Anti-ROR1 / anti-CD3 bispecific binding molecule

Abstract

The present invention relates to bispecific binding molecules that bind to ROR1 and CD3, and methods of using the same for treating diseases and disorders (such as cancer).

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Patent Application 62 / 852,039, filed on May 23, 2019. The disclosure of the priority application is incorporated herein by reference in its entirety.

[0003] Sequence listing

[0004] This application contains a sequence listing that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The electronic copy of the sequence listing was created on May 18, 2020, named 024651_WO004_SL.txt, and is 258,844 bytes in size. Background of the invention

[0005] Receptor tyrosine - kinase - like orphan receptor 1 (ROR1) is a cell - surface protein that mediates signals from its ligand, the secreted glycoprotein Wnt5a. Consistent with its role in influencing stem - cell fate during embryogenesis, ROR1 expression is observed in invasive malignancies that revert to an embryonic transcriptional program, but not in normal adult tissues, providing a favorable selectivity profile as a therapeutic target. ROR1 is ubiquitously expressed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), diffuse large B - cell lymphoma (DLBCL), and Richter transformation or Richter syndrome (RS). ROR1 is also present on the cell surface of multiple solid tumors and appears to be a marker of cancer stem cells. Because its expression level is not prominent in healthy adult tissues but shows high - level expression in multiple hematological and solid tumors, ROR1 is an attractive target for tumor - specific therapy.

[0006] Cluster of differentiation 3 (CD3) is a multimeric protein complex expressed on T cells that is associated with the T - cell receptor complex (TCR) and is essential for T - cell activation. Functional CD3 is formed by the dimeric association of two out of four different chains (ε, γ, δ, and ζ) to form one of the following three pairs of dimers: γ / ε, δ / ε, and ζ / ζ. Antibodies against CD3 have been shown to cluster CD3 on T cells, causing T - cell activation. Thus, anti - CD3 antibodies have been proposed for therapeutic purposes involving T - cell activation. For example, bispecific antibodies capable of binding CD3 and a target antigen have been proposed for therapeutic use, including T - cell immune responses against tissues and cells expressing the target antigen. The recently approved CD19 x CD3 bispecific T - cell engager (BiTE) blinatumomab has validated this approach.

[0007] Multiple forms and compositions of bispecific binding molecules have been described. Multiple variables affect the in vivo potency of these molecules, including PK, the targeted antigen epitope, the relative affinities of the antigen-binding components, and the valence and spatial configuration of the complementarity-determining regions. Currently, there is no reliable method to a priori design a single molecule to optimize all of these parameters of a bispecific binding molecule, such as an antibody that specifically binds to ROR1 and CD3. Bispecific constructs must be designed, made, and tested to systematically evaluate the effects of many of these parameters and to select the optimal bispecific binding molecule for therapeutic use, such as cancer treatment.

[0008] In view of the above, there is a need for new and improved cancer therapeutics that combine the tumor specificity of the ROR1 antigen with the potent activity of redirecting T cells. SUMMARY OF THE INVENTION

[0009] The present invention relates to novel bispecific binding molecules that target ROR1 and CD3, pharmaceutical compositions comprising one or more of these antibodies, and the use of the antibodies and pharmaceutical compositions for the treatment of cancer. Compared to currently available cancer treatments, including antibody therapies, the specific binding molecules of the present invention are expected to provide excellent clinical responses.

[0010] In some embodiments, the present disclosure provides a bispecific binding molecule comprising a first antigen-binding domain that specifically binds to the extracellular domain of human ROR1 and a second antigen-binding domain that specifically binds to the extracellular domain of human CD3.

[0011] In certain embodiments, the first antigen-binding domain competes with an antibody comprising the heavy-chain amino acid sequence shown in SEQ ID NO: 82 and the light-chain amino acid sequence shown in SEQ ID NO: 83 for binding to human ROR1, or binds to the same epitope on human ROR1 as an antibody comprising the heavy-chain amino acid sequence shown in SEQ ID NO: 82 and the light-chain amino acid sequence shown in SEQ ID NO: 83. In specific embodiments, the first antigen-binding domain comprises:

[0012] a) heavy-chain (H)-CDR1-3 and light-chain (L)-CDR1-3 comprising the amino acid sequences shown in SEQ ID NOs: 97, 61, 62, 63, 64, and 65, respectively;

[0013] b) H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences shown in SEQ ID NOs: 60, 61, 62, 63, 64, and 65, respectively;

[0014] c) A heavy chain variable domain (VH) comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:72 and a light chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:73;

[0015] d) A VH comprising the amino acid sequence of SEQ ID NO:74 and a VL comprising the amino acid sequence of SEQ ID NO:73;

[0016] e) A VH comprising the amino acid sequence of SEQ ID NO:72 and a VL comprising the amino acid sequence of SEQ ID NO:73;

[0017] f) A heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:84 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:83;

[0018] g) A HC comprising the amino acid sequence of SEQ ID NO:82 and a LC comprising the amino acid sequence of SEQ ID NO:83;

[0019] h) A HC comprising the amino acid sequence of SEQ ID NO:87 and a LC comprising the amino acid sequence of SEQ ID NO:83;

[0020] i) A HC comprising the amino acid sequence of SEQ ID NO:86 and a LC comprising the amino acid sequence of SEQ ID NO:83.

[0021] In certain embodiments, the second antigen-binding domain competes with an antibody comprising the following for binding to human CD3, or binds to the same epitope on human CD3 as an antibody comprising the following:

[0022] a) A heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:70 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO:71;

[0023] b) A VH comprising the amino acid sequence of SEQ ID NO:66 and a VL comprising the amino acid sequence of SEQ ID NO:67; or

[0024] c) A VH comprising the amino acid sequence of SEQ ID NO:68 and a VL comprising the amino acid sequence of SEQ ID NO:69. In certain embodiments, the second antigen-binding domain comprises:

[0025] a) Heavy chain (H)-CDR1-3 and light chain (L)-CDR1-3 comprising the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58, and 59, respectively;

[0026] b) H-CDR1-3 and L-CDR1-3 each comprising the amino acid sequences of SEQ ID NO: 47, 48, 49, 50, 51, and 52;

[0027] c) H-CDR1-3 and L-CDR1-3 each comprising the amino acid sequences of SEQ ID NO: 47, 53, 49, 50, 51, and 52;

[0028] d) A heavy chain variable domain (VH) comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 70 and a light chain variable domain (VL) comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 71;

[0029] e) A VH comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 66 and a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 67;

[0030] f) A VH comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 68 and a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 69;

[0031] g) A VH comprising the amino acid sequence of SEQ ID NO: 70 and a VL comprising the amino acid sequence of SEQ ID NO: 71;

[0032] h) A VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 67;

[0033] i) A VH comprising the amino acid sequence of SEQ ID NO: 68 and a VL comprising the amino acid sequence of SEQ ID NO: 69;

[0034] j) A VH comprising the amino acid sequence of SEQ ID NO: 80 and a VL comprising the amino acid sequence of SEQ ID NO: 81;

[0035] k) A VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 77; or

[0036] l) A VH comprising the amino acid sequence of SEQ ID NO: 78 and a VL comprising the amino acid sequence of SEQ ID NO: 79.

