domain antibody provided herein may comprise one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 72. The anti-DLL3 single-domain antibody provided herein may comprise one, two, or all three CDRs (CDR1, CDR2, and / or CDR3) of the amino acid sequence of SEQ ID NO: 73.

[0183] In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 1, respectively. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 2. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 3. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 4.The anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 5. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 6. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 7. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 8. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 9. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 10. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 40. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 41. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 42. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 43. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 44. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 44.CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 45. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 46. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 47. Specification 22 / 117 pages 27 CN 122003436 A In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 48. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 49. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 50. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 51. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 52. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 53. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 54. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 55. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 56.CDR1, CDR2, and CDR3 having the amino acid sequences of CDR2 and CDR3. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 57. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 58. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 59. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 60. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 61. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 62. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 63. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 64. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 65. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 66. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 67.The CDR1, CDR2, and CDR3 sequences are described herein. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 68. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 69. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 70. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 71. In some embodiments, the anti-DLL3 single-domain antibody provided in CN 122003436 A, page 23 / 117 of this specification, may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 72, respectively. In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 73, respectively.

[0184] The anti-DLL3 single-domain antibody provided herein may comprise:

[0185] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 11; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0186] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 21; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; An amino acid sequence with 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence; and / or

[0187] (iii) CDR3, comprising: (a) the amino acid sequence of SEQ ID NO: 30; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0188] The anti-DLL3 single-domain antibody provided herein may comprise:

[0189] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 12; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0190] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 22; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids); Amino acid sequences having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; and / or

[0191] (iii) CDR3 comprising: (a) the amino acid sequence of SEQ ID NO: 31; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0192] The anti-DLL3 single-domain antibody provided herein may comprise:

[0193] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 13; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c)Compared with it, it has an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids);

[0194] (ii) CDR2, which comprises: (a) the amino acid sequence of SEQ ID NO: 23; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared with it; and / or

[0195] (iii) CDR3, which comprises: (a) the amino acid sequence of SEQ ID NO: 32; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity ... An amino acid sequence with 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0196] The anti-DLL3 single-domain antibody provided herein may comprise:

[0197] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 14; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0198] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 22; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids);

[0197] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 22; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, Amino acid sequences with 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) amino acid sequences having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to; and / or

[0199] (iii) CDR3, comprising: (a) the amino acid sequence of SEQ ID NO: 33; (b) an amino acid sequence having at least 75%,An amino acid sequence with 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0200] The anti-DLL3 single-domain antibody provided herein may comprise:

[0201] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 15; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0202] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 24; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids);

[0202] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 24; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; Amino acid sequences having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; and / or

[0203] (iii) CDR3 comprising: (a) the amino acid sequence of SEQ ID NO: 34; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0204] The anti-DLL3 single-domain antibody provided herein may comprise:

[0205] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 16; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0206] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 25; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; and / or

[0207] (iii) CDR3, comprising: (a) the amino acid sequence of SEQ ID NO: 35; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or

[0207] (iii) CDR3, comprising: (a) the amino acid sequence of SEQ ID NO: 35; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; and / or

[0207] (iii) CDR3, comprising: (a) the amino acid sequence of SEQ ID NO: 35; (b) an amino acid sequence having at least 75 An amino acid sequence with 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0208] The anti-DLL3 single-domain antibody provided herein may comprise:

[0209] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 17; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0210] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 26; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; An amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; and / or

[0211] (iii) CDR3 comprising: (a) the amino acid sequence of SEQ ID NO: 36; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.The amino acid sequence of the same type; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0212] The anti-DLL3 single-domain antibody provided herein may comprise:

[0213] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 18; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it;

[0214] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 27; (b) an amino acid sequence having at least 75%, Amino acid sequences having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; and / or

[0215] (iii) CDR3 comprising: (a) the amino acid sequence of SEQ ID NO: 37; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0216] The anti-DLL3 single-domain antibody provided herein may comprise:

[0217] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 19; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0218] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 28; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids);

[0217] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 28; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1,Amino acid sequences with 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) amino acid sequences having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to it; and / or

[0219] (iii) CDR3, comprising: (a) the amino acid sequence of SEQ ID NO: 38; (b) amino acid sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to it; or (c) amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to it.

[0220] The anti-DLL3 single-domain antibody provided herein may comprise:

[0221] (i) CDR1, comprising: (a) the amino acid sequence of SEQ ID NO: 20; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0222] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 29; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids);

[0222] (ii) CDR2, comprising: (a) the amino acid sequence of SEQ ID NO: 29; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; or (c) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, Amino acid sequences with 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; or (c) amino acid sequences having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to; and / or

[0223] (iii) CDR3 comprising: (a) the amino acid sequence of SEQ ID NO: 39; (b) an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to; or (c) ...(or a sequence of 3 amino acids with substitutions, deletions, or additions).

[0224] In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 11, CDR2 containing the amino acid sequence of SEQ ID NO: 21, and CDR3 containing the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 12, CDR2 containing the amino acid sequence of SEQ ID NO: 22, and CDR3 containing the amino acid sequence of SEQ ID NO: 31. In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 13, CDR2 containing the amino acid sequence of SEQ ID NO: 23, and CDR3 containing the amino acid sequence of SEQ ID NO: 32. In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 14, CDR2 containing the amino acid sequence of SEQ ID NO: 22, and CDR3 containing the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 15, CDR2 containing the amino acid sequence of SEQ ID NO: 24, and CDR3 containing the amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 16, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 26, and CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 18, CDR2 containing the amino acid sequence of SEQ ID NO: 27, and CDR3 containing the amino acid sequence of SEQ ID NO: 37. In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 19, CDR2 containing the amino acid sequence of SEQ ID NO: 28, and CDR3 containing the amino acid sequence of SEQ ID NO: 38. In some embodiments, the anti-DLL3 single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO: 19, CDR2 containing the amino acid sequence of SEQ ID NO: 28, and CDR3 containing the amino acid sequence of SEQ ID NO: 28.CDR1 containing the amino acid sequence of SEQ ID NO: 20, CDR2 containing the amino acid sequence of SEQ ID NO: 29, and CDR3 containing the amino acid sequence of SEQ ID NO: 39.

[0225] The CDR sequence can be determined according to well-known numbering schemes. In some embodiments, the CDR can be determined according to the IMGT numbering scheme, Kabat numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or a combination thereof. In some embodiments, the CDR can be determined according to the AbM numbering scheme.

[0226] The anti-DLL3 single-domain antibody can be a camel antibody. The anti-DLL3 single-domain antibody can be a humanized antibody. Anti-DLL3 single-domain antibody can contain a recipient human frame, such as a human immunoglobulin frame or a human common frame.

[0227] The anti-DLL3 single-domain antibody may further comprise AS297272, AS300695, AS300724, AS301940, AS297693, AS300115, AS301207, AS302102, AS298907, AS301180, AS297272H4, AS297272H5, AS297272H6, AS297272H7, AS297272H8, AS297693H5, AS297693H6, AS297693H7, AS298907H7- 3. AS298907H10‑2, AS298907H10‑3, AS298907H13, AS300115H4, AS300115H5, AS300115H6, AS300115H7, AS300695H6, AS300695H8, AS300724H6, AS300724H7, AS301940H4, AS301940H5, AS301940H6, AS301940H7, AS301940H8, AS301940H9, AS301940H10, AS302102H7,

[0228] The anti-DLL3 single-domain antibody may contain one or more frame regions of AS302102H8, AS302102H9, AS301207H5, AS301207H6, AS301207H7 and / or AS301180H9.

[0229] Anti-DLL3 single-domain antibodies may include AS297272H4, AS297272H5, AS297272H6, AS297272H7, AS297272H8, AS297693H5, AS297693H6, AS297693H7, AS298907H7-3, AS298907H10-2, AS298907H10-3, AS298907H13, AS300115H4, AS300115H5, AS300115H6, and AS300115H7. One or more frame regions of AS300695H6, AS300695H8, AS300724H6, AS300724H7, AS301940H4, AS301940H5, AS301940H6, AS301940H7, AS301940H8, AS301940H9, AS301940H10, AS302102H7, AS302102H8, AS302102H9, AS301207H5, AS301207H6, AS301207H7 and / or AS301180H9.

[0230] In some embodiments, the anti-DLL3 single-domain antibody provided herein may comprise: (i) FR1 as shown in any one of SEQ ID NO: 1-10 and 40-73; (ii) FR2 as shown in any one of SEQ ID NO: 1-10 and 40-73; (iii) FR3 as shown in any one of SEQ ID NO: 1-10 and 40-73; and / or (iv) FR4 as shown in any one of SEQ ID NO: 1-10 and 40-73.

[0231] The anti-DLL3 single-domain antibody provided herein may comprise: (i) FR1 as shown in any one of SEQ ID NO: 1-10 and 40-73; (ii) FR2 as shown in any one of SEQ ID NO: 1-10 and 40-73; (iii) FR3 as shown in any one of SEQ ID NO: 1-10 and 40-73; and (iv) FR4 as shown in any one of SEQ ID NO: 1-10 and 40-73.

[0232] The anti-DLL3 single-domain antibody provided herein may comprise: (i) FR1 as shown in any one of SEQ ID NO: 1-10; (ii) FR2 as shown in any one of SEQ ID NO: 1-10; (iii) FR3 as shown in any one of SEQ ID NO: 1-10; and / or (iv) FR4 as shown in any one of SEQ ID NO: 1-10.

[0233] The anti-DLL3 single-domain antibody provided herein may comprise: (i) FR1 as shown in any one of SEQ ID NO: 40-73; (ii) FR2 as shown in any one of SEQ ID NO: 40-73; (iii) FR3 as shown in any one of SEQ ID NO: 40-73; and / or (iv) FR4 as shown in any one of SEQ ID NO: 40-73.

[0234] The anti-DLL3 single-domain antibody provided herein may comprise one or more FR regions selected from the group consisting of: FR1, FR2, FR3 and FR4 as shown in SEQ ID NO: 1. The anti-DLL3 single-domain antibody provided herein may comprise one or more FR regions selected from the group consisting of: FR1, FR2, FR3 and FR4 as shown in SEQ ID NO: 2. The anti-DLL3 single-domain antibody provided herein may comprise one or more FR regions selected from the group consisting of: FR1, FR2, FR3 and FR4 as shown in SEQ ID NO: 3. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 4. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 5. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 6. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 7. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 8. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 9. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 10.

[0235] The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: SEQ ID NO:The FR1, FR2, FR3, and FR4 shown in SEQ ID NO: 40. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 41. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 42. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 43. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 44. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of: FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 45. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 46. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 47. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 48. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 49. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 50. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 51. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 52. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 53. The anti-DLL3 single-domain antibody provided herein...The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 54. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 55. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 56. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 57. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 58. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 59. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 60. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 61. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 62. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 63. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 64. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 65. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 66.The anti-DLL3 single-domain antibody may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 67. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 68. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 69. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 70. The anti-DLL3 single-domain antibody provided herein may contain one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 71. The anti-DLL3 single-domain antibody provided herein may comprise one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 72. The anti-DLL3 single-domain antibody provided herein may comprise one or more FR regions selected from the group consisting of FR1, FR2, FR3, and FR4 as shown in SEQ ID NO: 73.

[0236] The frame region described herein is defined according to the boundaries of the CDR numbering scheme. In other words, if the CDR is defined by, for example, the Kabat, AbM, IMGT, Contact, or Chothia numbering scheme, the frame region is an amino acid residue in the variable region surrounding the CDR in the following form (from the N-terminus to the C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the N-terminal amino acid residue of CDR1, as defined by, for example, the Kabat numbering scheme, the AbM numbering scheme, the IMGT numbering scheme, the Contact numbering scheme, the Chothia numbering scheme, or a combination thereof; FR2 is defined as the amino acid residue between the amino acid residues of CDR1 and CDR2, as defined by, for example, the Kabat numbering scheme, the AbM numbering scheme, the IMGT numbering scheme, the Contact numbering scheme, the Chothia numbering scheme, or a combination thereof; FR3 is defined as the amino acid residue between the amino acid residues of CDR2 and CDR3, as defined by, for example, the Kabat numbering scheme, the AbM numbering scheme, the IMGT numbering scheme, the Contact numbering scheme, the Chothia numbering scheme, or a combination thereof; and FR4 is defined as the C-terminal amino acid residue of CDR3.Residues, as defined by, for example, the Kabat numbering scheme, the AbM numbering scheme, the IMGT numbering scheme, the Contact numbering scheme, the Chothia numbering scheme, or combinations thereof.

[0237] In some embodiments, the anti-DLL3 single-domain antibody provided herein comprises a VHH domain containing an amino acid sequence of any one of SEQ ID NO: 1-10 and 40-73; or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

[0238] In some embodiments, the anti-DLL3 single-domain antibody provided herein comprises a VHH domain containing an amino acid sequence of any one of SEQ ID NO: 1-10 and 40-73. The anti-DLL3 single-domain antibody provided herein may comprise a VHH domain containing an amino acid sequence of any one of SEQ ID NO: 1-10. The anti-DLL3 single-domain antibody provided herein may contain a VHH domain containing an amino acid sequence of any one of SEQ ID NO: 40-73.

[0239] The anti-DLL3 single-domain antibody or its antigen-binding fragment described herein contains an amino acid sequence having a certain percentage of identity with any of the following antibodies: AS297272, AS300695, AS300724, AS301940, AS297693, AS300115, AS301207, AS302102, AS298907, AS301180, AS297272H4, AS297272H5, AS297272H6, AS297272H7, AS297272H8, AS297693H5, AS297693H6, AS297693H7, AS298907H7- 3. AS298907H10-2, AS298907H10-3, AS298907H13, AS300115H4, AS300115H5, AS300115H6, AS300115H7, AS300695H6, AS300695H8, AS300724H6, AS300724H7, AS301940H4, AS301940H5, Instruction Manual 30 / 117 pages 35 CN 122003436 A AS301940H6, AS301940H7, AS301940H8, AS301940H9, AS301940H10, AS302102H7, AS302102H8, AS302102H9, AS301207H5, AS301207H6, AS301207H7 and AS301180H9.

