Single-domain antibodies that bind ROR1
Single-domain antibodies that bind receptor tyrosine kinase (ROR1) are described. The single-domain antibodies can be used for multiple purposes including in research, imaging, diagnosis, and treatment of ROR1-related conditions. The disclosed single-domain antibodies can be used as anti-cancer therapeutics such as antibody-drug conjugates, multi-domain binding molecules, or recombinant receptors or can be used as cancer imaging/diagnostic agents.
1 . A single-domain antibody that binds receptor tyrosine kinase (ROR1), the single domain antibody comprising a set of complementarity determining regions (CDRs) comprising:
(i) a CDR1 having the sequence as set forth in SEQ ID NO: 6, a CDR2 having the sequence as set forth in SEQ ID NO: 7, and a CDR3 having the sequence as set forth in SEQ ID NO: 8 according to IMGT;
(ii) a CDR1 having the sequence as set forth in SEQ ID NO: 9, a CDR2 having the sequence as set forth in SEQ ID NO: 10, and a CDR3 having the sequence as set forth in SEQ ID NO: 11 according to Kabat;
(iii) a CDR1 having the sequence as set forth in SEQ ID NO: 12, a CDR2 having the sequence as set forth in SEQ ID NO: 13, and a CDR3 having the sequence as set forth in SEQ ID NO: 11 according to Chothia;
(iv) a CDR1 having the sequence as set forth in SEQ ID NO: 14, a CDR2 having the sequence as set forth in SEQ ID NO: 15, and a CDR3 having the sequence as set forth in SEQ ID NO: 8 according to North;
(v) a CDR1 having the sequence as set forth in SEQ ID NO: 16, a CDR2 having the sequence as set forth in SEQ ID NO: 17, and a CDR3 having the sequence as set forth in SEQ ID NO: 18 according to Contact;
(vi) a CDR1 having the sequence as set forth in SEQ ID NO: 19, a CDR2 having the sequence as set forth in SEQ ID NO: 20, and a CDR3 having the sequence as set forth in SEQ ID NO: 21 according to IMGT;
(vii) a CDR1 having the sequence as set forth in SEQ ID NO: 22, a CDR2 having the sequence as set forth in SEQ ID NO: 23, and a CDR3 having the sequence as set forth in SEQ ID NO: 24 according to Kabat;
(viii) a CDR1 having the sequence as set forth in SEQ ID NO: 25, a CDR2 having the sequence as set forth in SEQ ID NO: 26, and a CDR3 having the sequence as set forth in SEQ ID NO: 24 according to Chothia;
(ix) a CDR1 having the sequence as set forth in SEQ ID NO: 27, a CDR2 having the sequence as set forth in SEQ ID NO: 28, and a CDR3 having the sequence as set forth in SEQ ID NO: 21 according to North;
(x) a CDR1 having the sequence as set forth in SEQ ID NO: 29, a CDR2 having the sequence as set forth in SEQ ID NO: 30, and a CDR3 having the sequence as set forth in SEQ ID NO: 31 according to Contact;
(xi) a CDR1 having the sequence as set forth in SEQ ID NO: 32, a CDR2 having the sequence as set forth in SEQ ID NO: 20, and a CDR3 having the sequence as set forth in SEQ ID NO: 33 according to IMGT;
(xii) a CDR1 having the sequence as set forth in SEQ ID NO: 34, a CDR2 having the sequence as set forth in SEQ ID NO: 23, and a CDR3 having the sequence as set forth in SEQ ID NO: 24 according to Kabat;
(xiii) a CDR1 having the sequence as set forth in SEQ ID NO: 35, a CDR2 having the sequence as set forth in SEQ ID NO: 26, and a CDR3 having the sequence as set forth in SEQ ID NO: 33 according to Chothia;
(xiv) a CDR1 having the sequence as set forth in SEQ ID NO: 36, a CDR2 having the sequence as set forth in SEQ ID NO: 28, and a CDR3 having the sequence as set forth in SEQ ID NO: 33 according to North;
(XV) a CDR1 having the sequence as set forth in SEQ ID NO: 29, a CDR2 having the sequence as set forth in SEQ ID NO: 30, and a CDR3 having the sequence as set forth in SEQ ID NO: 37 according to Contact;
(xvi) a CDR1 having the sequence as set forth in SEQ ID NO: 32, a CDR2 having the sequence as set forth in SEQ ID NO: 20, and a CDR3 having the sequence as set forth in SEQ ID NO: 21 according to IMGT;
(xvii) a CDR1 having the sequence as set forth in SEQ ID NO: 35, a CDR2 having the sequence as set forth in SEQ ID NO: 26, and a CDR3 having the sequence as set forth in SEQ ID NO: 24 according to Chothia; or
(xviii) a CDR1 having the sequence as set forth in SEQ ID NO: 36, a CDR2 having the sequence as set forth in SEQ ID NO: 28, and a CDR3 having the sequence as set forth in SEQ ID NO: 21 according to North
wherein the single-domain antibody is not linked to an IgM Fc region or multimerizing fragment thereof.