[0037] In some embodiments, the present disclosure provides a bispecific binding molecule comprising:

[0038] a) A first antigen-binding domain comprising heavy chain (H)-CDR1-3 and light chain (L)-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 97, 61, 62, 63, 64, and 65, respectively, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 54, 55, 56, 57, 58, and 59, respectively;

[0039] b) A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 60, 61, 62, 63, 64, and 65, respectively, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 54, 55, 56, 57, 58, and 59, respectively;

[0040] c) A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 60, 61, 62, 63, 64, and 65, respectively, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 47, 53, 49, 50, 51, and 52, respectively;

[0041] d) A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 97, 61, 62, 63, 64, and 65, respectively, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 47, 48, 49, 50, 51, and 52, respectively;

[0042] e) A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 97, 61, 62, 63, 64, and 65, respectively, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 47, 53, 49, 50, 51, and 52, respectively; or

[0043] f) A first antigen-binding domain, comprising H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:60, 61, 62, 63, 64 and 65, and a second antigen-binding domain, comprising H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51 and 52.

[0044] In some embodiments, the present disclosure provides a bispecific binding molecule comprising:

[0045] a) A first antigen-binding domain, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) respectively comprising the amino acid sequences of SEQ ID NO:74 and 73, and a second antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71;

[0046] b) A first antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:72 and 73, and a second antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71;

[0047] c) A first antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:72 and 73, and a second antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69;

[0048] d) A first antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:74 and 73, and a second antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67;

[0049] e) A first antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:74 and 73, and a second antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69; or

[0050] f) A first antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:72 and 73, and a second antigen-binding domain, comprising a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67.

[0051] In some embodiments, the present disclosure provides a bispecific binding molecule comprising:

[0052] a) a first antigen-binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) comprising the amino acid sequences set forth in SEQ ID NOs: 74 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 80 and 81, respectively;

[0053] b) a first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 72 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 80 and 81, respectively;

[0054] c) a first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 72 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 78 and 79, respectively;

[0055] d) a first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 74 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 76 and 77, respectively;

[0056] e) a first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 74 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 78 and 79, respectively; or

[0057] f) a first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 72 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 76 and 77, respectively.

[0058] In some embodiments, the present disclosure provides a bispecific binding molecule comprising:

[0059] a) a first antigen-binding domain comprising a heavy chain (HC) and a light chain (LC) comprising the amino acid sequences set forth in SEQ ID NOs: 84 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 70 and 71, respectively;

[0060] b) a first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:82 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71, respectively;

[0061] c) a first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:82 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69, respectively;

[0062] d) a first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:84 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67, respectively;

[0063] e) a first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:84 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69, respectively;

[0064] f) a first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:82 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67, respectively;

[0065] g) a first antigen-binding domain comprising an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107 or 109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71, respectively;

[0066] h) a first antigen-binding domain comprising an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106 or 109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71, respectively;

[0067] i) A first antigen-binding domain comprising: a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:86, a HC comprising the amino acid sequence of SEQ ID NO:106 or 109, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69, respectively;

[0068] j) A first antigen-binding domain comprising: a HC comprising the amino acid sequence of SEQ ID NO:87, a HC comprising the amino acid sequence of SEQ ID NO:107 or 109, and a LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67, respectively;

[0069] k) A first antigen-binding domain comprising: a HC comprising the amino acid sequence of SEQ ID NO:87, a HC comprising the amino acid sequence of SEQ ID NO:107 or 109, and a LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69, respectively; or

[0070] l) A first antigen-binding domain comprising: a HC comprising the amino acid sequence of SEQ ID NO:86, a HC comprising the amino acid sequence of SEQ ID NO:106 or 109, and a LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67, respectively.

[0071] In some embodiments, the present disclosure provides a bispecific binding molecule comprising:

[0072] a) A first antigen-binding domain comprising: a heavy chain (HC) and a light chain (LC) comprising the amino acid sequences of SEQ ID NO:84 and 83, respectively, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:80 and 81, respectively;

[0073] b) A first antigen-binding domain comprising: a HC and a LC comprising the amino acid sequences of SEQ ID NO:82 and 83, respectively, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:80 and 81, respectively;

[0074] c) A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:82 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:78 and 79, respectively;

[0075] d) A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:84 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:76 and 77, respectively;

[0076] e) A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:84 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:78 and 79, respectively;

[0077] f) A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:82 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:76 and 77, respectively;

[0078] g) A first antigen-binding domain comprising an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107 or 109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:80 and 81, respectively;

[0079] h) A first antigen-binding domain comprising an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106 or 109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:80 and 81, respectively;

[0080] i) A first antigen-binding domain comprising an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106 or 109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:78 and 79, respectively;

[0081] j) A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107 or 109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:76 and 77, respectively;

[0082] k) A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107 or 109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:78 and 79, respectively; or

[0083] l) A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106 or 109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:76 and 77, respectively.

[0084] In certain embodiments, the bispecific binding molecules of the present disclosure comprise the amino acid sequences of SEQ ID NO:14 and 19, SEQ ID NO:7 and 19, SEQ ID NO:7 and 18, or SEQ ID NO:7 and 23. In certain embodiments, the bispecific binding molecules of the present disclosure comprise:

[0085] a) The amino acid sequences of SEQ ID NO:1 and 16;

[0086] b) The amino acid sequences of SEQ ID NO:2 and 16;

[0087] c) The amino acid sequences of SEQ ID NO:3 and 16;

[0088] d) The amino acid sequences of SEQ ID NO:4 and 16;

[0089] e) The amino acid sequences of SEQ ID NO:5 and 16;

[0090] f) The amino acid sequences of SEQ ID NO:6 and 16;

[0091] g) The amino acid sequences of SEQ ID NO:7 and 17;

[0092] h) The amino acid sequences of SEQ ID NO:7 and 20;

[0093] i) The amino acid sequences of SEQ ID NO:7 and 21;

[0094] j) The amino acid sequences of SEQ ID NO:7 and 22;

[0095] k) The amino acid sequences of SEQ ID NO:8, 9 and 16;

[0096] l) The amino acid sequences of SEQ ID NO:8, 10 and 16;

[0097] m) The amino acid sequences of SEQ ID NO:8, 11 and 16;

[0098] n) The amino acid sequences of SEQ ID NO:12, 11 and 16;

[0099] o) The amino acid sequences of SEQ ID NO:12, 13 and 16; or

[0100] p) The amino acid sequences of SEQ ID NO:8, 15 and 16.

[0101] In some embodiments, the bispecific binding molecules described herein may have any valency of the following first and second antigen-binding domains:

[0102] a) The valency of the first antigen-binding domain is 2, and the valency of the second antigen-binding domain is 2;

[0103] b) The valency of the first antigen-binding domain is 1, and the valency of the second antigen-binding domain is 1; or

[0104] c) The valency of the first antigen-binding domain is 2, and the valency of the second antigen-binding domain is 1.

[0105] In some embodiments, the bispecific binding molecules described herein may have a human IgG1 constant region; the constant region may comprise the amino acid substitutions L234A, L235A and / or G237A, where the residues are numbered according to the EU system.

[0106] In some embodiments, the second antigen-binding domain of the bispecific binding molecules described herein is a scFv.