[0240] Mathematical algorithms can be used to determine the percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), which is modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST procedures of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set, for example, for score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameter set, for example, with a score of 50 and a word length of 3, to obtain amino acid sequences homologous to the protein molecules described herein. For obtaining vacancy alignments for comparative purposes, Gapped BLAST, as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997), can be used. Alternatively, PSI BLAST can be used for iterative searches to detect intermolecular distance relationships (ibid.). When using BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm 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 comparing amino acid sequences using the ALIGN program, a PAM 120 weighted residue table can be used with a vacancy length penalty of 12 and a vacancy penalty of 4. Whether vacancy is allowed or not, techniques similar to those described above can be used to determine the percentage of identity between two sequences. When calculating the percentage of identity, only exact matches are typically counted.

[0241] In another aspect, this document provides an anti-DLL3 antibody or its antigen-binding fragment that competitively and specifically binds to DLL3 with any of the anti-DLL3 single-domain antibodies described herein. Competitive binding can be determined using an ELISA assay. For example, a antibody containing SEQ IDNO: An anti-DLL3 single-domain antibody with an amino acid sequence of any one of 1-10 and 40-73 competitively and specifically binds to DLL3.

[0242] In another aspect, this document provides a DLL3-binding protein comprising any one of the above-described anti-DLL3 single-domain antibodies. The DLL3-binding protein may be a monoclonal antibody, including camel antibodies, chimeric antibodies, humanized antibodies, or human antibodies. The anti-DLL3 antibody may be an antibody fragment, such as a VHH fragment. The anti-DLL3 antibody may be a full-length heavy chain antibody containing the Fc region of any antibody class or isotype (e.g., IgG1 or IgG4). The Fc region may have reduced or minimized effector functions. The DLL3-binding protein may be a fusion protein comprising the anti-DLL3 single-domain antibody provided herein. The DLL3-binding protein may be a multispecific antibody comprising the anti-DLL3 single-domain antibody provided herein. Other exemplary DLL3-binding molecules are described in more detail in the following sections.

[0243] The DLL3-binding protein may be a multispecific antibody comprising the anti-DLL3 single-domain antibody provided herein. A multispecific antibody may comprise at least one of the above-described anti-DLL3 sdAbs, and / or another antibody or its antigen-binding fragment. A multispecific antibody may comprise two anti-DLL3 sdAbs, and in some embodiments, each of the two anti-DLL3 sdAbs is independently an anti-DLL3 sdAb as described above. The two anti-DLL3 sdAbs may comprise a first anti-DLL3 sdAb and a second anti-DLL3 sdAb, wherein the first anti-DLL3 sdAb comprises CDR1, CDR2, and CDR3 as shown in the anti-DLL3 sdAb comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 64; and the second anti-DLL3 sdAb comprises CDR1, CDR2, and CDR3 as shown in the anti-DLL3 sdAb comprising the amino acid sequence of SEQ ID NO: 68. The first anti-DLL3 sdAb may comprise: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31; or (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33; and / or, the second anti-DLL3 sdAb may comprise: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, a CDR3 ...CDR2 containing the amino acid sequence of SEQ ID NO: 37 and CDR3 containing the amino acid sequence of SEQ ID NO: 37.

[0244] The CDR sequence can be determined according to a well-known numbering scheme. The CDR can be determined according to the IMGT numbering scheme, the Kabat numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. In some embodiments, the CDR is determined according to the AbM numbering scheme.

[0245] The anti-DLL3 single-domain antibody can be a camel antibody. The anti-DLL3 single-domain antibody can be a humanized antibody. The anti-DLL3 single-domain antibody can contain a recipient human frame, such as a human immunoglobulin frame or a human common frame.

[0246] The first anti-DLL3 sdAb may comprise the amino acid sequence of SEQ ID NO: 56 or 64; or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; and / or, the second anti-DLL3 sdAb may comprise the amino acid sequence of SEQ ID NO: 68; or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

[0247] The first anti-DLL3 sdAb may be linked to the N-terminus or C-terminus of the second anti-DLL3 sdAb with or without a linker (e.g., a rigid peptide linker or a flexible peptide linker).

[0248] Multispecific antibodies may comprise the amino acid sequence of SEQ ID NO: 74 or 75; or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

[0249] Anti-DLL3 antibodies (e.g., anti-DLL3 single-domain antibodies) or antigen-binding proteins according to any of the foregoing aspects may include any features, alone or in combination, as described in sections 5.2.2 to 5.2.7 below.

[0250] 5.2.2 Humanized Single-Domain Antibodies

[0251] The single-domain antibodies described herein include humanized single-domain antibodies. General strategies for humanizing single-domain antibodies from camelid species have been described (see, for example, Vincke et al., J. Biol. Chem., 284(5):3273-3284 (2009)) and can be used to generate the humanized VHH domain disclosed in this paper. Humanized single-domain antibodies from camelid speciesThe design may include marker residues in VHH, such as residues 11, 37, 44, 45 and 47 (residues numbered according to Kabat) (Muyldermans, Reviews Mol Biotech 74:277-302 (2001)).

[0252] Humanized antibodies, such as the humanized single-domain antibodies disclosed herein, can also be generated using a variety of techniques known in the art, including but not limited to CDR transplantation (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and US Patent Nos. 5,225,539, 5,530,101 and 5,585,089), faceting or surface repair (European Patent Nos. EP 592,106 and EP 519,596; 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 truncation (US Patent No. 5,565,332) and, for example, US Patent No. 6,407,213, US Patent No. 5,766,886, WO 9317105, 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), Bacae et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer 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 the U.S. Patent Publication No. 32 / 117, page 37, CN 122003436 A, US 2005 / 0042664 A1 (February 24, 2005), each incorporated herein by reference in its entirety.

[0253] The single-domain antibodies provided herein can be humanized single-domain antibodies that bind to DLL3 (including human DLL3). For example, the humanized single-chain antibodies disclosed herein may comprise one or more CDRs shown in SEQ ID NO: 1-10 and 40-73. Various methods for humanizing non-human antibodies are known in the art. For example, humanized antibodies may have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as “input” residues, and these residues are typically derived from “input” variable domains. Humanization can be performed, for example, by replacing the corresponding sequence of a human antibody with a hypervariable region sequence, as described in 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). The humanization of the single-domain antibodies provided herein can be performed as described in Section 6 below.

[0254] Humanized antibodies can be constructed via CDR transplantation, in which the amino acid sequence of the CDR of a parental non-human antibody is transplanted onto 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 referred to these as “specificity-determining residues” or SDRs (Padlan et al., FASEB J 9:133-39 (1995)). In SDR transplantation techniques, only SDR residues are transplanted onto the human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34 (2005)).

[0255] Selecting the human variable domain for the preparation of humanized antibodies can be important for reducing antigenicity. For example, sequences of variable domains of non-human antibodies are screened against an entire library of known human variable domain sequences according to the so-called “best fit” method. A human frame for a humanized antibody can be chosen that most closely resembles the human sequence of the non-human antibody (Sims et al., J. Immunol. 151:2296–308 (1993); and Chothia et al., J. Mol. Biol. 196:901–17 (1987)). Another approach uses a specific frame derived from a specific subgroup of the light or heavy chain, based on a shared sequence of all human antibodies. The same frame can 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)). The frame can...The concordant sequences are derived from the most abundant human subclasses, VL6 subgroup I (VL6I) ​​and VH subgroup III (VHIII). In another approach, human germline genes are used as the source of the frame region.

[0256] In an alternative paradigm based on CDR comparison, known as hyperhumanization, FR homology is irrelevant. This method involves comparing non-human sequences with a functional human germline gene pool. Then, those genes encoding canonical structures that are identical or closely related to mouse sequences are selected. Next, among the genes sharing canonical structures with non-human antibodies, those genes with the highest homology within the CDR are selected as FR donors. Finally, the non-human CDR is transplanted onto these FRs (see, for example, Tan et al., J. Immunol. 169:1119-25 (2002)).

[0257] It is also generally desirable to humanize antibodies to retain their affinity for antigens and other advantageous biological properties. To achieve this goal, humanized antibodies are prepared according to a method that analyzes parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that illustrate and visualize possible three-dimensional conformations 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)). Examining these visualizations allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, such as analyzing residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the receptor and input sequence to obtain desired antibody characteristics, such as increased affinity for one or more target antigens. Typically, hypervariable residues are directly and most significantly involved in influencing antigen binding. Specification 33 / 117 pages 38 CN 122003436 A

[0258] Another approach to antibody humanization is based on an antibody humanity metric called Human String Content (HSC). This method compares mouse sequences to a human germline gene pool and scores the differences as HSC. Target sequences are then humanized to generate a variety of different humanized variants by maximizing their HSCs rather than using a global identity metric (Lazar et al., Mol. Immunol. 44:1986-98 (2007)).

[0259] In addition to the methods described above, empirical methods can also be used to generate and select humanized antibodies. These methods include those based on generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries as well as by screening bacterial colonies (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)).

[0260] In the FR library method, a batch 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 CDR for transplantation. The residues to be replaced may include some or all of the “cursor” residues identified as potentially contributing to the CDR structure (see, for example, Foote and Winter, J Mol. Biol. 224:487–99 (1992)), or a more limited set of target residues identified by Baca et al., J. Biol. Chem. 272:10678–84 (1997).

[0261] In FR shuffling, the entire FR is combined with a nonhuman CDR rather than creating a combinatorial library of selected residue variants (see, for example, Dall'Acqua et al., Methods 36:4360 (2005)). A one-step FR shuffling process can be used. Such a method has proven effective because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, for example, Damschroder et al., Mol. Immunol. 44:3049–60 (2007)).

[0262] The “humanizing” approach is based on the experimental identification of the basic minimum specificity determinant (MSD) and on the sequential replacement of non-human fragments into a human FR library and evaluation of binding. This approach typically results in epitope preservation and identification of antibodies from multiple subclasses with different human V-segment CDRs.

[0263] The “human engineering” approach involves modifying non-human antibodies or antibody fragments by specifically altering the amino acid sequence of the antibody, thereby producing modified antibodies with reduced immunogenicity in humans, which still retain the desired binding properties of the original non-human antibody. Typically, this technique involves replacing the non-human antibody with a specific amino acid sequence of the antibody.Amino acid residues are categorized as “low-risk,” “intermediate-risk,” or “high-risk” residues. This categorization is performed using an overall risk / reward calculation that assesses the predicted benefit of a specific substitution (e.g., for human immunogenicity) versus the risk that the substitution would affect the resulting antibody folding. Specific human amino acid residues to be substituted at a given position (e.g., low-risk or intermediate-risk) in the non-human antibody sequence can be selected by comparing the amino acid sequence from the variable region of the non-human antibody with the corresponding region of the specific or shared human antibody sequence. Amino acid residues at low-risk or intermediate-risk positions in the non-human sequence can be substituted for corresponding residues in the human antibody sequence based on the alignment. Techniques for preparing human engineered proteins are described in more detail in Studnicka et al., Protein Engineering 7:805-14 (1994); U.S. Patents 5,766,886, 5,770,196, 5,821,123, and 5,869,619; and PCT Publication WO 93 / 11794.

[0264] Composite human antibodies can be generated using, for example, Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To generate composite human antibodies, variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T-cell epitopes, thereby minimizing the immunogenicity of the resulting antibody.

[0265] Deimmunized antibodies are antibodies in which T-cell epitopes have been removed. Methods for preparing deimmunized antibodies have been 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 comprise a variable region lacking T-cell epitopes (see specification 34 / 117 pages 39 CN 122003436 A) and a human constant region. Briefly, the variable region of the antibody is cloned, and the T-cell epitopes are subsequently identified by testing overlapping peptides derived from the antibody variable region in a T-cell proliferation assay. T-cell epitopes were identified using computational methods to identify peptides that bind to human MHC class II. Mutations were introduced into the variable region to eliminate binding to human MHC class II. The mutated variable region was then used to generate deimmunizing antibodies.

[0266] 5.2.3 Single-domain antibody variants

[0267] One or more amino acid sequence modifications of single-domain antibodies that bind to DLL3 as described herein may be considered. For example, it may be necessary to optimize the binding affinity and / or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Therefore, in addition to those described herein,In addition to DLL3-binding single-domain antibodies, variants of DLL3-binding single-domain antibodies described herein are also considered. For example, single-domain antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or peptide. Those skilled in the art will understand that amino acid alterations can change the post-translational process of a single-domain antibody.

[0268] Chemical Modification

[0269] The single-domain antibodies provided herein can be chemically modified, for example, by covalently linking any type of molecule to the single-domain antibody. Antibody derivatives may include antibodies that have already been chemically modified, for example by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, or conjugation to one or more immunoglobulin domains (e.g., Fc or a portion of Fc). Any of a variety of chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, antibodies may contain one or more non-classical amino acids.

[0270] The antibodies provided herein can be modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of antibody glycosylation sites can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0271] When the single-domain antibody provided herein is fused to the Fc region, the carbohydrate linked thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are typically linked to Asn297 of the CH2 domain of the Fc region via an N-bond. See, for example, Wright et al. TIBTECH 15:2632 (1997). Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the “stem” of the biantennary oligosaccharide structure. The oligosaccharides in the binding molecules provided herein can be modified to produce variants with certain improved properties.

[0272] When the single-domain antibody provided herein is fused to the Fc region, the antibody variants provided herein may have a carbohydrate structure lacking (directly or indirectly) fucose linked to said Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the glycan chain at Asn 297 relative to the sum of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) linked to Asn 297, as described, for example, in WO 2008 / 077546. Asn 297 refers to the asparagine residue (EU number of Fc region residues) located at approximately position 297 in the Fc region; however, due to the small amount of fucose in the antibody...Sequence variation: Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between position 294 and position 300. Such fucosylated variants can possess improved ADCC function. See, for example, U.S. Patent Publications US 2003 / 0157108 and US 2004 / 0093621. Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Specification 35 / 117 pages 40 CN 122003436 A Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108; and WO 2004 / 056312, especially in Example 11), as well as knockout cell lines such as α-1,6-fucosylation 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 WO 2003 / 085107).