2 . The single-domain antibody of claim 1 , wherein the single-domain antibody comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
3 . The single-domain antibody of claim 1 , wherein the single-domain antibody comprises the sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
4 . A heavy chain only antibody (HcAb) comprising the single-domain antibody of claim 1 linked to an IgG, IgA, IgD, or IgE Fc region of an antibody.
5 . A multi-domain binding molecule comprising at least two binding domains wherein at least one binding domain comprises a single-domain antibody of claim 1 and wherein the multi-domain binding molecule does not comprise an IgM Fc region or multimerizing fragment thereof.
6 . The multi-domain binding molecule of claim 5 , wherein the multi-domain binding molecule comprises a CD19 binding domain.
7 . The multi-domain binding molecule of claim 6 , wherein the CD19 binding domain comprises an anti-CD19 scFv.
8 . The multi-domain binding molecule of claim 6 , wherein the CD19 binding domain comprises a variable heavy chain sequencing comprising a CDRH1 having the sequence as set forth in SEQ ID NO: 66, a CDRH2 having the sequence as set forth in SEQ ID NO: 67, and a CDRH3 having the sequence as set forth in SEQ ID NO: 68; and a variable light chain sequence comprising a CDRL1 having the sequence as set forth in SEQ ID NO: 63, a CDRL2 having the sequence as set forth in SEQ ID NO: 64, and a CDRL3 having the sequence as set forth in SEQ ID NO: 65; or a variable heavy chain sequence comprising a CDRH1 having the sequence as set forth in SEQ ID NO: 72, a CDRH2 having the sequence as set forth in SEQ ID NO: 73, and a CDRH3 having the sequence as set forth in SEQ ID NO: 74; and a variable light chain sequence comprising a CDRL1 having the sequence as set forth in SEQ ID NO: 69, a CDRL2 having the sequence as set forth in SEQ ID NO: 70, and a CDRL3 having the sequence as set forth in SEQ ID NO: 71.
9 . The multi-domain binding molecule of claim 5 , wherein the multi-domain binding molecule comprises an immune cell engaging molecule.
10 . The multi-domain binding molecule of claim 9 , wherein the immune cell engaging molecule activates a B cell, T cell, natural killer (NK) cell, or macrophage.
11 . The multi-domain binding molecule of claim 10 , wherein the T cell is a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and/or a naïve T cell.
12 . The multi-domain binding molecule of claim 9 , wherein a binding domain of the immune cell engaging molecule binds CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1, CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4/80, IFGR2, Toll-like receptors 1-9, IL-4Rα, or MARCO.
13 . The multi-domain binding molecule of claim 9 , wherein a binding domain of the immune cell engaging molecule binds CD3.
14 . The multi-domain binding molecule of claim 13 , wherein the CD3 binding domain is derived from the OKT3 antibody.
15 . The multi-domain binding molecule of claim 13 , wherein the binding domain that binds CD3 has a variable heavy chain sequence comprising a CDRH1 having the sequence as set forth in SEQ ID NO: 82, a CDRH2 having the sequence as set forth in SEQ ID NO: 83, and a CDRH3 having the sequence as set forth in SEQ ID NO: 84; and a variable light chain sequence comprising a CDRL1 having the sequence as set forth in SEQ ID NO: 79, a CDRL2 having the sequence as set forth in SEQ ID NO: 80, and a CDRL3 having the sequence as set forth in SEQ ID NO: 81.
16 . The multi-domain binding molecule of claim 9 , wherein a binding domain of the immune cell engaging molecule binds CD28.
17 . The multi-domain binding molecule of claim 16 , wherein the CD28 binding domain is derived from the TGN1412 antibody.
18 . The multi-domain binding molecule of claim 5 , wherein the single-domain antibody is linked to a first Fc region of the multi-domain binding molecule and a binding domain that binds CD3 is linked to the second Fc region of the multi-domain binding molecule.
19 . A method of treating a subject in need thereof having an ROR1-related cancer, the method comprising administering a therapeutically effective amount of the single-domain antibody of claim 1 to the subject thereby treating the subject in need thereof having the ROR1-related cancer.
20 . The method of claim 19 , wherein the ROR1-related cancer comprises chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), multiple myeloma (MM), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), breast cancer, ovarian cancer, pancreatic cancer, lung cancer, or neuroblastoma.