[0107] In some embodiments of the bispecific binding molecules described herein, the heavy or light chain amino acid sequence of the second antigen-binding domain is fused to the heavy or light chain amino acid sequence of the first antigen-binding domain via a peptide linker. In certain embodiments, the peptide linker has the amino acid sequence GGGGSGGGGS (SEQ ID NO:93). In specific embodiments, the heavy or light chain amino acid sequence of the second antigen-binding domain can be fused to, for example:

[0108] a) the carboxyl terminus of the light chain of the first antigen-binding domain; or

[0109] b) the amino terminus of the light chain of the first antigen-binding domain.

[0110] In certain embodiments, the bispecific binding molecules described herein have at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the following characteristics:

[0111] a) The KD for immobilized ROR1 as determined by ELISA is 0.5 mM or lower;

[0112] b) The KD for soluble b-ROR1 as determined by ELISA is 0.4 nM or lower;

[0113] c) It is demonstrated that there is a reduction in internalization in ROR1-transfected MEC cells and / or Jurkat cells compared to an antibody comprising the heavy and light chain amino acid sequences of SEQ ID NOs: 82 and 83, respectively;

[0114] d) Induces LDH release in ROR1-transfected MEC cells exposed to PBMC at 1 μg / mL or lower;

[0115] e) Induces LDH release in JeKo-1 cells exposed to PBMC at 1 μg / mL or lower;

[0116] f) Induces LDH release in Mino cells exposed to PBMC at 1 μg / mL or lower;

[0117] g) Induces LDH release in MDA-MB-468 cells exposed to PBMC at 1 μg / mL or lower;

[0118] h) Upregulates CD69 on the surface of T cells co-cultured with ROR1-transfected MEC cells at 1 μg / mL or lower as determined by flow cytometry;

[0119] i) Upregulates CD69 on the surface of T cells co-cultured with ROR1-transfected JeKo-1 cells at 1 μg / mL or lower as determined by flow cytometry;

[0120] j) As determined by flow cytometry, upregulating CD69 on the surface of T cells co-cultured with ROR1-transfected Mino cells at 1 μg / mL or lower;

[0121] k) As determined by flow cytometry, upregulating CD69 on the surface of T cells co-cultured with ROR1-transfected MDA-MB-468 cells at 1 μg / mL or lower; and

[0122] l) Inducing T cells co-cultured with JeKo-1 or ROR1-transfected MEC cells to release IFN-γ, IFN-α, IL-10, IL-6, IL-4, and IL-2 at 1 μg / mL or lower.

[0123] The present disclosure also provides an immunoconjugate comprising a bispecific binding molecule described herein, wherein the bispecific binding molecule is conjugated to a cytotoxic agent.

[0124] The present disclosure also provides a pharmaceutical composition comprising a bispecific binding molecule described herein and a pharmaceutically acceptable excipient.

[0125] The present disclosure also provides one or more isolated nucleic acid molecules comprising nucleotide sequences encoding the heavy and light chain variable domains (VH and VL) of the first antigen-binding domain of a bispecific binding molecule described herein, and further comprising nucleotide sequences encoding the VH and VL of the second antigen-binding domain of a bispecific binding molecule described herein.

[0126] In some embodiments, the isolated nucleic acid molecule comprises:

[0127] a) The nucleotide sequences of SEQ ID NO: 37 and 42;

[0128] b) The nucleotide sequences of SEQ ID NO: 30 and 42;

[0129] c) The nucleotide sequences of SEQ ID NO: 30 and 41;

[0130] d) The nucleotide sequences of SEQ ID NO: 30 and 46;

[0131] e) The nucleotide sequences of SEQ ID NO: 24 and 39;

[0132] f) The nucleotide sequences of SEQ ID NO: 25 and 39;

[0133] g) The nucleotide sequences of SEQ ID NO: 26 and 39;

[0134] h) The nucleotide sequences of SEQ ID NO: 27 and 39;

[0135] i) The nucleotide sequences of SEQ ID NO: 28 and 39;

[0136] j) The nucleotide sequences of SEQ ID NO: 29 and 39;

[0137] k) The nucleotide sequences of SEQ ID NO: 30 and 40;

[0138] l) The nucleotide sequences of SEQ ID NO: 30 and 43;

[0139] m) The nucleotide sequences of SEQ ID NO: 30 and 44;

[0140] n) The nucleotide sequences of SEQ ID NO: 30 and 45;

[0141] o) The nucleotide sequences of SEQ ID NO: 31, 32 and 39;

[0142] p) The nucleotide sequences of SEQ ID NO: 31, 33 and 39;

[0143] q) The nucleotide sequences of SEQ ID NO: 31, 34 and 39;

[0144] r) The nucleotide sequences of SEQ ID NO: 35, 34 and 39;

[0145] s) The nucleotide sequences of SEQ ID NO: 35, 36 and 39; or

[0146] t) The nucleotide sequences of SEQ ID NO: 31, 38 and 39.

[0147] The present disclosure also provides a vector comprising the isolated nucleic acid molecule described herein.

[0148] The present disclosure also provides a host cell comprising nucleotide sequences encoding the heavy and light chain variable domains (VH and VL) of the first antigen-binding domain of the bispecific binding molecule described herein, and further comprising nucleotide sequences encoding the VH and VL of the second antigen-binding domain of the bispecific binding molecule described herein. In some embodiments, the host cell comprises the following nucleotide sequences:

[0149] a) SEQ ID NO: 37 and 42;

[0150] b) SEQ ID NO: 30 and 42;

[0151] c) SEQ ID NO: 30 and 41;

[0152] d) SEQ ID NO: 30 and 46;

[0153] e) SEQ ID NO: 24 and 39;

[0154] f) SEQ ID NO: 25 and 39;

[0155] g) SEQ ID NO: 26 and 39;

[0156] h) SEQ ID NO: 27 and 39;

[0157] i) SEQ ID NO: 28 and 39;

[0158] j) SEQ ID NO: 29 and 39;

[0159] k) SEQ ID NO: 30 and 40;

[0160] l) SEQ ID NO: 30 and 43;

[0161] m) SEQ ID NO: 30 and 44;

[0162] n) SEQ ID NO: 30 and 45;

[0163] o) SEQ ID NO: 31, 32 and 39;

[0164] p) SEQ ID NO: 31, 33 and 39;

[0165] q) SEQ ID NO: 31, 34 and 39;

[0166] r) SEQ ID NO: 35, 34 and 39;

[0167] s) SEQ ID NO: 35, 36 and 39; or

[0168] t) SEQ ID NO: 31, 38 and 39.

[0169] The present invention also provides a method for generating the bispecific binding molecules described herein, which comprises providing the host cells described herein, culturing the host cells under conditions suitable for the expression of the bispecific binding molecules, and isolating the resulting bispecific binding molecules.

[0170] The present disclosure also provides a method for treating cancer in a patient, which comprises administering to the patient a bispecific binding molecule as described herein. In addition, the present disclosure provides the use of the bispecific binding molecule as described herein in the preparation of a medicament for treating cancer in a patient. In some embodiments, the cancer is ROR1-positive cancer. In some embodiments, the cancer is leukemia, lymphoma or solid tumor. In some embodiments, the cancer is acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, T-cell leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, T-cell non-Hodgkin lymphoma, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, multiple myeloma, marginal zone lymphoma, small lymphocytic lymphoma or non-Hodgkin lymphoma that has undergone Richter transformation. In some embodiments, the cancer is colon cancer, non-small cell lung cancer, glioblastoma, hepatocellular carcinoma, pancreatic cancer, Ewing sarcoma, osteosarcoma, head and neck cancer, ovarian cancer, breast cancer or triple-negative breast cancer. In certain embodiments, the patient can be treated with one or more additional therapeutic agents, such as Bruton's tyrosine kinase (BTK) inhibitors, B-cell lymphoma-2 (Bcl-2) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and phosphatidylinositol 3-kinase (PI3K) inhibitors. In a particular embodiment, the additional therapeutic agent is ibrutinib, acalabrutinib, venetoclax, everolimus, sapanisertib or idelalisib.