[0273] Binding molecules comprising the single-domain antibodies provided herein may further be provided with aliquoted oligosaccharides, for example, wherein the biantennary oligosaccharide linked to the Fc region is aliquoted with GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No.6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variations having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such variants may have improved CDC function. Such variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0274] In molecules comprising the single-domain antibody of the present invention and the Fc region, one or more amino acid modifications may be introduced into the Fc region to generate Fc region variants. Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0275] This disclosure may consider variants having some (but not all) effector functions that make these variants ideal candidates for applications where the in vivo half-life of the binding molecule is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the binding molecule lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding capacity. Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Patent Nos. 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 Bruggemann, M. et al., J. Exp. Med. 166:1351–1361 (1987)). Alternatively, non-radioactive assays may be used, see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA); and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo, for example in animal models, such as those described in Clynes et al., Proc. Nat'l Acad. Sci. USA.As disclosed in 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. 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 determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0276] Binding molecules with reduced effector function include those that substitute one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the Fc mutant known as “DANA” (US Patent No. 7,332,581) in which residues 265 and 297 are substituted with alanine. Specification 36 / 117 pages 41 CN 122003436 A

[0277] describes certain variants that enhance or degrade binding with FcR. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0278] Variants may contain an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (EU numbers of residues) within the Fc region). Alterations can occur within the Fc region, leading to changes in C1q binding and / or complement-dependent cytotoxicity (CDC) (i.e., increases or decreases), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0279] Binding molecules with prolonged half-life and improved binding to neonatal Fc receptors (FcRn) (which are responsible for binding maternal...)The transfer of IgG from the body to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) is described in US 2005 / 0014934 A1 (Hinton et al.). Those molecules contain an Fc region with one or more amino acid substitutions, where these substitutions improve the binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of the following 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, substituted Fc region residue 434 (US Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, which provides for other examples of Fc region variants.

[0280] It may be necessary to produce cysteine-engineered antibodies in which one or more residues of the antibody are replaced by cysteine ​​residues. The substituted residues may appear at accessible sites on the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby located at accessible sites on the antibody and can be used to conjugate the antibody to other parts.

[0281] Substitution, Deletion, or Insertion

[0282] Mutation can 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. Target sites for substitution mutagenesis include CDR and FR.

[0283] Amino acid substitution can be the result of replacing one amino acid with another that has similar structure and / or chemical properties, such as replacing leucine with serine, for example, a conserved amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the molecule provided herein, including, for example, site-directed mutagenesis leading to amino acid substitutions and PCR-mediated mutagenesis. Insertions or deletions may optionally range from about 1 to 5 amino acids. Substitutions, deletions, or insertions may include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. Substitutions may be conserved amino acid substitutions at one or more predicted non-essential amino acid residues. Permissible variations can be determined by systematically inserting, deleting, or substituting amino acids in the sequence and testing the activity exhibited by a parent antibody of the resulting variant.

[0284] Amino acid sequence insertions include N-terminal and / or C-terminal fusions, lengths ranging from one residue to a polypeptide containing multiple residues, and insertions into sequences of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue.

[0285] This disclosure includes single-domain antibodies generated by conserved amino acid substitution. In conserved amino acid substitution, the amino acid residue is replaced by an amino acid residue having a side chain with a similar charge. As described above, families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be randomly introduced along all or part of the coding sequence, for example, through saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed, and the protein's activity can be determined. Conservative substitutions (e.g., within groups of amino acids with similar properties and / or side chains) can be made to maintain or not significantly alter the properties. Exemplary substitutions are shown in Table 3 below.

[0286] Table 3. Amino Acid Substitutions

[0287]

[0288] Amino acids can be grouped according to the similarity of their side chain properties (see, for example, Lehninger, Biochemistry 73-75 (2nd edition 1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Uncharged polar: 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 can be grouped according to common side-chain characteristics: (1) Hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) Neutral-hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic:(4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe. For example, any cysteine ​​residue that does not participate in maintaining the correct conformation of a single-domain antibody may also be replaced, for example, with another amino acid such as alanine or serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Non-conservative substitutions would require exchanging members of one of these categories for another.

[0289] One type of substitution variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variants selected for further research will have modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (e.g., those described herein). In short, one or more CDR residues are mutated, and the variant antibody is displayed on the phage and screened for specific biological activities (e.g., binding affinity).

[0290] Changes (e.g., substitutions) can be made in the CDR, for example, to improve antibody affinity. Such changes can be made in the CDR “hotspots,” which are residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179–196 (2008)) and / or the SDR (a-CDR), where the binding affinity of the resulting variant antibody or fragment thereof is tested. Affinity maturation by constructing and reselecting from secondary libraries is described, for example, Hoogenboom et al. Methods in Molecular Biology 178:1–37 (edited by O'Brien et al., specification 38 / 117 pages 43 CN 122003436 A Human Press, Totowa, NJ, (2001)). For affinity maturation, diversity can be introduced into the variable gene selected for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, where several CDR residues (e.g., 4–6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. A more detailed description of affinity maturation is provided in the following sections.

[0291] Substitution, insertion, or deletion can occur within one or more CDRs, as long as such changes do not substantially reduce the antibody’s ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions as provided herein) can be made in CDRs that do not substantially reduce binding affinity. Each CDR of the variant VHH sequence provided herein can be unchanged or can contain no more than one, two, or three amino acid substitutions.

[0292] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called “alanine scan mutagenesis,” as described in Cunningham and Wells, Science, 244:1081–1085 (1989). In this method, a residue or group of residues of a target residue (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Additional substitutions can be introduced at amino acid positions to demonstrate functional sensitivity to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex can be determined to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain the desired properties.

[0293] Amino acid sequence insertions include fusion of the amino terminus and / or carboxyl terminus, lengths ranging from one residue to peptides containing one hundred or more residues, and insertions within sequences of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or peptide that increases the serum half-life of the antibody.

[0294] Variations can be performed using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, for example, Carter, Biochem J. 237:1-7 (1986); and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, for example, Wells et al., Gene 34:315-23 (1985)) or other known techniques can be used to produce single-domain antibody variant DNA.

[0295] 5.2.4 In vitro affinity maturation

[0296] Antibody variants with improved properties such as affinity, stability or expression levels compared to parental antibodies can be prepared by in vitro affinity maturation. As with the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. Antibody libraries are displayed on the surface of an organism (e.g., bacteriophage, bacteria, yeast or mammalian cells) or with their encoding mRNA.Or DNA association (e.g., covalent or non-covalent). Affinity selection of the displayed antibody allows the isolation of an organism or complex carrying the genetic information encoding the antibody. Two or three rounds of mutation and selection using display methods such as phage display typically produce antibody fragments with affinity in the low nanomolar range. Affinity-matured antibodies can have nanomolar or even picomolar affinity for the target antigen.

[0297] Phage display is a common method for displaying and selecting antibodies. Antibodies are displayed as fusions with phage coat proteins on the surface of an Fd or M13 phage. Selection involves exposing the antigen to allow the antibodies displayed by the phage to bind to their targets; this process is called “panning.” Phages that bind to the antigen are recovered and used to infect bacteria to produce phages for further rounds of selection. For reviews, see, for example, Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002); and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004). Specification 39 / 117 pages 44 CN 122003436 A

[0298] In yeast display systems (see, for example, Boder et al., Nat. Biotech. 15:553-57 (1997); and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies can be fused to the adhesion subunit of the yeast lectin protein Aga2p, which is linked to the yeast cell wall via a disulfide bond with Aga1p. Displaying the protein via Aga2p causes the protein to protrude from the cell surface, thereby minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen libraries to select antibodies with improved affinity or stability. The binding to the target soluble antigen is determined by labeling yeast with a biotinylated antigen and a second reagent, such as streptavidin, conjugated with a fluorophore. Changes in antibody surface expression can be measured by immunofluorescence labeling of hemagglutinin or c-Myc epitope tags (e.g., scFv) flanking single-chain antibodies. Expression has been shown to correlate with the stability of the displayed protein, and therefore antibodies can be selected to improve both stability and affinity (see, for example, Shusta et al., J. Mol. Biol. 292:949-56 (1999)). Another advantage of yeast display is the use of endoplasmic reticulum chaperone proteins and quality control mechanisms, where the displayed protein folds within the endoplasmic reticulum of eukaryotic yeast cells. Once maturation is complete, antibody affinity can be conveniently “tied” simultaneously with the display on the yeast surface, eliminating the need for expression and purification of each clone. The theoretical limitation of yeast surface display is that it is less efficient than other display methods.Smaller functional library sizes are possible; however, recent methods use a yeast cell mating system to generate combinatorial diversity with an estimated size of 10¹⁴ (see, for example, U.S. Patent Publication 2003 / 0186374; and Blaise et al., Gene 342:211-18 (2004)).

[0299] 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 fused to a spacer sequence lacking a stop codon. This spacer sequence remains attached to the peptidyl tRNA and occupies the ribosome channel during translation, thereby causing the target protein to protrude from the ribosome and fold. The resulting complex of mRNA, ribosome, and protein can bind to a surface-bound ligand, allowing simultaneous separation of the antibody and its encoding mRNA by affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed back into cDNA, which can then be mutagenized and used for the next round of selection (see, for example, Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). In mRNA display, puromycin is used as an adaptor molecule to establish a covalent bond between the antibody and the mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0300] Because these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, the diversity of the library is not limited by the efficiency of bacterial cell transformation, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be easily introduced after each round of selection, for example, by non-proofreading polymerase, since no library needs to be transformed after any diversification step.

[0301] Mammalian display systems can be used.

[0302] Diversity can also be introduced into the CDRs of an antibody library either by targeting or by random introduction. The former involves targeting all CDRs of the antibody by high or low levels of mutagenic sequence or targeting isolated hotspots of somatic hypermutation (see, for example, Ho et al., J. Biol. Chem. 280:607-17 (2005)) or residues suspected of affecting affinity for experimental or structural reasons. Diversity can also be introduced by replacing naturally diverse regions with DNA shuffling or similar techniques (see, for example, Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Patents 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops extending into the framework region residues (see, for example, Bond et al., J. Mol. Biol. 348:699-709).(2005)), using loop deletions and insertions or hybridization-based diversification in CDRs (see, for example, U.S. Patent Publication No. 2004 / 0005709). Other methods for generating diversification in CDRs are disclosed, for example, in U.S. Patent No. 7,985,840. Other methods that can be used to generate antibody libraries and / or mature antibody affinity are disclosed, for example, in U.S. Patent Nos. 8,685,897 and 8,603,930 and U.S. Publications Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855 and 2009 / 0075378, each of which is incorporated herein by reference. Specification 40 / 117 pages 45 CN 122003436 A

[0303] The screening of libraries can be accomplished by various techniques known in the art. For example, single-domain antibodies can be immobilized on solid supports, columns, needles, or cellulose / poly(vinylidene fluoride) membranes / other filter membranes, expressed on host cells attached to an adsorption plate or used for cell sorting, conjugated with biotin to capture with streptavidin-coated beads, or used in any other method to pan for display libraries.

[0304] For reviews 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 references therein.

[0305] 5.2.5 Modification of Single-Domain Antibodies

[0306] Covalent modification of single-domain antibodies is included within the scope of this disclosure. Covalent modification includes reacting the target amino acid residues of the single-domain antibody with an organic derivatizer capable of reacting with selected side chains or N-terminal or C-terminal residues of the single-domain antibody. Other modifications include deamidation of glutamine acyl and asparagyl residues to the corresponding glutamine acyl and asparagyl residues, hydroxylation of proline and lysine, hydroxyphosphorylation of serine or threonyl residues, methylation of the α-amino group of the lysine, arginine, and histidine side chains (see, for example, Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0307] Other types of covalent modifications of single-domain antibodies included within the scope of this disclosure include altering the native glycosylation pattern of the antibody or peptide as 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 antibodies can be linked to one of a variety of non-protein polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyethylene, in a manner as described, for example, in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. Single-domain antibodies binding to the DLL3 disclosed herein can also be fused or conjugated to one or more immunoglobulin constant regions or portions thereof (e.g., Fc) genes to prolong half-life and / or confer known Fc-mediated effector functions.

[0308] Single-chain antibodies that bind to DLL3 disclosed herein can also be modified to form chimeric molecules comprising a DLL3-binding single-chain antibody fused to another heterologous polypeptide or amino acid sequence, such as an epitope tag (see, for example, Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)) or the Fc region of an IgG molecule (see, for example, Aruffo, Antibody Fusion Proteins 221-42 (edited by Chamow and Ashkenazi, 1999)). DLL3-binding single-chain antibodies can also be used to generate chimeric antigen receptors (CARs) that bind to DLL3, as described in more detail below.

[0309] Fusion proteins comprising a single-chain antibody that binds to DLL3 disclosed herein, and a heterologous polypeptide. Heterologous polypeptides fused to or chemically conjugated to antibody genes can be used to target antibodies to cells having DLL3 expressed on their cell surface.

[0310] A set of antibodies that bind to the DLL3 antigen is also provided herein. The antibody set may have different association rates, different dissociation rates, different affinities for the DLL3 antigen, and / or different specificities for the DLL3 antigen. The set contains about 10 to about 1000 or more antibodies or is composed of about 10 to about 1000 or more antibodies. The antibody set can be used in, for example, 96-well or 384-well plates for assays such as ELISA.