[0171] The present disclosure also provides a kit comprising a bispecific binding molecule as described herein. In addition, the present disclosure provides an article comprising a bispecific binding molecule as described herein, wherein the article is suitable for treating cancer in a patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0172] Figure 1 is a schematic diagram of an exemplary configuration of the bispecific binding molecule of the present disclosure.

[0173] Figure 2 is a pair of graphs illustrating the binding of parental Ab1 Fab (WT) and three heavy chain variants (Ab2(T32A), Ab3(T32E) and W110Y) to human ROR1, as determined by ELISA (Figure A) and flow cytometry (Figure B) for binding to live ROR1-transfected MEC cells.

[0174] Figure 3 is a binding graph of parental Ab1 Fab (WT) and three heavy chain variants (Ab2(T32A), Ab6(T32A(HC)+A25P(LC)) and Ab7(T32A(HC)+T69R(LC))) to human ROR1, determined by ELISA using extended wash conditions.

[0175] Figure 4 These are a pair of graphs showing the selectivity of bispecific constructs 1 - 3, 7 - 9, and 13 - 15 for ROR1, as determined by flow cytometry of binding to ROR1 - transfected MEC cells (Figure A) and control MEC cells (Figure B). Nonspecific human IgG was used as a control ("Control").

[0176] Figure 5 These are a set of graphs showing the effect of ROR1 expression level on the binding of bispecific constructs 1 - 3, 7 - 9, and 13 - 15 (Figure A), 4 - 6 and 10 - 12 (Figure B), and 3, 9, and 15 - 20 (Figure C). Binding was evaluated by flow cytometry using JeKo - 1 cells, which express ROR1 at a lower level (~13,000 copies / cell) than ROR1 - transfected MEC cells (~56,000 copies / cell).

[0177] Figure 6 These are a pair of graphs showing the binding of bispecific constructs to immobilized human ROR1, characterized by ELISA.

[0178] Figure 7 These show the binding of bispecific constructs to soluble biotinylated human ROR1, characterized by ELISA.

[0179] Figure 8 These are a set of graphs showing the cell - surface levels of ROR1 - binding bispecific binding molecules ("surface biAb"), the surface level of free ROR1 not bound by bispecific binding molecules ("unoccupied epitope"), and total cell - surface ROR1 ("total ROR1") in ROR1 - transfected MEC cells incubated with construct 1 (Figure A), construct 7 (Figure B), and construct 13 (Figure C).

[0180] Figure 9 These are a set of graphs showing the cell - surface levels of ROR1 - binding bispecific binding molecules ("surface biAb"), the surface level of free ROR1 not bound by bispecific binding molecules ("unoccupied epitope"), and total cell - surface ROR1 ("total ROR1") in ROR1 - transfected MEC cells incubated with construct 19 (Figure A) and construct 20 (Figure B).

[0181] Figure 10 These show the quantification of bispecific constructs 1, 7, and 13 on the surface of ROR - 1 - transfected MEC cells after 24 - hour incubation.

[0182] Figure 11A set of graphs showing the binding of bispecific constructs 1-6 (Figure A), 7-12 (Figure B), and 13-15 (Figure C) to Jurkat cells by flow cytometry. Nonspecific human IgG was used as a control ("Control").

[0183] Figure 12 A set of graphs showing the binding of bispecific constructs 1, 7, and 13 (Figure A) and bispecific constructs 3, 9, and 15-10 (Figure B) to Jurkat cells by flow cytometry. Nonspecific human IgG was used as a control in one experiment (Figure A, "Control"), while an irrelevant ROR1 x CD3 bispecific construct (U.S. Patent Publication 2017 / 0233472) was used as a control in a second experiment (Figure B, "Control 1").

[0184] Figure 13 Graphs quantifying the levels of bispecific constructs 1-3, 7-9, and 13-15 on the surface of Jurkat cells over time after binding to CD3 in the absence of ROR1.

[0185] Figure 14 A set of graphs quantifying the LDH released from ROR1-transfected MEC cells (Figure A) and the cell surface levels of CD69 on CD8+ cells (Figure B), where ROR1-transfected cells were incubated with (1) human PBMCs and (2) bispecific constructs 1, 3, 7, 9, 13, and 15, and a control anti-CD19 / anti-CD3 bispecific binding molecule ("CD19 x CD3").

[0186] Figure 15 A set of graphs quantifying the LDH released from ROR1-transfected MEC cells (Figure A) and the cell surface levels of CD69 on CD8 + cells (Figure B), where ROR1-transfected cells were incubated with human PBMCs and bispecific constructs 3, 9, and 15-20.

[0187] Figure 16 Graphs comparing the LDH release between ROR1-transfected and non-transfected MEC cells incubated with (1) PBMCs and (2) bispecific constructs 1-3, 7-9, and 13-15 and a control anti-CD19 / anti-CD3 bispecific binding molecule. LDH release was also evaluated in samples without target cells ("PBMCs") and samples without bispecific constructs ("no biAb").

[0188] Figure 17 A set of graphs evaluating the CD69 levels on T cells where ROR1 - / CD19 + MEC cells (Figure A) or ROR1 + / CD19 + MEC cells (Figure B) were incubated with (1) T cells and (2) a bispecific construct 7 or a control anti-CD19 / anti-CD3 bispecific binding molecule (“CD19xCD3”).

[0189] Figure 18 Is a set of graphs quantifying the release of LDH (Figure A) and the cell surface levels of CD69 on CD8 + cells (Figure B) where JeKo-1 cells were incubated with human PBC and bispecific constructs 2, 3, 8, 9, 14, and 15.

[0190] Figure 19 Is a set of graphs quantifying the release of LDH (Figure A) and the cell surface levels of CD69 on CD8 + cells (Figure B) where JeKo-1 cells were incubated with human PBC and bispecific constructs 3, 7, 9, and 15 - 20.

[0191] Figure 20 Is a set of graphs quantifying the release of LDH (Figure A) and the cell surface levels of CD69 on CD8 + cells (Figure B) where Mino cells were incubated with PBMC and bispecific constructs 7 - 9.

[0192] Figure 21 Is a set of graphs quantifying the release of LDH (Figure A) and the cell surface levels of CD69 on CD8 + cells (Figure B) where MDA-MB-468 cells were incubated with human PBC and bispecific constructs 2, 3, 8, 9, 12, and 14.

[0193] Figure 22 Is a set of graphs quantifying the release of LDH (Figure A) and the cell surface levels of CD69 on CD8 + cells (Figure B) where MDA-MB-468 cells were incubated with human PBC and bispecific constructs 9, 19, and 20.

[0194] Figure 23 Is a set of graphs quantifying the release of TNF-α from JeKo-1 cells (Figure A), ROR1-transfected MEC cells (Figure B), and mock-transfected ROR1-MEC cells (Figure C) after activation of T cells with the indicated target cells and bispecific constructs 7, 9, 18, and 20 (Figure A) or 9, 19, 20 (Figure B and C). Mock-transfected ROR1-MEC cells (Figure C) were treated with a single concentration (1 μg / mL) of the bispecific construct. "Control 1" is an irrelevant ROR1 x CD3 bispecific construct.