[0311] 5.2.6 Preparation of Single-Domain Antibodies

[0312] Methods for preparing single-domain antibodies have been described. See, for example, Els Pardon et al., Nature Protocol, 9(3): 674 (2014). Single-domain antibodies (such as VHH) can be obtained using methods known in the art, such as by immunizing camelid species (such as camels or llamas) and obtaining hybridomas therefrom, or by cloning a single-domain antibody library using molecular biology techniques known in the art and subsequently selecting it by ELISA with a single clone of an unselected library or by using phage display. Instruction manual, pages 41 / 117, 46 CN 122003436 A

[0313] The single-domain antibodies described herein can be produced by culturing or transfecting cells with a vector containing nucleic acid encoding the single-domain antibody. The polynucleotide sequence encoding the polypeptide component of the antibody disclosed herein can be obtained using standard recombinant techniques. The desired polynucleotide sequence can 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 technology. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a host cell. 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 in the vector to be inserted and the specific host cell for transformation with the vector. Suitable host cells for expressing the antibodies disclosed herein include prokaryotes such as archaea and eubacteria, including Gram-negative or Gram-positive organisms; eukaryotic microorganisms such as filamentous fungi or yeast; invertebrate cells such as insect or plant cells; and vertebrate cells such as mammalian host cell lines. The host cells are transformed with the expression vector described above and cultured in a conventional nutrient medium appropriately modified to induce promoters, select transformants, or amplify genes encoding the desired sequence. Antibodies produced from host cells were purified using standard protein purification methods known in the art.

[0314] Methods for antibody production, including vector construction, expression, and purification, are further described in the following literature: Plückthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (edited by McCafferty et al., 1996); Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, Current Protocols in Protein Science (2009); Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, Antibody Expression and Production (edited by Al-Rubeai, 2011); and Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009).

[0315] Of course, alternative methods well known in the art can be used to prepare anti-DLL3 single-domain antibodies. For example, suitableAmino acid sequences or portions thereof can be generated using solid-phase techniques via direct peptide synthesis (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 can be performed using artificial techniques or by automation. Individual portions of an anti-DLL3 antibody can be chemically synthesized individually and combined using chemical or enzymatic methods to produce the desired anti-DLL3 antibody. Alternatively, antibodies can be purified from cells or body fluids such as milk of transgenic animals engineered to express antibodies, as disclosed in U.S. Patent Nos. 5,545,807 and 5,827,690.

[0316] In particular, the single-domain antibodies or other DLL3-binding proteins provided herein can be generated by immunizing llamas, sorting individual B cells, extracting the V gene, cloning DLL3-binding proteins (such as the VHH-Fc fusion), and then expressing and purifying them on a small scale. Further screening can be performed on single-domain antibodies and other molecules that bind to DLL3, including selecting one or more of ELISA positivity, BLI positivity, and KD less than 100 nM. These selection criteria can be combined as described in Section 6 below. In addition, the ability of individual VHH binding proteins (and other molecules that bind to DLL3) to bind to cells expressing DLL3 can be determined. Such determinations can be performed using FACS analysis of cells expressing DLL3 and measuring the mean fluorescence intensity (MFI) of fluorescently labeled VHH molecules. The above aspects will be described in more detail below.

[0317] Polyclonal Antibodies

[0318] Polyclonal antibodies are usually produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Using bifunctional agents or derivatizing agents, such as maleimide benzoyl sulfosuccinimide ester (conjugated via cysteine ​​residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR (where R and R1 are independently lower alkyl groups), it may be useful to conjugate the relevant antigen with a protein (e.g., keyhole hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) that is immunogenic in the species to be immunized. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, a synthetic mycobacterium trehalose ester). The immunization regimen can be selected by those skilled in the art without excessive experimentation.

[0319] For example, by conjugating, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes ofFreund's complete adjuvant combination and intradermal injection solution at multiple sites are used to immunize animals against antigens, immunogenic conjugates, or derivatives. One month later, animals are boosted with 1 / 5 to 1 / 10 of the original amount of peptides or conjugates in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, blood is taken from the animals and serum antibody titers are determined. Animals are boosted until the titer plateaus. Conjugates can also be prepared as protein fusions in recombinant cell cultures. In addition, aggregating agents such as alum are suitable for enhancing the immune response.

[0320] Monoclonal Antibodies

[0321] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising this population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies.

[0322] For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by a recombinant DNA method (US Patent No. 4,816,567).

[0323] In the hybridoma method, a suitable host animal is immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to proteins used for immunization. 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)).

[0324] Immunizing agents typically comprise an antigen protein or a fusion variant thereof. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103. Immortalized cell lines are typically transformed mammalian cells. The hybridoma cells thus prepared are seeded and grown in a suitable culture 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 effectively, support stable high levels of antibody production by selected antibody-producing cells, and are sensitive to the culture medium (e.g., HAT medium).

[0325] The production of monoclonal antibodies against the antigen in the culture medium in which the hybridoma cells are grown is determined. The presence of monoclonal antibodies against the desired antigen in the culture medium in which the hybridoma cells are cultured can be determined. Such techniques and determinations are known in the art. For example, binding affinity can be determined by Munson et al.,The determination was made using Scatchard analysis of Anal. Biochem., 107:220 (1980).

[0326] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clonal subclones can be grown using a limiting dilution procedure and standard methods (Goding, ibid.). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 media. Alternatively, hybridoma cells can be grown as tumors in mammals.

[0327] Monoclonal antibodies secreted by the subclones are appropriately separated from the culture medium, ascites, or serum using routine immunoglobulin purification procedures such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0328] Monoclonal antibodies can also be prepared using recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 and as described above. DNA encoding monoclonal antibodies can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or non-immunoglobulin-producing myeloma cells to synthesize monoclonal antibodies in such recombinant host cells. Review articles 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).

[0329] Antibodies can be isolated from antibody phage libraries generated using the techniques 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 strategies for constructing very large phage libraries, including chain tamponade (Marks et al., Bio / Technology, 10:779-783 (1992)) and the generation of high-affinity (nM range) human antibodies through combined infection and in vivo recombination (Waterhouse et al., Nucl. Acids Res., 21: 2265-2266).(1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0330] DNA can also be modified, for example by substituting the coding sequence (US Patent No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently linking 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 chimeric bivalent antibodies containing one antigen-specific antigen-binding site and another antigen-binding site specific to a different antigen.

[0331] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutyrylimide.

[0332] Recombinant Production in Prokaryotic Cells

[0333] The polynucleotide sequence encoding the antibody disclosed herein can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells such as hybridoma cells. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing the heteropolynucleotide in a prokaryotic host. Many vectors available in the art and known 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 in the vector to be inserted and the specific host cell to which the vector is to be transformed. Each vector contains different components depending on its function (amplification or expression of the heteropolynucleotide, or both) and its compatibility with the specific host cell in which it is located. Vector components typically 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 heteronucleotide insert, and a transcription termination sequence.

[0334] Typically, plasmid vectors containing replicons and control sequences derived from species compatible with the host cell are used in conjunction with these hosts. Vectors typically carry a replication site and a marker sequence that provides phenotypic selection in transformed cells. For example, *E. coli* is typically transformed using pBR322 (a plasmid derived from the *E. coli* species). Examples of pBR322 derivatives used to express specific antibodies are described in detail in U.S. Patent No. 5,648,237 to Carter et al.

[0335] Additionally, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors associated with these hosts. For example, phages such as GEM™-11 can be used to prepare recombinant vectors that can be used to transform susceptible host cells such as *E. coli* LE392.

[0336] The expression vectors disclosed herein may contain two or more promoter-cistron pairs encoding each polypeptide component. A promoter is a non-translational regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters are generally classified into two classes: inducible and constitutive. An inducible promoter is a promoter that initiates an increased level of transcription of the cistron under its control in response to changes in culture conditions, such as the presence or absence of nutrients or changes in temperature.

[0337] A large number of promoters recognized by a variety of potential host cells are well known. Selected promoters can be operatively linked to cistron DNA encoding the antibodies of the present invention by digestion of the source DNA with restriction enzymes to remove the promoter and inserting the isolated promoter sequence into the vector disclosed herein. Natural promoter sequences and many heterologous promoters can be used to direct the amplification and / or expression of target genes. Heterologous promoters can be used because they generally allow for greater transcription and higher yield of the expressed target gene compared to natural target polypeptide promoters.

[0338] Promoters suitable for prokaryotic hosts include the PhoA promoter, β-galactosidase and lactose promoter systems, tryptophan (trp) promoter systems, and hybrid promoters such as the tac or trc promoters. However, other promoters that function in bacteria, such as other known bacterial or bacteriophage promoters, are also suitable. Their nucleic acid sequences have been disclosed, allowing technicians to operatively link them to cistrons encoding target peptides using adapters or adaptors (Siebenlist et al., Cell 20: 269 (1980)) to provide any desired restriction site.

[0339] In one aspect, each cistron within the recombinant vector contains a secretory signal sequence component that directs the transmembrane translocation of the expressed polypeptide. Typically, the signal sequence can be an integral part of the vector, or it can be a portion of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of this disclosure should be a signal sequence that can be recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence can be replaced by a prokaryotic signal sequence selected, for example, from the group consisting of alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA, and MBP.

[0340] Antibody production according to this disclosure can occur in the cytoplasm of the host cell and therefore does not require the presence of a secretory signal sequence in each cistron. Certain host strains (e.g., *Escherichia coli* trxB- strain) provide cytoplasmic conditions favorable for disulfide bond formation, thereby allowing the expressed protein subunits to fold and assemble correctly.

[0341] Suitable prokaryotic host cells for expressing the antibodies disclosed herein include archaea and eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia coli (e.g., *Escherichia coli*), Bacillus (e.g., *Bacillus subtilis*), Enterobacter, Pseudomonas (e.g., *Pseudomonas aeruginosa*), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Hyaluronic acid bacteria, or Paracoccus. Gram-negative cells can be used. *Escherichia coli* cells can be used as a 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 Accession No. 27,325) and its derivatives, including strain 33D3 (US Patent No. 5,639,635) with the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A(nmpc-fepE) degP41 kanR). Other strains and their derivatives, such as *E. coli* 294 (ATCC 31,446), *E. coli* B, *E. coli* 1776 (ATCC 31,537), and *E. coli* RV308 (ATCC 31,608), are also suitable. These examples are illustrative and not limiting. Methods for constructing any of the above-described bacterial derivatives with a defined genotype are known in the art and described, for example, in Bass et al., Proteins, 8:309-314 (1990). Given the reproducibility of the replicon in bacterial cells, it is generally necessary to select suitable bacteria. For example, *Escherichia coli*, *Serratia*, or *Salmonella* species may be suitable as hosts when replicons are provided using well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410.

[0342] Typically, the host cell should secrete a minimal amount of proteolytic enzymes, and additional protease inhibitors may ideally be incorporated into the cell culture.

[0343] The host cells are transformed with the above-described expression vector and cultured in a conventional nutrient medium appropriately modified to induce promoters, select transformants, or amplify genes encoding desired sequences, as per specification page 45 / 117, 50 CN 122003436 A. Transformation refers to the introduction of DNA into a prokaryotic host, enabling the DNA to replicate as an extrachromosomal element or via chromosomal integrons. Transformation is performed using standard techniques suitable for the host cells used. Calcium treatment with calcium chloride is commonly used in environments with significant cell wall barriers.Bacterial cells. Another transformation method uses polyethylene glycol / DMSO. Another technique used is electroporation.

[0344] Prokaryotic cells used to generate the antibodies disclosed herein are grown in media known in the art and adapted to culture selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. The media may also contain selectants chosen based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the media to allow the growth of cells expressing an ampicillin resistance gene.

[0345] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources may also be introduced individually at appropriate concentrations or as a mixture with another supplement or medium 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, mercaptoglycolate, dithioerythritol, and dithiothreitol. Prokaryotic host cells are cultured at suitable temperature and pH.

[0346] If an inducible promoter is used in the expression vector of this disclosure, protein expression is induced under conditions suitable for promoter activation. In one aspect of this disclosure, the PhoA promoter is used to control the transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-restricted medium for induction. For example, the phosphate-restricted medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods 263:133-147 (2002)). Depending on the vector construct used, a variety of other inducers known in the art can be used.

[0347] The expressed antibody of this disclosure is secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves destroying the microorganism, usually by methods such as osmotic shock, sonication, or lysis. Once the cells are destroyed, cell debris or intact cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transferred to a culture medium and isolated therein. Cells can be removed from the culture, and the culture supernatant can be filtered and concentrated for further purification of the resulting protein. The expressed peptides can be further separated and identified using known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0348] Alternatively, protein production can be carried out in large quantities by fermentation. Various large-scale fed-batch fermentation programs can be used to produce recombinant proteins. To improve the yield and quality of the antibodies disclosed herein, various fermentation conditions can be modified. For example, chaperone proteins have been shown to contribute to the proper folding and dissolution 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).

[0349] In order to minimize the proteolysis of expressed heterologous proteins, especially those sensitive to proteolysis, certain host strains lacking proteolytic enzymes can be used in this disclosure, such as, for example, U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996). E. coli strains lacking proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins can be used as host cells in the expression system encoding the antibodies disclosed herein.

[0350] The antibodies produced herein can be further purified to obtain substantially homogeneous formulations for further assays and uses. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica or cation exchange resins such as DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75. Protein A immobilized on a solid phase can, for example, be used for the immunoaffinity purification of the binding molecules disclosed herein. The solid phase immobilizing protein A can be a column comprising a glass or silica surface, or a controlled-pore glass column or silica column. The column may have been coated with a reagent such as glycerol in an attempt to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants that are nonspecifically bound to the solid phase. Finally, the target antibody is recovered from the solid phase by elution.

[0351] Recombinant Production in Eukaryotic Cells

[0352] For eukaryotic expression, the vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0353] The vector used for the eukaryotic host may also be an insert of another polypeptide that encodes a signal sequence or has a specific cleavage site at the N-terminus of a mature protein or polypeptide. The selected heterologous signal sequence may be a heterologous signal sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In mammalian cell expression, mammalian signals can be obtained.Sequences and viral secretion leader sequences, such as the herpes simplex gD signal. DNA from such precursor regions can be linked in the reading frame to DNA encoding the disclosed antibody.

[0354] Typically, mammalian expression vectors do not require a replication origin component (the SV40 origin is typically only used because it contains an early promoter).

[0355] Expression and cloning vectors may contain selection genes, also known as selection markers. Selection genes may encode proteins that confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline); supplement auxotrophic deficiencies; or provide key nutrients unavailable from complex culture media.

[0356] One example of a selection scheme utilizes a drug to inhibit the growth of host cells. Cells successfully transformed with a heterologous gene produce a drug-conferring protein and thus survive in the selection scheme. Examples of such advantageous selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0357] Another example of a suitable selection marker for mammalian cells is one capable of identifying those cells that can take up nucleic acids encoding the disclosed antibody. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx) (a competitive antagonist of DHFR). When using wild-type DHFR, an exemplary suitable host cell is a DHFR-deficient Chinese hamster ovary (CHO) cell line. Alternatively, host cells transformed or co-transformed with a polypeptide encoding a DNA sequence, wild-type DHFR protein, and another selective marker such as aminoglycoside 3'-phosphotransferase (APH) (especially wild-type hosts containing endogenous DHFR) can be selected by cell growth in a medium containing a selective agent for selective labeling, such as an aminoglycoside antibiotic.

[0358] Expression and cloning vectors typically contain a promoter that is recognized by the host organism and operatively linked to a nucleic acid encoding the desired polypeptide sequence. Eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Another sequence may be included, located 70 to 80 bases upstream of the transcription start site of many genes. The 3' end of most eukaryotes can be a signal for adding a polyadenylated tail to the 3' end of a coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0359] Transcription of polypeptides from vectors in mammalian host cells can be controlled, for example, by promoters obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenoviruses (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40)), heterologous mammalian promoters (e.g., actin promoters or immunoglobulin promoters), and heat shock promoters, provided that these promoters are compatible with the host cell system.

[0360] Transcription of DNA encoding the antibodies disclosed herein in higher eukaryotes is typically amplified by inserting enhancer sequences into vectors. Many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are now known. Examples include the SV40 enhancer (bp 100-270) located late at the origin of replication, the early promoter enhancer of cytomegalovirus (CMV) on page 52 of CN 122003436 A, the polyoma enhancer located late at the origin of replication, and the adenovirus enhancer. For enhancing elements used to activate eukaryotic promoters, see also Yaniv, Nature 297:17-18 (1982). Enhancers can be spliced ​​into vectors at the 5' or 3' position of the polypeptide coding sequence, but are preferably located at the 5' site of the promoter.

[0361] Expression vectors for eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also contain sequences required to terminate transcription and stabilize mRNA. Such sequences are typically obtained from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide fragments that are transcribed into polyadenylated fragments within the untranslated portion of a polypeptide encoding mRNA. A useful transcription termination component is the bovine growth hormone polyadenylated region.

[0362] Suitable host cells for cloning or expressing DNA in the vectors described herein include higher eukaryotic cells, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include: monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cell subclones used for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Support cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); and human cervical cancer cells (HELA, ATCC CCL 1651). 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep...G2, HB 8065; mouse mammary tumors (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad . Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2).

[0363] Host cells can be transformed with the expression or cloning vectors described above for antibody production and cultured in conventional nutrient media that are appropriately modified to induce promoters, select transformants, or amplify genes encoding desired sequences.

[0364] Host cells used to produce the antibodies disclosed herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Duchenne Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. Additionally, any culture medium described in Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Reissue Patent 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the GENTAMYCIN™ drug), trace elements (defined as inorganic compounds typically present in micromolar final concentrations), and glucose or equivalent energy. It may also include any other necessary supplements at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc., are those previously used with the host cells selected for expression and will be obvious to those skilled in the art.

[0365] When using recombinant technology, antibodies can be produced intracellularly, in the interstitial space, or secreted directly into the culture medium. If antibodies are produced intracellularly, as a first step, particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. When antibodies are secreted into the culture medium, the supernatant from such expression systems is typically concentrated first using a commercially available protein concentrator filter (e.g., an Amicon or Millipore Pellicon ultrafiltration device). Protease inhibitors, such as those described on pages 48 / 117 of the specification, are also included.122003436 A PMSF may be included in any of the foregoing steps to inhibit proteolysis and may include antibiotics to prevent the growth of foreign contaminants.

[0366] Protein compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The matrix to which the affinity ligand is attached is typically agarose, but other matrices are also available. Mechanically stable matrices such as controlled-pore glass or poly(styrene-divinyl)benzene allow for faster flow rates and shorter processing times compared to agarose. Depending on the antibody to be recovered, other protein purification techniques may also be used, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin SEPHAROSE™ chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation. After any one or more preliminary purification steps, the mixture containing the target antibody and contaminants can be subjected to low-pH hydrophobic interaction chromatography.

[0367] 5.2.7 Binding molecule comprising a single-domain antibody

[0368] In another aspect, this document provides a binding molecule comprising a single-domain antibody (e.g., targeting the VHH domain of DLL3) provided herein. In addition to the chimeric antigen receptor (CAR) provided herein as described in Section 5.3 below, the single-domain antibody targeting DLL3 provided herein is also part of other binding molecules. Exemplary binding molecules of this disclosure are described herein.

[0369] Fusion protein

[0370] The single-domain antibody provided herein can be fused to or chemically conjugated to the gene of another agent (e.g., a protein-based entity). The single-domain antibody can be chemically conjugated to the agent or otherwise non-covalently conjugated to the agent. The agent can be a peptide or antibody (or a fragment thereof).

[0371] Therefore, what is provided herein can be single-domain antibodies (e.g., VHH domains) that are recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) with a heterologous protein or polypeptide (or fragments thereof, such as about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 amino acids or more than 500 amino acids) to generate fusion proteins, and their uses. In particular, this document provides fusion proteins comprising antigen-binding fragments (e.g., CDR1, CDR2, and / or CDR3) of the single-domain antibodies provided herein and heterologous proteins, polypeptides, or peptides.

[0372] Furthermore, the antibodies provided herein can be fused with labeled or “tag” sequences (such as peptides) to facilitate purification. The labeled or tagged amino acid sequence can be a hexahistine peptide, a hemagglutinin (“HA”) tag, or a “FLAG” tag.

[0373] Methods of fusing or conjugating portions (including peptides) with antibodies are known (see, for example, Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, Monoclonal Antibodies and Cancer Therapy 243-56 (edited by Reisfeld et al., 1985); Hellstrom et al., Antibodies for Drug Delivery, Controlled Drug Delivery 623-53 (edited by Robinson et al., 2nd edition, 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, Monoclonal Antibodies: Biological and Clinical Applications 475-506 (edited by Pinchera et al., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, Monoclonal Antibodies for Cancer Detection and Therapy 303-16 (edited by Baldwin et al.)). 1985); Thorpe et al., Immunol. Rev. 62:119-58 (1982); U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT Publication WO 91 / Specification 49 / 117 pages 54 CN 122003436 A 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631 and WO 99 / 04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-39 (1991); Traunecker et al.,Nature, 331:84-86 (1988); Zheng et al., J. Immunol. 154:5590-600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-41 (1992).

[0374] Fusion proteins can be generated by, for example, gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively, “DNA shuffling”) techniques. DNA shuffling can be used to alter the activity of the single-domain antibodies presented herein, including, for example, antibodies with high affinity and low dissociation rates (see, for example, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patten et al., Curr. Opinion Biotechnol. 8:724–33 (1997); Harayama, Trends Biotechnol. 16(2):76–82 (1998); Hansson et al., J. Mol. Biol. 287:265–76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308–13 (1998)). Antibodies or their encoded antibodies can be altered prior to recombination by random mutagenesis using error-prone PCR, random nucleotide insertion, or other methods. The polynucleotide encoding the antibodies provided herein can be recombined with one or more components, motifs, segments, parts, domains, fragments, etc., of one or more heterologous molecules.

[0375] The single-domain antibodies (e.g., VHH domains) provided herein can be conjugated to a second antibody to form an antibody heteroconjugate.

[0376] The single-domain antibody can be fused to a drug gene. Gene fusion can be achieved by placing a linker (e.g., a polypeptide) between the single-domain antibody and the drug. The linker can be a flexible linker.

[0377] The single-domain antibody can be conjugated to a therapeutic molecule gene, wherein a hinge region links the single-domain antibody to the therapeutic molecule.

[0378] Methods for preparing the various fusion proteins provided herein are also provided herein. The various methods described in Section 5.2.6 above can also be used to prepare the fusion proteins provided herein.

[0379] The fusion proteins provided herein can be recombinantly expressed. Recombinant expression of the fusion proteins provided herein may require the construction of an expression vector containing a polynucleotide encoding the protein or a fragment thereof. Once the polynucleotide encoding the protein or a fragment thereof provided herein is obtained, a vector for generating the molecule can be produced using recombinant DNA technology known in the art. Therefore, this paper describes a method for preparing proteins by expressing polynucleotides containing encoding nucleotide sequences.Methods well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Reproducible vectors are also provided that contain nucleotide sequences operably linked to a promoter encoding a fusion protein provided herein or a fragment thereof or a CDR.

[0380] Expression vectors can be transferred to host cells using conventional techniques, and then the transfected cells can be cultured using conventional techniques to produce the fusion protein provided herein. Therefore, host cells containing polynucleotides operably linked to a heterologous promoter encoding a fusion protein provided herein or a fragment thereof are also provided herein.

[0381] A variety of host expression vector systems can be used to express the fusion proteins provided herein. Such host expression systems represent media through which the target coding sequence can be generated and subsequently purified, but also represent cells that can express the fusion proteins provided herein in situ when transformed or transfected with a suitable nucleotide coding sequence. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing coding sequences (e.g., *Escherichia coli* and *Bacillus subtilis*); yeast transformed with recombinant yeast expression vectors containing coding sequences (e.g., *Pichia pastoris*); insect cell systems infected with recombinant viral expression vectors containing coding sequences (e.g., baculoviruses); plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing coding sequences (e.g., Ti plasmids); or mammalian cell systems containing recombinant expression constructs (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). Bacterial cells such as *Escherichia coli*, or eukaryotic cells, particularly those used for expressing complete recombinant antibody molecules, can be used to express recombinant fusion proteins. For example, mammalian cells such as Chinese hamster ovary cells (CHO) bound to a major intermediate early gene promoter element from a vector such as human cytomegalovirus are an efficient expression system for antibodies or variants thereof. Expression of the nucleotide sequence encoding the fusion protein provided herein can be regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0382] In bacterial systems, a variety of expression vectors can be advantageously selected depending on the intended use of the expressed fusion protein. For example, when producing large quantities of such fusion proteins, a pharmaceutical composition for producing the fusion protein may be required.Vectors that guide the expression of easily purified, high-level fusion protein products. These vectors include, but are not limited to, the *E. coli* expression vector pUR278 (Ruther et al., EMBO 12:1791 (1983)), in which the coding sequence can be individually linked to the vector along with the lacZ coding region within the frame to produce the fusion protein; pIN vectors (Inouye & Inouye, *Nucleic Acids Res.* 13:3101–3109 (1985); Van Heeke & Schuster, *J. Biol. Chem.* 24:5503–5509 (1989)); and so on. pGEX vectors can also be used to express exogenous peptides as fusion proteins with glutathione 5-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site, allowing the cloned target gene product to be released from the GST portion.

[0383] In mammalian host cells, many virus-based expression systems can be used. In the case of adenovirus as the expression vector, the target coding sequence can be linked to the adenovirus transcription / translation control complex, such as a late promoter and a triple leader sequence. The chimeric gene can then be inserted into the adenovirus genome via in vitro or in vivo recombination. Insertion in non-essential regions of the viral genome (e.g., regions E1 or E3) will produce a viable recombinant virus capable of expressing the fusion protein in an infected host (see, for example, Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)). Efficient translation of the inserted coding sequence may also require specific initiation signals. These signals include the ATG start codon and adjacent sequences. Furthermore, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire inserted fragment. These exogenous translation control signals and start codons can have a variety of sources, including natural and synthetic. Expression efficiency can be improved by including appropriate transcriptional enhancer elements, transcription terminators, etc. (see, for example, Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0384] Additionally, host cell lines that regulate the expression of the inserted sequence or modify and process the gene product in a desired specific manner can be selected. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products.The appropriate cell line or host system can be selected to ensure proper modification and processing of the expressed exogenous protein. For this purpose, eukaryotic host cells with appropriate cellular mechanisms for processing primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells.

[0385] For long-term, high-yield production of recombinant proteins, stable expression can be utilized. For example, cell lines that stably express fusion proteins can be engineered. Besides using expression vectors containing viral replication origins, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectivity markers. After introducing exogenous DNA, engineered cells can be grown in enrichment media for 1–2 days and then transferred to selective media. Selectivity markers in the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing fusion proteins. Such engineered cell lines may be particularly useful in screening and evaluating compositions that interact directly or indirectly with binding molecules.

[0386] Many selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:8-17 (1980)) genes that can be used for tk-, hgprt- or aprt- cells, respectively. Furthermore, antimetabolite resistance can be used as a basis for selecting the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); and gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072).(1981)); neo, which confers resistance to aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Well-known methods in the field of recombinant DNA technology can be routinely applied to select desired recombinant clones, and such methods are described in, for example, the following literature: Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Dracopoli et al. (eds.), Current Protocols in Human Genetics, Chapters 12 and 13, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated herein by reference in their entirety.

[0387] The expression level of the fusion protein can be increased by vector amplification (for the review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When the marker in the vector system expressing the fusion protein is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the fusion protein gene,Therefore, the production of fusion proteins will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0388] Host cells can be co-transfected with a variety of expression vectors provided herein. Vectors can contain the same selective markers that enable the corresponding encoded polypeptides to be expressed equally. Alternatively, a single vector encoding and capable of expressing multiple polypeptides can be used. The coding sequence can contain cDNA or genomic DNA.

[0389] Once the fusion proteins provided herein have been produced by recombinant expression, they can be purified by any method known in the art for purifying polypeptides (e.g., immunoglobulin molecules), such as by chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after protein A, size column chromatography, and Kappa select affinity chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the fusion protein molecules provided herein can be fused with heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0390] Immunoconjugates

[0391] This disclosure also provides immunoconjugates that, with or without a linker, are conjugated to one or more agents (e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof) or radioisotopes) described herein (e.g., anti-DLL3 single-domain antibodies).