[0195] Figure 24 A set of graphs quantifying TNF-γ released from JeKo-1 cells (Figure A), ROR1-transfected MEC cells (Figure B), and mock-transfected ROR1-MEC cells (Figure C) after activation of T cells with the indicated target cells and bispecific constructs 7, 9, 18, and 20 (Figure A) or 9, 18, and 20 (Figure B and C). Mock-transfected ROR1-MEC cells (Figure C) were treated with a single concentration (1 μg / mL) of the bispecific construct. "Control 1" is an irrelevant ROR1 x CD3 bispecific construct.

[0196] Figure 25 A set of graphs quantifying IL-2 released from JeKo-1 cells (Figure A), ROR1-transfected MEC cells (Figure B), and mock-transfected ROR1-MEC cells (Figure C) after activation of T cells with the indicated target cells and bispecific constructs 7, 9, 18, and 20 (Figure A) or 9, 18, and 20 (Figure B and C). Mock-transfected ROR1-MEC cells (Figure C) were treated with a single concentration (1 μg / mL) of the bispecific construct. "Control 1" is an irrelevant ROR1 x CD3 bispecific construct.

[0197] Figure 26 A set of graphs quantifying IL-2 released from JeKo-1 cells (Figure A), ROR1-transfected MEC cells (Figure B), and mock-transfected ROR1-MEC cells (Figure C) after activation of T cells with the indicated target cells and bispecific constructs 7, 9, 18, and 20 (Figure A) or 9, 18, and 20 (Figure B and C). Mock-transfected ROR1-MEC cells (Figure C) were treated with a single concentration (1 μg / mL) of the bispecific construct. "Control 1" is an irrelevant ROR1 x CD3 bispecific construct.

[0198] Figure 27 A set of graphs quantifying IL-6 released from JeKo-1 cells (Figure A), ROR1-transfected MEC cells (Figure B), and mock-transfected ROR1-MEC cells (Figure C) after activation of T cells with the indicated target cells and bispecific constructs 7, 9, 18, and 20 (Figure A) or 9, 18, and 20 (Figure B and C). Mock-transfected ROR1-MEC cells (Figure C) were treated with a single concentration (1 μg / mL) of the bispecific construct. "Control 1" is an irrelevant ROR1 x CD3 bispecific construct.

[0199] Figure 28A set of graphs showing the quantification of IL-10 released from JeKo-1 cells (Figure A), ROR1-transfected MEC cells (Figure B), and mock-transfected ROR1-MEC cells (Figure C) after activation of T cells with the indicated target cells and bispecific constructs 7, 9, 18, and 20 (Figure A) or 9, 18, and 20 (Figure B and C). Mock-transfected ROR1-MEC cells (Figure C) were treated with a single concentration (1 μg / mL) of the bispecific construct. "Control 1" is an irrelevant ROR1 x CD3 bispecific construct.

[0200] Figure 29 A table showing the SEQ ID NOs of the specified sequences of the anti-ROR1 and anti-CD3 antigen-binding domains. Detailed Description

[0201] The present disclosure provides novel bispecific binding molecules that bind to ROR1 and CD3. In some embodiments, binding of the bispecific binding molecule to ROR1 and CD3 brings T cells into proximity with ROR1-positive tumor cells, thereby treating cancer by invoking the cytotoxicity of T cells. Unless otherwise indicated, as used herein, "ROR1" refers to human ROR1. The human ROR1 polypeptide sequence is available under UniProt accession number Q01973-1 (SEQ ID NO: 89). Unless otherwise indicated, as used herein, "CD3" refers to human CD3. CD3 consists of one γ chain, one δ chain, and two ε chains. The human CD3γ polypeptide sequence is available under GenBank accession number NP_000064.1 (SEQ ID NO: 90). The human CD3δ polypeptide sequence is available under GenBank accession number NP_000723.1 (SEQ ID NO: 91). The human CD3ε polypeptide sequence is available under GenBank accession number NP_000724.1 (SEQ ID NO: 92).

[0202] In some embodiments, the antigen-binding domains of the bispecific binding molecules of the present disclosure are derived from an anti-ROR1 antibody and an anti-CD3 antibody. As used herein, the term "antibody" (Ab) or "immunoglobulin" (Ig) can refer to a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) interconnected by disulfide bonds. Each heavy chain consists of a heavy-chain variable domain (VH) and a heavy-chain constant region (CH). Each light chain consists of a light-chain variable domain (VL) and a light-chain constant region (CL). The VH and VL domains can be further subdivided into hypervariable regions, called "complementary determining regions" (CDRs), interspersed with more conserved regions, called "framework regions" (FRs). Each VH and VL consists of three CDRs (H-CDRs herein denote CDRs from heavy chains; and L-CDRs herein denote CDRs from light chains) and four FRs, arranged from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0203] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of several well-known schemes, including those described in Kabat et al., 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) ("Kabat" numbering scheme); Al-Lazikani et al., JMB 273, 927-948 (1997) ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996) ("contact" numbering scheme); Lefranc et al., Dev Comp Immunol. 27(1):55-77 (2003) ("IMGT" numbering scheme); and Honegger and Plückthun, J Mol Biol, 309(3):657-70 (2001) ("Aho" numbering scheme).

[0204] The boundaries of given CDRs or FRs can vary according to the scheme used for identification. For example, the Kabat scheme is based on sequence alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with inserted letters for insertions, e.g., "30a". These two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numberings. The contact scheme is based on the molecule of the complex crystal structure and is similar to the Chothia numbering scheme in many respects. Unless otherwise specified, the CDRs of antibodies referred to herein can be identified according to any one of the Kabat, Chothia, IMGT, contact, and Aho methods.

[0205] In some embodiments, the anti-ROR1 antibody and / or anti-CD3 antibody from which the bispecific binding molecule of the present disclosure is derived are monoclonal antibodies. In some embodiments, the anti-ROR1 antibody and / or anti-CD3 antibody are chimeric, humanized, or fully humanized antibodies.

[0206] The term "affinity" refers to a measure of the attraction between an antigen and an antibody or its antigen-binding fragment or related molecule (e.g., a bispecific binding molecule). The intrinsic attraction of an antibody for an antigen is typically expressed as the binding affinity equilibrium constant (K D ) of a particular antibody-antigen interaction. When K D ≤ 1 mM, preferably ≤ 100 nM, the antibody is considered to specifically bind to the antigen. K D The binding affinity constant can be measured by, for example, surface plasmon resonance (BIAcore TM ) or biolayer interferometry techniques, such as using the IBIS MX96 SPR system from IBIS Technologies or the Octet TM system from ForteBio.

[0207] The term "complementary site" refers to the antigen-binding site of an antibody (i.e., the part of the antibody that recognizes and binds to an epitope).