[0392] Immunoconjugates are antibody-drug conjugates (ADCs) in which an antibody is conjugated to one or more drugs, including but not limited to maytansine (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); olistatin, such as monomethyl olistatin drug portions DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588 and 7,498,298); saccharin; calcitrinin or derivatives thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336–3342 (1993); and Lode et al., Cancer Res. 58: 2925–2928 (1998); anthracyclines such as doxorubicin or doxycycline (see Kratz et al., Current Med. Chem. 13:477–523).(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358–362 (2006); Torgov et al., Bioconj. Chem. 16:717–721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829–834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529–1532 (2002); King et al., J. Med. Chem. 45:4336–4343 (2002); and US Patent No. 6,630,579); methotrexate; vindesin; taxanes, such as docetaxel, paclitaxel, larostaxate, testathaxate, and oxathaxate; trichothecene; and CC1065.

[0393] Immunoconjugates may comprise antibodies as described herein conjugated to an enzyme-active toxin or a fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from *Pseudomonas aeruginosa*), ricin A chain, abrin A chain, modisocyanate A chain, α-triscarcinone, tung oil protein, carnation toxin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, toxin protein, croton toxin, soapwort inhibitor, white tree toxin, mitomycin, localized aspergillin, phenolmycin, enoxacin, and teloflavin.

[0394] Immunoconjugates may comprise antibodies as described herein conjugated to a radioactive atom to form a radioconjugate. Various radioisotopes can be used to prepare radioconjugates. Examples include radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu. When radioactive conjugates are used for detection, they can contain radioactive atoms used for scintillation studies, such as tc99m or I123, or spin labels used for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0395] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyl dithio)propionate (SPDP), succinimide-4-(N-maleimide methyl)cyclohexane-1-carboxylic acid ester (SMCC), iminothiones (IT), and bifunctional derivatives of imine esters (such as adipamide dimethyl)Ester (HCl), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazido derivatives (such as bis(p-diazidobenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See WO 94 / 11026.

[0396] The linker can be a “cleavable linker” that promotes the release of the conjugate in the cell, but non-cleavable linkers are also considered herein. Linkers used in the conjugates disclosed herein include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), light-labile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to circumvent multidrug transporter-mediated resistance.

[0397] The immunoconjugates discussed herein are considered, but not limited to, such conjugates prepared with cross-linking agents, including but not limited to commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfonyl-EMCS, sulfonyl-GMBS, sulfonyl-KMUS, sulfonyl-MBS, sulfonyl-SIAB, sulfonyl-SMCC, and sulfonyl-SMPB and SVSB (succinimide-(4-vinyl sulfone)benzoate).

[0398] The antibodies provided herein may be conjugated or recombinantly fused with, for example, diagnostic molecules. Such diagnosis and detection can be accomplished, for example, by conjugating antibodies to detectable substances, including but not limited to various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin / biotin or avidin / biotin; fluorescent materials such as, but not limited to, umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, or jellyfish luminescent protein; and chemiluminescent materials such as 225AcγRadioactive isotopes that emit radiation, Auger emission, beta emission, alpha emission, or positron emission.

[0399] 5.3 Chimeric Antigen Receptor

[0400] In another aspect, this document provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising one or more single-domain antibodies (e.g., VHH) that bind to DLL3 as provided herein. Exemplary CARs comprising the VHH domain of the present invention (i.e., VHH-based CARs) are described in Section 6 below.

[0401] The chimeric antigen receptor (CAR) provided herein may comprise a polypeptide comprising: (a) an extracellular antigen-binding domain comprising one or more single-domain antibodies (sdAbs) that specifically bind to DLL3 as provided herein, and optionally one or more additional binding domains; (b) a transmembrane domain; and (c) an intracellular signal transduction domain. Each component and additional region are described in more detail below.

[0402] 5.3.1. Extracellular Antigen Binding Domain

[0403] The extracellular antigen binding domain of the CAR described herein comprises one or more single-domain antibodies (such as any one of 1, 2, 3, 4, 5, 6 or more). The single-domain antibodies may be directly fused to each other via peptide bonds or via peptide linkers.

[0404] Single-Domain Antibodies

[0405] The CAR disclosed herein comprises an extracellular antigen binding domain comprising one or more single-domain antibodies. The sdAbs may have the same or different sources and have the same or different sizes. Exemplary sdAbs include, but are not limited to, heavy chain variable domains (e.g., VHH or VNAR) from heavy chain-only antibodies, naturally light chain-deficient binding molecules, single-domains (such as VH or VL) derived from conventional 4-chain antibodies, humanized heavy chain-only antibodies, human single-domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single-domain scaffolds other than those derived from antibodies. Any sdAb known in the art or developed by this disclosure, including the single-domain antibodies described above in this disclosure, can be used to construct the CARs described herein. sdAbs can be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, fish, sharks, goats, rabbits, and cattle. Single-domain antibodies considered herein also include naturally occurring single-domain antibody molecules from species other than camelidae and sharks.

[0406] sdAbs can be derived from naturally occurring single-domain antigen-binding molecules, referred to as heavy-chain antibodies lacking the light chain (also referred to herein as “heavy-chain-only antibodies”). For example, such single-domain molecules are disclosed in WO 94 / 04678 and Hamers-Casterman, C. et al., Nature 363:446-448 (1993). For clarity, sdAbs derived from naturally occurring heavy-chain molecules lacking the light chain are referred to as heavy-chain antibodies.The variable domain is referred to herein as VHH to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules, as described in specification 54 / 117 pages 59 CN 122003436 A, can be derived from antibodies produced in camelid species (e.g., camels, llamas, vicuñas, dromedaries, alpacas, and guanacos). Other species outside the camelid family can produce naturally occurring heavy-chain molecules lacking the light chain, and such VHHs are within the scope of this disclosure. Additionally, humanized versions of VHHs, as well as other modifications and variants, are also considered within the scope of this disclosure.

[0407] VHH molecules from camelids are approximately one-tenth the size of IgG molecules. They are single polypeptides and can be very stable, tolerating extreme pH and temperature conditions. Furthermore, they can be resistant to the action of proteases, unlike conventional four-chain antibodies. Moreover, in vitro expression of VHHs yields high yields of properly folded functional VHHs. Additionally, antibodies generated in camelids can recognize epitopes other than those recognized by antibodies generated in vitro using antibody libraries or via immunization of mammals other than camelids (see, for example, WO 9749805). Therefore, multispecific or multivalent CARs containing one or more VHH domains can interact more effectively with targets compared to multispecific or multivalent CARs containing antigen-binding fragments derived from conventional 4-chain antibodies. Since VHHs are known to bind to “unusual” epitopes (such as cavities or grooves), CARs containing such VHHs may have a higher affinity for therapeutic purposes than conventional multispecific peptides.

[0408] sdAbs can be derived from variable regions of immunoglobulins found in cartilaginous fish. For example, sdAbs can be derived from an immunoglobulin isotype called a novel antigen receptor (NAR) found in shark serum. Methods for generating single-domain molecules with variable regions derived from NAR (“lgNAR”) are described in WO 03 / 014161 and Streltsov, Protein Sci. 14:2901-2909 (2005).

[0409] sdAb is a recombinant antibody, CDR transplantation antibody, humanized antibody, camel-derived antibody, deimmunized antibody, and / or an in vitro generated antibody (e.g., selected by phage display). The amino acid sequence of the frame region can be altered by “camelization” of specific amino acid residues in the frame region. Camelization refers to the substitution or replacement of one or more amino acid residues in the amino acid sequence of the (naturally occurring) VH domain of a conventional 4-chain antibody with one or more amino acid residues at one or more corresponding positions in the VHH domain of a heavy chain antibody. This can be done in a manner known in the art and will be clear to those skilled in the art. Amino acids that form the VH-VL interface and / or are present at the VH-VL interface can be used to modify the amino acid sequence.Such "camelization" substitutions are inserted at acid sites and / or at so-called cameloid marker residues, as defined herein (see, for example, WO 94 / 04678, Davies and Riechmann FEBS Letters 339: 285–290 (1994); Davies and Riechmann, Protein Engineering 9 (6): 531–537 (1996); Riechmann, J. Mol. Biol. 259: 957–969 (1996); and Riechmann and Muyldermans, J. Immunol. Meth. 231:25–38 (1999)).

[0410] The sdAb can be a human single-domain antibody produced by transgenic mice or rats expressing human heavy chain segments. See, for example, US 20090307787, US Patent No. 8,754,287, US 20150289489, US 20100122358, and WO 2004049794. The sdAb may be affinity mature.

[0411] Naturally occurring VHH domains targeting specific antigens or targets can be obtained from (naïve or immunized) libraries of camel VHH sequences. Such methods may or may not involve using the said antigen or target, or at least a portion thereof, fragment, antigenic determinant, or epitope, to screen such libraries using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from (naïve or immunized) VHH libraries can be used, such as VHH libraries obtained from (naïve or immunized) VHH libraries by techniques described, for example, in WO 00 / 43507 (e.g., random mutagenesis and / or CDR shuffling).

[0412] Single-domain antibodies can be generated from conventional four-chain antibodies. See, for example, EP 0 368 684; Ward et al., specification 55 / 117 pages 60 CN 122003436 A Nature, 341 (6242): 544-6 (1989); Holt et al., Trends Biotechnol., 21(11):484-490 (2003); WO 06 / 030220; and WO 06 / 003388.

[0413] The extracellular antigen-binding domains provided herein may include at least one binding domain, and the at least one binding domain may include a single-domain antibody that binds to DLL3 as provided herein, for example, an anti-DLL3 single-domain antibody described in Section 5.2 above.

[0414] The CAR provided herein may comprise a polypeptide comprising: (a) an extracellular antigen-binding domain comprising one or more anti-DLL3 sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein each of the one or more anti-DLL3 sdAbs is independently an anti-DLL3 sdAb as described in Section 5.2 above.

[0415] The CAR provided herein may comprise a polypeptide comprising: (a) an extracellular antigen-binding domain comprising one or more anti-DLL3 sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein each of the one or more anti-DLL3 sdAbs is independently an anti-DLL3 sdAb as described in Section 5.2 above, including, for example, the VHH domains in Table 2 and those having one, two, or all three CDRs in any of those VHH domains in Table 2. The anti-DLL3 sdAb may be a camel antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0416] In some embodiments, the CAR provided herein may comprise a polypeptide comprising: (a) an extracellular antigen-binding domain comprising one or more anti-DLL3 sdAbs; (b) a transmembrane domain; and (c) an intracellular signal transduction domain, wherein each of the one or more anti-DLL3 sdAbs independently comprises (1) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 1; (2) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 2; (3) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 3; (4) having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 3; (4) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 5; (5) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 5; (6) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 6; (7) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 7; (8) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 8; (9) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 8;(9) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 10; (10) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 10; (11) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 40; (12) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 41; (13) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 42; (14) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 42; (15) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 44; (16) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 45; (17) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 46; (18) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 47; (19) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 48. (20) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 49; (21) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 50; (22) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 51; (23) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 52; (24) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 51;(25) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 54; (26) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 55; (27) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 56; (28) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 57; (29) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 58. CDR2 and CDR3; (30) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 59; (31) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 60; (32) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 61; (33) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 62; (34) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 63; (35) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 63; (36) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 65; (37) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 66; (38) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 67; (39) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 65.(40) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 69; (41) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 70; (42) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 71; (43) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 72; or (44) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 69. The amino acid sequences of CDR1, CDR2 and CDR3 shown in 73.

[0417] In some embodiments, the CAR provided herein may comprise a polypeptide comprising: (a) an extracellular antigen-binding domain comprising one or more anti-DLL3 sdAbs; (b) a transmembrane domain; and (c) an intracellular signal transduction domain, wherein each of the one or more anti-DLL3 sdAbs independently comprises (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 30; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 32; (4) comprising the amino acid sequence of SEQ ID NO: (5) CDR1 containing the amino acid sequence of SEQ ID NO: 14, CDR2 containing the amino acid sequence of SEQ ID NO: 22, and CDR3 containing the amino acid sequence of SEQ ID NO: 33; (6) CDR1 containing the amino acid sequence of SEQ ID NO: 16, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO: 14.(7) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 26 and CDR3 containing the amino acid sequence of SEQ ID NO: 36; (8) CDR1 containing the amino acid sequence of SEQ ID NO: 18, CDR2 containing the amino acid sequence of SEQ ID NO: 27 and CDR3 containing the amino acid sequence of SEQ ID NO: 37; (9) CDR1 containing the amino acid sequence of SEQ ID NO: 19, CDR2 containing the amino acid sequence of SEQ ID NO: 28 and CDR3 containing the amino acid sequence of SEQ ID NO: 38; or (10) CDR1 containing the amino acid sequence of SEQ ID NO: 20, CDR2 containing the amino acid sequence of SEQ ID NO: 29 and CDR3 containing the amino acid sequence of SEQ ID NO: 39.

[0418] The CDR1, CDR2, or CDR3 of the anti-DLL3 sdAb can be 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.

[0419] In some embodiments, the CAR provided herein may comprise a polypeptide comprising: (a) an extracellular antigen-binding domain comprising one or more anti-DLL3 sdAbs; (b) a transmembrane domain; and (c) an intracellular signal transduction domain, wherein each of the one or more anti-DLL3 sdAbs independently comprises or consists of an amino acid sequence of any one of SEQ ID NO: 1-10 and 40-73; or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

[0420] The extracellular antigen-binding domain may comprise two anti-DLL3 sdAbs, and in some embodiments, the two anti-DLL3 sdAbs are each independently an anti-DLL3 sdAb as described in section 5.2 above.

[0421] In some embodiments, the extracellular antigen-binding domain comprises two anti-DLL3 sdAbs, wherein the two anti-DLL3 sdAbs comprise a first anti-DLL3 sdAb and a second anti-DLL3 sdAb, and wherein:

[0422] (1) the first anti-DLL3 sdAb comprises, for example, SEQ ID NO: 56 or SEQ IDThe second anti-DLL3 sdAb contains CDR1, CDR2 and CDR3 as shown in the anti-DLL3 sdAb containing the amino acid sequence of SEQ ID NO: 64; and

[0423] (2) the second anti-DLL3 sdAb contains CDR1, CDR2 and CDR3 as shown in the anti-DLL3 sdAb containing the amino acid sequence of SEQ ID NO: 68.