[0208] As used herein, the term "epitope" refers to a part (determinant) of an antigen to which an antibody or related molecule (e.g., a bispecific binding molecule) specifically binds. Epitope determinants are usually composed of the chemically reactive surface groups of a molecule, such as amino acids or carbohydrates or sugar side chains, and usually have specific three-dimensional structural features as well as specific charge characteristics. Epitopes can be "linear" or "conformational". In a linear epitope, all the interaction points between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction points occur on amino acid residues on the protein that are separated from each other in the primary amino acid sequence. Once the desired epitope on an antigen has been identified, it is possible to generate antibodies against that epitope using techniques well known in the art. For example, antibodies against a linear epitope can be generated, e.g., by immunizing an animal with a peptide having the amino acid residues of the linear epitope. Antibodies against a conformational epitope can be generated, e.g., by immunizing an animal with a small domain containing the relevant amino acid residues of the conformational epitope. Antibodies against a specific epitope can also be generated, e.g., by immunizing an animal with a target molecule (e.g., ROR1 or CD3) or a related portion thereof and then screening for binding to the epitope.

[0209] It is possible to determine whether an antibody binds or competes for binding to the same epitope of ROR1 or CD3 as the bispecific binding molecules of the present disclosure by using methods known in the art, including but not limited to competition assays, epitope mapping, and alanine scanning. In one embodiment, the bispecific binding molecules of the present disclosure are allowed to bind to ROR1 or CD3 under saturated conditions, and then the ability of a test antibody to bind to the antigen is measured. If the test antibody is able to bind to the antigen simultaneously with a reference specific binding molecule, the test antibody binds to an epitope different from that of the reference bispecific binding molecule. However, if the test antibody is unable to bind to the antigen simultaneously, the test antibody binds to the same epitope, an overlapping epitope, or an epitope very close to the epitope to which the bispecific binding molecules of the present disclosure bind. Such an experiment can be performed using, for example, ELISA, RIA, BIACORE TM 、SPR, biolayer interferometry, or flow cytometry. To test whether the bispecific binding molecules of the present disclosure cross-compete with another antibody for binding to ROR1 or CD3, the above competition methods can be used in both directions, i.e., determining whether a known antibody blocks the test antibody and vice versa. Such cross-competition experiments can be performed, for example, using an IBIS MX96 SPR instrument or an Octet TM system.

[0210] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human ROR1 or CD3 or portions thereof). It has been shown that certain fragments of full-length antibodies can perform the antigen-binding function of the antibody. Examples of binding fragments included within the term "antigen-binding portion" include (i) Fab fragment: a monovalent fragment consisting of the V L and V H domains, C L and C H 1; (ii) F(ab')2 fragment: a divalent fragment containing two Fab fragments linked by a hinge region disulfide bond; (iii) Fd fragment consisting of V H and C H 1 domains; (iv) Fv fragment consisting of the V L and V H domains of a single arm of the antibody; (v) a dAb fragment consisting of the V H domain; and (vi) isolated complementarity-determining regions (CDRs) capable of specifically binding to an antigen. In addition, although the two domains of the Fv fragment, V L and V H , are encoded by different genes, they can be joined together using recombinant methods by a synthetic linker such that they can be made into a single protein chain, wherein the V L and V H domains pair to form a monovalent molecule (referred to as a single-chain variable fragment (scFv)). The present disclosure also includes antigen-binding molecules comprising V H and / or V L . In the case of VH, the molecule may also comprise one or more of the CH1, hinge, CH2 or CH3 regions. Such single-chain antibodies are also intended to be included within the term "antigen-binding portion" of an antibody. Also included are other forms of single-chain antibodies, such as diabodies. A diabody is a bivalent bispecific antibody in which the V H and V L domains are expressed on a single peptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains on the other chain and creating two antigen-binding sites.

[0211] Unless otherwise specified, all antibody amino acid residue numberings referred to in the present disclosure are those under the Honegger numbering scheme (Honegger and Plückthun, J Mol Biol, 309(3):657-70(2001)).

[0212] Anti-ROR1 / anti-CD3 bispecific binding molecule

[0213] The present disclosure relates to a bispecific binding molecule comprising a first antigen-binding domain that specifically binds to the extracellular domain of human ROR1 and a second antigen-binding domain that specifically binds to the extracellular domain of human CD3.

[0214] In some embodiments, the first antigen-binding domain binds to an epitope on the extracellular portion of the ROR1 protein, such as an epitope in one or more of the immunoglobulin (Ig)-like, cysteine-rich, and kringle domains. In certain embodiments, the first antigen-binding domain binds to the amino acid sequence of ROR1 shown in SEQ ID NO: 94 or 95 (excluding the terminal cysteine, which is added for convenience of conjugation).

[0215] In some embodiments, the first antigen-binding domain competes with the anti-ROR1 antibody described in PCT patent application PCT / US2013 / 32572 for binding to human ROR1, and / or binds to the same epitope on human ROR1 as the anti-ROR1 antibody described in PCT patent application PCT / US2013 / 32572. In some embodiments, the first antigen-binding domain competes with an antibody ("Ab1") comprising the heavy chain amino acid sequence shown in SEQ ID NO: 82 and the light chain amino acid sequence shown in SEQ ID NO: 83 for binding to human ROR1, and / or binds to the same epitope on human ROR1 as an antibody ("Ab1") comprising the heavy chain amino acid sequence shown in SEQ ID NO: 82 and the light chain amino acid sequence shown in SEQ ID NO: 83.

[0216] In some embodiments, the first antigen-binding domain comprises heavy chain (H)-CDR1-3 in the amino acid sequence of SEQ ID NO: 82 and light chain (L)-CDR1-3 in the amino acid sequence of SEQ ID NO: 83, wherein the CDRs are determined by the Kabat, Chothia, IMGT, or contact methods or any combination thereof.

[0217] In some embodiments, the first antigen-binding domain comprises H-CDR1-3 and L-CDR1-3 of the following amino acid sequences:

[0218] - SEQ ID NOs: 60, 61, 62, 63, 64, and 65, respectively;

[0219] - SEQ ID NOs: 97, 61, 62, 63, 64, and 65, respectively;

[0220] - SEQ ID NOs: 98, 61, 62, 63, 64, and 65, respectively;

[0221] - SEQ ID NO:60, 61, 62, 99, 64, 65 respectively;

[0222] - SEQ ID NO:60, 61, 62, 63, 102, 65 respectively;

[0223] - SEQ ID NO:97, 61, 62, 99, 64, 65 respectively;

[0224] - SEQ ID NO:97, 61, 62, 63, 102, 65 respectively;

[0225] - SEQ ID NO:98, 61, 62, 99, 64, 65 respectively; or

[0226] - SEQ ID NO:98, 61, 62, 63, 102, 65 respectively.

[0227] In some embodiments, the first antigen-binding domain comprises a heavy-chain variable domain (VH) and a light-chain variable domain (VL), the heavy-chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:72, and the light-chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:73.

[0228] In some embodiments, the first antigen-binding domain comprises:

[0229] - VH comprising the amino acid sequence of SEQ ID NO:72 and VL comprising the amino acid sequence of SEQ ID NO:73;

[0230] - VH comprising the amino acid sequence of SEQ ID NO:74 and VL comprising the amino acid sequence of SEQ ID NO:73;

[0231] - VH comprising the amino acid sequence of SEQ ID NO:75 and VL comprising the amino acid sequence of SEQ ID NO:73;

[0232] - VH comprising the amino acid sequence of SEQ ID NO:72 and VL comprising the amino acid sequence of SEQ ID NO:100;

[0233] - VH comprising the amino acid sequence of SEQ ID NO:72 and VL comprising the amino acid sequence of SEQ ID NO:103;

[0234] - a VH comprising the amino acid sequence of SEQ ID NO:74 and a VL comprising the amino acid sequence of SEQ ID NO:100;

[0235] - a VH comprising the amino acid sequence of SEQ ID NO:74 and a VL comprising the amino acid sequence of SEQ ID NO:103;

[0236] - a VH comprising the amino acid sequence of SEQ ID NO:75 and a VL comprising the amino acid sequence of SEQ ID NO:100; or

[0237] - a VH comprising the amino acid sequence of SEQ ID NO:75 and a VL comprising the amino acid sequence of SEQ ID NO:103;

[0238] In some embodiments, the first antigen-binding domain comprises a heavy chain (HC) and a light chain (LC), the heavy chain comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:82, and the light chain comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:83.