[0424] The first anti-DLL3 sdAb may comprise: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31; or (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33; and / or, the second anti-DLL3 sdAb may comprise: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 37.

[0425] The first anti-DLL3 sdAb may comprise the amino acid sequence of SEQ ID NO: 56 or 64; or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it; and / or, the second anti-DLL3 sdAb may comprise the amino acid sequence of SEQ ID NO: 68; or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

[0426] The first anti-DLL3 sdAb may be linked to the N-terminus or C-terminus of the second anti-DLL3 sdAb with or without a linker (e.g., a rigid peptide linker or a flexible peptide linker).

[0427] The extracellular antigen-binding domain may comprise the amino acid sequence of SEQ ID NO: 74 or 75; or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

[0428] The extracellular antigen-binding domain may further comprise one or more additional antigen-binding domains.

[0429] One or more additional antigens targeted by the CAR disclosed herein may be cell surface molecules, such as cell surface markers on target cells associated with a specific disease state. The antigen may be a tumor antigen. Tumors express a variety of proteins that can serve as target antigens for immune responses, particularly T-cell-mediated immune responses. CAR-targeted antigens may be antigens on a single lesion cell, or antigens expressed on different cells, each of which may affect the disease. CAR-targeted antigens may be directly or indirectly involved in the disease.

[0430] Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T-cell-mediated immune response. The selection of additional targeting antigens disclosed herein will depend on the specific type of cancer to be treated. Exemplary tumor antigens include, but are not limited to, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, HER2 / neu, survival proteins and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, liver glycoside B2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0431] Tumor antigens may contain one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a number of proteins that can be used as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and gp100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecular group, such as the oncogene HER2 / Neu / ErbB-2. Another group of target antigens is oncoemulsification antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute true tumor-specific immunoglobulin antigens unique to the individual tumor. In addition to CD19, B-cell differentiation antigens (such as CD20 and CD37) are other candidates for target antigens in B-cell lymphoma.

[0432] Tumor antigens can be tumor-specific antigens (TSA) or tumor-associated antigens (TAA). TSA is specific to tumor cells and is not present on other cells in the body. TAA-associated antigens are not specific to tumor cells; on the contrary, they are alsoAntigens are expressed on normal cells under conditions that do not induce immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during embryonic development, when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0433] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / Melan A (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0434] Other protein-based large antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0435] In some more specific embodiments, one or more additional antigens are selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that bind to CD83, and combinations thereof.

[0436] In addition to one or more antigen-binding domains provided herein, the CARs provided herein may further include one or more of the following: a linker (e.g., a peptide linker), a transmembrane domain, a hinge domain, a signal peptide, an intracellular signal transduction domain, and a co-stimulatory signal transduction domain, each of which is described in more detail below.

[0437] For example, the intracellular signal transduction domain may comprise a primary intracellular signal transduction domain of an immune effector cell (e.g., a T cell). The primary intracellular signal transduction domain may be derived from CD3ζ. The intracellular signal transduction domain may comprise a co-stimulatory signal transduction domain. The co-stimulatory signal transduction domain may be derived from a co-stimulatory molecule selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that binds to CD83, and combinations thereof. The co-stimulatory signal transduction domain may be derived from CD137. The DLL3 CAR may further include a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. The DLL3 CAR may further include a signal peptide (e.g., a CD8α signal peptide) located at the N-terminus of the polypeptide. The polypeptide may include, from the N-terminus to the C-terminus: a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signal transduction domain derived from CD137, and a primary intracellular signal transduction domain derived from CD3ζ. The DLL3 CAR may be monospecific. The DLL3 CAR may be monovalent. The DLL3 CAR may be bivalent. The DLL3 CAR provided herein may be bispecific.

[0438] Peptide Linker

[0439] In the case where multiple antibodies (e.g., multiple antibody fragments) are present in the CAR of the present invention, the various antibodies may be fused to each other via a peptide linker. Antibodies can fuse directly with each other without any peptide linkers. Peptide linkers connecting different antibodies can be the same or different. Different domains of a CAR can also fuse with each other via peptide linkers.

[0440] Depending on the structural and / or functional characteristics of the antibody and / or various domains, each peptide linker in a CAR can have the same or different lengths and / or sequences. Each peptide linker can be selected and optimized independently. The length, flexibility, and / or other properties of one or more peptide linkers used in a CAR can have some influence on properties, including but not limited to affinity, specificity, or affinity for one or more specific antigens or epitopes. For example, longer peptide linkers can be selected to ensure that two adjacent domains do not spatially interfere with each other. Transmembrane domains of a CAR can be coupled with intracellular signal transduction domains.Short peptide linkers are provided between adjacent domains. The peptide linkers may contain flexible residues (such as glycine and serine) that allow adjacent domains to move freely relative to each other. For example, a glycine-serine dinucleotide may be a suitable peptide linker.

[0441] The peptide linkers may have any suitable length. The length of the peptide linker may be at least about any of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. The length of the peptide linker may not exceed about any of the following: 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids. The length of the peptide linker can be any of the following: about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0442] The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy-chain-only antibody can be used as a linker. See, for example, WO 1996 / 34103. The peptide linker can be a flexible linker. Exemplary flexible joints include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible joints known in the art. Other linkers known in the art (e.g., WO 2016014789; WO 2015158671; WO 2016102965; US 20150299317; WO 2018067992; US 7741465; Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990); and Bird et al., Science 242:423-426, specification 60 / 117 pages 65 CN 122003436 A (1988)) may also be included in the CAR provided herein, the disclosure of each of these documents being incorporated herein by reference.

[0443] The peptide linker may comprise the amino acid sequence of SEQ ID NO: 129.

[0444] 5.3.2.Transmembrane Domain

[0445] The CAR disclosed herein comprises a transmembrane domain that can be fused directly or indirectly to an extracellular antigen-binding domain. The transmembrane domain may be derived from natural or synthetic sources. As used herein, “transmembrane domain” means any protein structure that is thermodynamically stable in a cell membrane, such as a eukaryotic cell membrane. The transmembrane domain suitable for the CAR described herein may be derived from a naturally occurring protein. Alternatively, it may be a synthetic, non-naturally occurring protein segment, such as a thermodynamically stable hydrophobic protein segment in a cell membrane.

[0446] Transmembrane domains are classified according to their three-dimensional structure. For example, a transmembrane domain may form an α-helix, a complex of more than one α-helix, a β-barrel, or any other stable structure capable of crossing the phospholipid bilayer of the cell. In addition, transmembrane domains may also be classified, or alternatively, according to their topological structure, including the number of times the transmembrane domain crosses the membrane and the orientation of the protein. For example, single-transmembrane proteins cross the cell membrane once, while multi-transmembrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times). Membrane proteins can be defined as type I, type II, or type III, depending on the topology of their terminals and one or more membrane-transfer segments relative to the cell interior and exterior. Type I membrane proteins have a single transmembrane region oriented such that the N-terminus of the protein is located on the extracellular side of the lipid bilayer of the cell, while the C-terminus of the protein is located on the intracellular side. Type II membrane proteins also have a single transmembrane region, but are oriented such that the C-terminus of the protein is located on the extracellular side of the lipid bilayer of the cell, while the N-terminus of the protein is located on the intracellular side. Type III membrane proteins have multiple transmembrane segments and can be further classified according to the number of transmembrane segments and the positions of the N-terminus and C-terminus.

[0447] The transmembrane domains of the CARs described herein can be derived from type I single-transmembrane proteins. Transmembrane domains derived from multi-transmembrane proteins are also applicable to the CARs described herein. Multiple transmembrane proteins may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) α-helix or β-sheet structure. The N-terminus and C-terminus of a multiple transmembrane protein may be located on opposite sides of a lipid bilayer; for example, the N-terminus of the protein may be located on the intracellular side of the lipid bilayer, while the C-terminus of the protein may be located on the extracellular side.

[0448] The transmembrane domain of a CAR may comprise transmembrane domains selected from: α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR,CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, claudin‑6, IL‑2R β, IL‑2R γ, IL‑7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA‑6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA‑1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA‑1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domains are derived from molecules selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0449] In some specific embodiments, the transmembrane domains are derived from CD8α. In some embodiments, the transmembrane domain is a transmembrane domain of CD8α comprising the amino acid sequence of SEQ ID NO: 126.

[0450] The transmembrane domain used in the CAR described herein may also comprise at least a portion of a synthetic, non-naturally occurring protein segment. The transmembrane domain may be a synthetic, non-naturally occurring α-helix or β-sheet. The protein segment may be at least about 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0451] The transmembrane domain provided herein may comprise a transmembrane region and an intracellular region located on the C-terminal side of the transmembrane domain. The intracellular region of the transmembrane domain may comprise three or more amino acids and may contribute to enabling the transmembrane domain to function in lipids.Oriented in the bilayer. One or more cysteine ​​residues may be present in the transmembrane region of the transmembrane domain. One or more cysteine ​​residues may be present in the intracellular region of the transmembrane domain. The intracellular region of the transmembrane domain may contain positively charged amino acids. The intracellular region of the transmembrane domain may contain the amino acids arginine, serine, and lysine.

[0452] The transmembrane region of the transmembrane domain may contain hydrophobic amino acid residues. The transmembrane domain of the CAR provided herein may contain artificially hydrophobic sequences. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. The transmembrane region may primarily contain hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. The transmembrane region may be hydrophobic. The transmembrane region may contain a polyleucine-alanine sequence. The hydrophilic or hydrophobic or hydrophilic characteristics of the protein or protein segment may be assessed by any method known in the art, such as Kyte and Doolittle hydrophilicity analysis.

[0453] 5.3.3. Intracellular Signaling Domain

[0454] The CAR disclosed herein includes an intracellular signaling domain. The intracellular signaling domain is responsible for activating at least one normal effector function of an immune effector cell expressing the CAR. The term "effector function" refers to a specific function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including the secretion of cytokines. Therefore, the term "cytoplasmic signaling domain" refers to a portion of a protein that transduces effector function signals and directs the cell to perform a specific function. While the entire cytoplasmic signaling domain can generally be used, in many cases it is not necessary to use the entire chain. With regard to the use of a truncated portion of the cytoplasmic signaling domain, such a truncated portion may be used instead of the complete chain, as long as it transduces effector function signals. Therefore, the term cytoplasmic signaling domain means any truncated portion of the cytoplasmic signaling domain that is sufficient to transduce effector function signals.

[0455] In some embodiments, the intracellular signaling domain includes the primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain that is essentially composed of primary intracellular signaling domains of immune effector cells. A “primary intracellular signaling domain” refers to a cytoplasmic signaling sequence that functions in a stimulatory manner to induce the function of an immune effector. The primary intracellular signaling domain may contain a signaling motif called an immune receptor tyrosine-based activation motif or ITAM. As used herein, “ITAM” is a conserved protein motif that is commonly found at the tail of signaling molecules expressed in many immune cells. The motif may contain an amino group separated by 6–8 amino acids.Two repeats of the acid sequence YxxL / I, where each x is independently any amino acid, produce the conserved motif YxxL / Ix(6-8) YxxL / I. The ITAM within the signal transduction molecule is important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM after activation of the signal transduction molecule. The ITAM can also serve as a docking site for other proteins involved in signal transduction pathways. Exemplary primary cytoplasmic signal transduction sequences containing ITAM include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (Fcε Rib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0456] In some embodiments, the primary intracellular signal transduction domain is derived from CD3ζ. The intracellular signal transduction domain may consist of the cytoplasmic signal transduction domain of CD3ζ. The primary intracellular signal transduction domain of CD3ζ may contain the amino acid sequence of SEQ ID NO: 128. The primary intracellular signal transduction domain may be a cytoplasmic signal transduction domain of wild-type CD3ζ. The primary intracellular signal transduction domain may be a functional mutant of the cytoplasmic signal transduction domain of CD3ζ containing one or more mutations (e.g., Q65K). Specification 62 / 117 pages 67 CN 122003436 A

[0457] 5.3.4. Co-stimulatory signal transduction domain

[0458] In addition to stimulating antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell. In some embodiments, the CAR includes at least one co-stimulatory signal transduction domain. As used herein, the term "co-stimulatory signal transduction domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response (e.g., effector function). The costimulatory signal transduction domain of the chimeric receptor described herein may be a cytoplasmic signal transduction domain derived from a costimulatory protein, which transduces signals and regulates responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils. A “costimulatory signal transduction domain” may be an intracellular portion of a costimulatory molecule. The term “costimulatory molecule” refers to a homologous binding partner on an immune cell (e.g., a T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the immune cell, such as, but not limited to, proliferation and survival.

[0459] An intracellular signal transduction domain may contain a single costimulatory signal transduction domain. An intracellular signal transduction domain may contain two or more (e.g., any one of about 2, 3, 4, or more) costimulatory signal transduction domains. An intracellular signal transduction domain may contain two or more identical costimulatory signal transduction domains. Intracellular signal transduction domainA domain may contain two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. An intracellular signaling domain comprises a primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ) and one or more costimulatory signaling domains. One or more costimulatory signaling domains and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ) may be fused to each other via optional peptide linkers. The primary intracellular signaling domain and one or more costimulatory signaling domains may be arranged in any suitable order. One or more costimulatory signaling domains may be located between a transmembrane domain and a primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ). Multiple costimulatory signaling domains may provide additive or synergistic stimulation.

[0460] Activation of costimulatory signaling domains in host cells (e.g., immune cells) can induce increased or decreased cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule may be applicable to the CAR described herein. One or more types of costimulatory signaling domains are selected based on factors such as the type of immune effector cells in which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect). Examples of co-stimulatory signaling domains for CARs can be cytoplasmic signaling domains of co-stimulatory proteins, including but not limited to members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α and TNF RII / TNFRSF1B; members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6 and SLAM / CD150); and any other co-stimulatory molecules, such as CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C. (Instructions for use, pages 63 / 117, CN 122003436 A)

[0461] One or more costimulatory signal transduction domains may be selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, and combinations thereof.