[0239] In some embodiments, the first antigen-binding domain comprises:

[0240] - an HC comprising the amino acid sequence of SEQ ID NO:82 and an LC comprising the amino acid sequence of SEQ ID NO:83;

[0241] - an HC comprising the amino acid sequence of SEQ ID NO:84 and an LC comprising the amino acid sequence of SEQ ID NO:83;

[0242] - an HC comprising the amino acid sequence of SEQ ID NO:85 and an LC comprising the amino acid sequence of SEQ ID NO:83;

[0243] - an HC comprising the amino acid sequence of SEQ ID NO:82 and an LC comprising the amino acid sequence of SEQ ID NO:101;

[0244] - an HC comprising the amino acid sequence of SEQ ID NO:82 and an LC comprising the amino acid sequence of SEQ ID NO:104;

[0245] - an HC comprising the amino acid sequence of SEQ ID NO:84 and an LC comprising the amino acid sequence of SEQ ID NO:101;

[0246] - an HC comprising the amino acid sequence of SEQ ID NO:84 and an LC comprising the amino acid sequence of SEQ ID NO:104;

[0247] - an HC comprising the amino acid sequence of SEQ ID NO:85 and an LC comprising the amino acid sequence of SEQ ID NO:101; or

[0248] - an HC comprising the amino acid sequence of SEQ ID NO:85 and an LC comprising the amino acid sequence of SEQ ID NO:104;

[0249] In certain embodiments, the HC sequence is modified to reduce or eliminate effector function. For example, the IgG1 portion of the HC sequence may contain an "LALA" (L234A / L235A) or "LALAGA" (L234A / L235A / G237A) mutation (where all residues are numbered according to the EU numbering scheme).

[0250] In some embodiments, the first antigen-binding domain comprises:

[0251] - an HC comprising the amino acid sequence of SEQ ID NO:7 and an LC comprising the amino acid sequence of SEQ ID NO:83;

[0252] - an HC comprising the amino acid sequence of SEQ ID NO:14 and an LC comprising the amino acid sequence of SEQ ID NO:83;

[0253] - an HC comprising the amino acid sequence of SEQ ID NO:105 and an LC comprising the amino acid sequence of SEQ ID NO:83;

[0254] - an HC comprising the amino acid sequence of SEQ ID NO:7 and an LC comprising the amino acid sequence of SEQ ID NO:101;

[0255] - an HC comprising the amino acid sequence of SEQ ID NO:7 and an LC comprising the amino acid sequence of SEQ ID NO:104;

[0256] - an HC comprising the amino acid sequence of SEQ ID NO:14 and an LC comprising the amino acid sequence of SEQ ID NO:101;

[0257] - an HC comprising the amino acid sequence of SEQ ID NO:14 and an LC comprising the amino acid sequence of SEQ ID NO:104;

[0258] - an HC comprising the amino acid sequence of SEQ ID NO:105 and an LC comprising the amino acid sequence of SEQ ID NO:101; or

[0259] - an HC comprising the amino acid sequence of SEQ ID NO:105 and an LC comprising the amino acid sequence of SEQ ID NO:104;

[0260] In some embodiments, the first antigen-binding domain comprises:

[0261] - an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:83;

[0262] - an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:83;

[0263] - an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:83;

[0264] - an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:101;

[0265] - an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:104;

[0266] - an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:101;

[0267] - an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:104;

[0268] - an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:101; or

[0269] - HC comprising the amino acid sequence of SEQ ID NO:88, HC comprising the amino acid sequence of SEQ ID NO:108, and LC comprising the amino acid sequence of SEQ ID NO:104.

[0270] In some embodiments, the first antigen-binding domain comprises:

[0271] - HC comprising the amino acid sequence of SEQ ID NO:86, HC comprising the amino acid sequence of SEQ ID NO:109, and LC comprising the amino acid sequence of SEQ ID NO:83;

[0272] - HC comprising the amino acid sequence of SEQ ID NO:87, HC comprising the amino acid sequence of SEQ ID NO:109, and LC comprising the amino acid sequence of SEQ ID NO:83;

[0273] - HC comprising the amino acid sequence of SEQ ID NO:88, HC comprising the amino acid sequence of SEQ ID NO:109, and LC comprising the amino acid sequence of SEQ ID NO:83;

[0274] - HC comprising the amino acid sequence of SEQ ID NO:86, HC comprising the amino acid sequence of SEQ ID NO:109, and LC comprising the amino acid sequence of SEQ ID NO:101;

[0275] - HC comprising the amino acid sequence of SEQ ID NO:86, HC comprising the amino acid sequence of SEQ ID NO:109, and LC comprising the amino acid sequence of SEQ ID NO:104;

[0276] - HC comprising the amino acid sequence of SEQ ID NO:87, HC comprising the amino acid sequence of SEQ ID NO:109, and LC comprising the amino acid sequence of SEQ ID NO:101;

[0277] - HC comprising the amino acid sequence of SEQ ID NO:87, HC comprising the amino acid sequence of SEQ ID NO:109, and LC comprising the amino acid sequence of SEQ ID NO:104;

[0278] - HC comprising the amino acid sequence of SEQ ID NO:88, HC comprising the amino acid sequence of SEQ ID NO:109, and LC comprising the amino acid sequence of SEQ ID NO:101; or

[0279] -HCs comprising the amino acid sequence of SEQ ID NO:89, HCs comprising the amino acid sequence of SEQ ID NO:106, and LCs comprising the amino acid sequence of SEQ ID NO:104.

[0280] In some embodiments, the first antigen-binding domain comprises the six CDRs, heavy and light chain variable domains, and / or heavy and light chain amino acid sequences of an anti-ROR1 antibody as described in PCT / US2013 / 32572.

[0281] In some embodiments, the second antigen-binding domain binds to a Group I or Group II epitope of CD3, as defined in Tunnacliffe et al., International Immunology 1:546-550 (1989).

[0282] In some embodiments, the second antigen-binding domain competes with the anti-CD3 antibody OKT3 or SP34 for binding to human CD3, and / or binds to the same epitope of human CD3 as the anti-CD3 antibody OKT3 or SP34. In some embodiments, the second antigen-binding domain competes with an antibody comprising:

[0283] -VH comprising the amino acid sequence of SEQ ID NO:66 and VL comprising the amino acid sequence of SEQ ID NO:67;

[0284] -VH comprising the amino acid sequence of SEQ ID NO:68 and VL comprising the amino acid sequence of SEQ ID NO:69; or

[0285] -VH comprising the amino acid sequence of SEQ ID NO:70 and VL comprising the amino acid sequence of SEQ ID NO:71.