[0462] The intracellular signal transduction domain in the CAR disclosed herein may include a costimulatory signal transduction domain derived from CD137 (i.e., 4-1BB). The intracellular signal transduction domain may include a cytoplasmic signal transduction domain of CD3ζ and a costimulatory signal transduction domain of CD137. The intracellular signal transduction domain may include a costimulatory signal transduction domain of CD137 containing the amino acid sequence of SEQ ID NO: 127.

[0463] Any variants of the costimulatory signal transduction domains described herein are also within the scope of this disclosure, such that the costimulatory signal transduction domains are capable of modulating the immune response of immune cells. Compared to the wild-type control, the co-stimulatory signal transduction domain can contain up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8). It may contain one or more amino acids.Such a modified costimulatory signaling domain may be referred to as a variant. Mutations of amino acid residues in a costimulatory signaling domain, relative to a non-mutated costimulatory signaling domain, can lead to an increase in signal transduction and enhanced stimulation of the immune response. Mutations of amino acid residues in a costimulatory signaling domain, relative to a non-mutated costimulatory signaling domain, can lead to a decrease in signal transduction and reduced stimulation of the immune response.

[0464] 5.3.5. Hinge Domain

[0465] The CAR disclosed herein may comprise a hinge domain located between an extracellular antigen-binding domain and a transmembrane domain. A hinge domain is an amino acid segment typically found between two domains of a protein and can allow for the flexibility of the protein and movement of one or both domains relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule may be used.

[0466] The hinge domain may contain about 10-100 amino acids, for example, any one of about 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. The length of the hinge domain may be any one of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

[0467] The hinge domain may be a hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art that contains a hinge domain is suitable for the chimeric receptor described herein. The hinge domain may be at least a portion of a hinge domain of a naturally occurring protein and imparts flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. The hinge domain may be a portion of the hinge domain of CD8α, for example, a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In some embodiments, the hinge domain of CD8α comprises the amino acid sequence of SEQ ID NO: 125.

[0468] The hinge domain of an antibody (such as an IgG, IgA, IgM, IgE, or IgD antibody) is also suitable for the pH-dependent chimeric receptor system described herein. The hinge domain may be a hinge domain connecting the constant domains CH1 and CH2 of an antibody. The hinge domain may be a hinge domain of an antibody and a hinge domain comprising the antibody and one or more constant regions of the antibody. The hinge domain may comprise the antibody and a hinge domain of the CH3 constant region of the antibody. The hinge domain may comprise the antibody and a hinge domain of the CH2 and CH3 constant regions of the antibody. The antibody may be an IgG, IgA, IgM, IgE, or IgD antibody.Optionally, the antibody is an IgG antibody. The antibody may be an IgG1, IgG2, IgG3, or IgG4 antibody. The hinge region may include the hinge region of the IgG1 antibody and the CH2 and CH3 constant regions. The hinge region may include the hinge region of the IgG1 antibody and the CH3 constant region.

[0469] Non-naturally occurring peptides may also be used as the hinge domain of the chimeric receptor described herein. Extracellular ligand specification for Fc receptor 64 / 117 pages 69 CN 122003436 A The hinge domain between the C-terminus of the binding domain and the N-terminus of the transmembrane domain may be a peptide linker, such as a (GxS)n linker, where x and n may be independently integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater.

[0470] 5.3.6. Signal peptide

[0471] The CAR disclosed herein may include a signal peptide (also referred to as a signal sequence) at the N-terminus of the polypeptide. Typically, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. Signal peptides can target effector molecules to cellular secretory pathways and allow effector molecules to integrate and anchor into the lipid bilayer. Signal peptides suitable for the CARs described herein and comprising signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences will be apparent to those skilled in the art. Signal peptides can be derived from molecules selected from the group consisting of CD8α, GM-CSF receptor α, and the IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the CD8α signal peptide comprises the amino acid sequence of SEQ ID NO: 124.

[0472] 5.3.7. Exemplary CAR

[0473] Exemplary CARs incorporating DLL3 are generated as shown in Section 6 below, such as DCAR001, DCAR002, DCAR003, DCAR004, DCAR005, DCAR006, DCAR007, DCAR008, DCAR009, DCAR010, DCAR012, DCAR013, DCAR014, DCAR015, DCAR016, DCAR017, DCAR018, DCAR019, DCAR020, DCAR021, DCAR022, DCAR023, DCAR024, DCAR025, DCAR026, DCAR027, DCAR028, DCAR029, DCAR030, DCAR031, DCAR032, DCAR033, DCAR034, DCAR035, DCAR036, DCAR037, DCAR038, DCAR039, DCAR040, DCAR041, DCAR042, DCAR043, DCAR044, DCAR045, DCAR046, DCAR047.

[0474] In some embodiments, the CAR provided herein may comprise an amino acid sequence of any one of SEQ ID NO: 77-86, 88-123, and ...

Claims

1. An anti-DLL3 (δ-like ligand 3) single-domain antibody (sdAb), comprising: (1) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1 respectively; (2) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 2 respectively; (3) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 3 respectively; (4) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 4 respectively; (5) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 5 respectively; (6) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 6 respectively; (7) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 7 respectively; (8) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 8, respectively; (9) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 9, respectively; or (10) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 10, respectively.

2. The anti-DLL3 sdAb according to claim 1, wherein CDR1, CDR2 or CDR3 is determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme or a combination thereof.

3. The anti-DLL3 sdAb according to claim 1 or claim 2, wherein the anti-DLL3 sdAb comprises: (1) CDR1 containing the amino acid sequence of SEQ ID NO: 11, CDR2 containing the amino acid sequence of SEQ ID NO: 21 and CDR3 containing the amino acid sequence of SEQ ID NO: 30; (2) CDR1 containing the amino acid sequence of SEQ ID NO: 12, CDR2 containing the amino acid sequence of SEQ ID NO: 22 and CDR3 containing the amino acid sequence of SEQ ID NO: 31; (3) CDR1 containing the amino acid sequence of SEQ ID NO: 13, CDR2 containing the amino acid sequence of SEQ ID NO: 23, and CDR3 containing the amino acid sequence of SEQ ID NO: 32; (4) CDR1 containing the amino acid sequence of SEQ ID NO: 14, CDR2 containing the amino acid sequence of SEQ ID NO: 22 and CDR3 containing the amino acid sequence of SEQ ID NO: 33; (5) CDR1 containing the amino acid sequence of SEQ ID NO: 15, CDR2 containing the amino acid sequence of SEQ ID NO: 24, and CDR3 containing the amino acid sequence of SEQ ID NO: 34; (6) CDR1 containing the amino acid sequence of SEQ ID NO: 16, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO: 35; (7) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 26 and CDR3 containing the amino acid sequence of SEQ ID NO: 36; (8) CDR1 containing the amino acid sequence of SEQ ID NO: 18, CDR2 containing the amino acid sequence of SEQ ID NO: 27 and CDR3 containing the amino acid sequence of SEQ ID NO: 37; (9) CDR1 containing the amino acid sequence of SEQ ID NO: 19, CDR2 containing the amino acid sequence of SEQ ID NO: 28, and CDR3 containing the amino acid sequence of SEQ ID NO: 38; or (10) CDR1 containing the amino acid sequence of SEQ ID NO: 20, CDR2 containing the amino acid sequence of SEQ ID NO: 29 and CDR3 containing the amino acid sequence of SEQ ID NO:

39.

4. The anti-DLL3 sdAb according to any one of claims 1-3, wherein the anti-DLL3 sdAb further comprises one or more FR regions selected from the group consisting of FR1, FR2, FR3 and FR4 as shown in any one of SEQ ID NO: 1-10 and 40-73.

5. The anti-DLL3 sdAb according to any one of claims 1-4, wherein the anti-DLL3 sdAb is a camel antibody, a chimeric antibody, a human antibody, or a humanized antibody.

6. The anti-DLL3 sdAb according to any one of claims 1-5, wherein the anti-DLL3 sdAb comprises a VHH structure.

7. The anti-DLL3 sdAb according to any one of claims 1-6, wherein the anti-DLL3 sdAb comprises an amino acid sequence of any one of SEQ ID NO: 1-10 and 40-73, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

8. The anti-DLL3 sdAb according to any one of claims 1-7, wherein the anti-DLL3 sdAb is genetically fused or chemically conjugated with the agent.

9. The anti-DLL3 sdAb according to any one of claims 1-8, wherein the anti-DLL3 sdAb further comprises an immunoglobulin heavy chain constant region or a fragment thereof; Preferably, the immunoglobulin is IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgM or IgA.

10. A multispecific antibody comprising at least one anti-DLL3sdAb according to any one of claims 1-9, and / or another antibody or an antigen-binding fragment thereof.

11. The multispecific antibody of claim 10, wherein the multispecific antibody comprises two anti-DLL3 sdAbs according to any one of claims 1-9.

12. The multispecific antibody according to claim 10 or 11, wherein the multispecific antibody comprises the amino acid sequence of any one of SEQ ID NO:74-75, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

13. An isolated nucleic acid encoding an anti-DLL3 sdAb according to any one of claims 1-9, or a multispecific antibody according to any one of claims 10-12.

14. A vector comprising the isolated nucleic acid according to claim 13.

15. A host cell comprising the isolated nucleic acid according to claim 13 or the vector according to claim 14.

16. A chimeric antigen receptor (CAR) comprising a polypeptide, said polypeptide comprising: (a) An extracellular antigen-binding domain comprising one or more anti-DLL3 sdAbs according to any one of claims 1-9; (b) Transmembrane domains; and (c) Intracellular signal transduction domains.

17. The CAR of claim 16, wherein the extracellular antigen-binding domain comprises two anti-DLL3sdAbs.

18. The CAR of claim 16 or 17, wherein the two anti-DLL3 sdAbs comprise a first anti-DLL3 sdAb and a second anti-DLL3 sdAb, and wherein: (1) The first anti-DLL3 sdAb comprises CDR1, CDR2, and CDR3 as shown in the anti-DLL3 sdAb containing the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 64; and (2) The second anti-DLL3 sdAb contains CDR1, CDR2 and CDR3 as shown in the anti-DLL3 sdAb containing the amino acid sequence of SEQ ID NO:

68.

19. The CAR according to any one of claims 16-18, wherein the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains.

20. The CAR of claim 19, wherein the one or more additional antigen-binding domains bind to one or more antigens selected from the group consisting of: CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77.

21. The CAR according to any one of claims 16-20, wherein the transmembrane domain is derived from a molecule selected from the group consisting of: CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD-1; Preferably, the transmembrane domain is derived from CD8α.

22. The CAR according to any one of claims 16-21, wherein the intracellular signal transduction domain comprises the primary intracellular signal transduction domain of an immune effector cell.

23. The CAR of claim 22, wherein the primary intracellular signal transduction domain is derived from CD3-ζ.

24. The CAR according to any one of claims 16-23, wherein the intracellular signal transduction domain further comprises a co-stimulatory signal transduction domain.

25. The CAR of claim 24, wherein the costimulatory signal transduction domain is derived from a costimulatory molecule selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands binding to CD83, and combinations thereof.

26. The CAR of claim 25, wherein the co-stimulatory signal transduction domain is derived from CD137.

27. The CAR according to any one of claims 16-26, wherein the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

28. The CAR of claim 27, wherein the hinge domain is derived from CD8α.

29. The CAR according to any one of claims 16-28, wherein the CAR further comprises a signal peptide located at the N-terminus of the polypeptide.

30. The CAR of claim 29, wherein the signal peptide is derived from CD8α.

31. The CAR according to any one of claims 16-30, wherein the CAR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 77-86, 88-123 and 130-175.

32. An isolated nucleic acid comprising a nucleic acid sequence encoding a CAR according to any one of claims 16-31.

33. A vector comprising the isolated nucleic acid according to claim 32.

34. An engineered immune cell comprising a CAR according to any one of claims 16-31, an isolated nucleic acid according to claim 32, or a vector according to claim 33.

35. The engineered immune cell according to claim 34, wherein the immune cell is a T cell, NK cell, peripheral blood mononuclear cell (PBMC), hematopoietic stem cell, pluripotent stem cell, embryonic stem cell, or any combination thereof.

36. A method for producing engineered immune cells, the method comprising introducing the vector according to claim 33 into the cells.

37. A pharmaceutical composition comprising an anti-DLL3 sdAb according to any one of claims 1-9, a multispecific antibody according to any one of claims 10-12, a CAR according to any one of claims 16-31, an isolated nucleic acid according to claim 13 or 32, a vector according to claim 14 or 33, or engineered immune cells according to claim 34 or 35, and a pharmaceutically acceptable excipient.

38. A method of treating a disease or condition of a subject, the method comprising administering to the subject an effective amount of an anti-DLL3 sdAb according to any one of claims 1-9, a multispecific antibody according to any one of claims 10-12, engineered immune cells according to claim 34 or 35, or a pharmaceutical composition according to claim 37.

39. The method of claim 38, wherein the disease or symptom is a DLL3-related disease or symptom.

40. The method of claim 38 or 39, wherein the disease or symptom is a tumor or cancer.

41. The method according to any one of claims 38-40, wherein the disease or condition is selected from the group consisting of neuroendocrine tumors (NETs) or neuroendocrine carcinomas (NECs). Preferably, the disease or condition is selected from the group consisting of: lung cancer (e.g., small cell lung cancer (SCLC)), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), bladder cancer (e.g., small cell bladder cancer (SCBC)), neuroendocrine prostate cancer (NEPC), melanoma, glioma (e.g., low-grade glioma), glioblastoma, medullary thyroid carcinoma, neuroendocrine bladder cancer (NEBC), testicular cancer, and pancreatic neuroendocrine tumor (PNET).

42. Use of the anti-DLL3 sdAb according to any one of claims 1-9, the multispecific antibody according to any one of claims 10-12, the CAR according to any one of claims 16-31, the isolated nucleic acid according to claim 13 or 32, the vector according to claim 14 or 33, the engineered immune cell according to claim 34 or 35, or the pharmaceutical composition according to claim 37 in the manufacture of a medicament for use in treating a disease or condition (e.g., cancer) in a subject.

Images