[0286] In some embodiments, the second antigen-binding domain comprises H-CDR1-3 and L-CDR1-3 of the following amino acid sequences:

[0287] -SEQ ID NO:66 and 67, respectively;

[0288] -SEQ ID NO:68 and 69, respectively; or

[0289] -SEQ ID NO:70 and 71, respectively;

[0290] wherein the CDRs are determined by the Kabat, Chothia, IMGT, or contact methods or any combination thereof.

[0291] In some embodiments, the second antigen-binding domain comprises H-CDR1-3 and L-CDR1-3 of the following amino acid sequences:

[0292] - SEQ ID NO:47, 48, 49, 50, 51, and 52, respectively;

[0293] - SEQ ID NO:47, 53, 49, 50, 51, and 52, respectively; or

[0294] - SEQ ID NO:54, 55, 56, 57, 58, and 59, respectively.

[0295] In some embodiments, the second antigen-binding domain comprises:

[0296] - a VH comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:66, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:67;

[0297] - a VH comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:68, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:69; or

[0298] - a VH comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:70, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:71.

[0299] In some embodiments, the second antigen-binding domain comprises:

[0300] - a VH comprising the amino acid sequence of SEQ ID NO:66 and a VL comprising the amino acid sequence of SEQ ID NO:67;

[0301] - a VH comprising the amino acid sequence of SEQ ID NO:68 and a VL comprising the amino acid sequence of SEQ ID NO:69;

[0302] - A VH comprising the amino acid sequence of SEQ ID NO:70 and a VL comprising the amino acid sequence of SEQ ID NO:71;

[0303] - A VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:77;

[0304] - A VH comprising the amino acid sequence of SEQ ID NO:78 and a VL comprising the amino acid sequence of SEQ ID NO:79; or

[0305] - A VH comprising the amino acid sequence of SEQ ID NO:80 and a VL comprising the amino acid sequence of SEQ ID NO:81.

[0306] In some embodiments, the second antigen-binding domain comprises the six CDRs and / or the variable domains of the heavy and light chains of the anti-CD3 antibody described in U.S. Patent Publication 2018 / 0112011.

[0307] The present disclosure also contemplates any combination of the above-described first and second antigen-binding domains.

[0308] In some embodiments, the bispecific binding molecule of the present disclosure comprises:

[0309] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:97, 61, 62, 63, 64 and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58 and 59;

[0310] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:60, 61, 62, 63, 64 and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58 and 59;

[0311] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:60, 61, 62, 63, 64 and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 53, 49, 50, 51 and 52;

[0312] - The first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:97, 61, 62, 63, 64 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51 and 52;

[0313] - The first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:97, 61, 62, 63, 64 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 53, 49, 50, 51 and 52;

[0314] - The first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:60, 61, 62, 63, 64 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51 and 52;

[0315] - The first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:98, 61, 62, 63, 64 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58 and 59;

[0316] - The first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:98, 61, 62, 63, 64 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51 and 52;

[0317] - A first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:98, 61, 62, 63, 64 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51 and 52;

[0318] - A first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:60, 61, 62, 99, 64 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58 and 59;

[0319] - A first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:60, 61, 62, 99, 64 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51 and 52;

[0320] - A first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:60, 61, 62, 99, 64 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 53, 49, 50, 51 and 52;

[0321] - A first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:60, 61, 62, 63, 102 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58 and 59;

[0322] - A first antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:60, 61, 62, 63, 102 and 65, and a second antigen-binding domain, which comprises: H-CDR1-3 and L-CDR1-3 respectively comprising the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51 and 52;

[0323] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:60, 61, 62, 63, 102, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51, and 52;

[0324] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:97, 61, 62, 99, 64, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58, and 59;

[0325] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:97, 61, 62, 99, 64, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51, and 52;

[0326] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:97, 61, 62, 99, 64, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:47, 53, 49, 50, 51, and 52;

[0327] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:97, 61, 62, 63, 102, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58, and 59;

[0328] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:97, 61, 62, 63, 102, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51, and 52;

[0329] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:97, 61, 62, 63, 102, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:47, 53, 49, 50, 51, and 52;

[0330] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:98, 61, 62, 99, 64, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58, and 59;

[0331] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:98, 61, 62, 99, 64, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51, and 52;

[0332] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:98, 61, 62, 99, 64, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:47, 53, 49, 50, 51, and 52;

[0333] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:98, 61, 62, 63, 102, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58, and 59;

[0334] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:98, 61, 62, 63, 102, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:47, 48, 49, 50, 51, and 52; or

[0335] - A first antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:98, 61, 62, 63, 102, and 65, and a second antigen-binding domain comprising H-CDR1-3 and L-CDR1-3 that respectively comprise the amino acid sequences of SEQ ID NO:47, 53, 49, 50, 51, and 52.

[0336] In some embodiments, the bispecific binding molecule of the present disclosure comprises:

[0337] - A first antigen-binding domain comprising VH and VL that are respectively at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:74 and 73, and a second antigen-binding domain comprising VH and VL that are respectively at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:70 and 71;

[0338] - A first antigen-binding domain comprising VH and VL that are respectively at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:72 and 73, and a second antigen-binding domain comprising VH and VL that are respectively at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:70 and 71;

[0339] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:72 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:68 and 69, respectively;

[0340] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:74 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:66 and 67, respectively;

[0341] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:74 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:68 and 69, respectively;

[0342] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:72 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:66 and 67, respectively;

[0343] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:75 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:70 and 71, respectively;

[0344] - A first antigen-binding domain comprising VH and VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:75 and 73 respectively, and a second antigen-binding domain comprising VH and VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:66 and 67 respectively;

[0345] - A first antigen-binding domain comprising VH and VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:75 and 73 respectively, and a second antigen-binding domain comprising VH and VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:68 and 69 respectively;

[0346] - A first antigen-binding domain comprising VH and VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:72 and 100 respectively, and a second antigen-binding domain comprising VH and VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:70 and 71 respectively;

[0347] - A first antigen-binding domain comprising VH and VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:72 and 100 respectively, and a second antigen-binding domain comprising VH and VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:66 and 67 respectively;

[0348] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:72 and 100, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:68 and 69, respectively;

[0349] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:72 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:70 and 71, respectively;

[0350] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:72 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:66 and 67, respectively;

[0351] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:72 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:68 and 69, respectively;

[0352] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:74 and 100, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:70 and 71, respectively;

[0353] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:74 and 100 respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:66 and 67 respectively;

[0354] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:74 and 100 respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:68 and 69 respectively;

[0355] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:74 and 103 respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:70 and 71 respectively;

[0356] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:74 and 103 respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:66 and 67 respectively;

[0357] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:74 and 103 respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:68 and 69 respectively;

[0358] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:75 and 100 respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:70 and 71 respectively;

[0359] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:75 and 100 respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:66 and 67 respectively;

[0360] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:75 and 100 respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:68 and 69 respectively;

[0361] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:75 and 103 respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the amino acid sequences of SEQ ID NO:70 and 71 respectively;

[0362] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:75 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:66 and 67, respectively; or

[0363] - A first antigen-binding domain comprising a VH and a VL that are at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:75 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL that are at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NO:68 and 69, respectively.

[0364] In some embodiments, the bispecific binding molecule of the present disclosure comprises:

[0365] - A first antigen-binding domain comprising a VH and a VL that respectively comprise the amino acid sequences of SEQ ID NO:74 and 73, and a second antigen-binding domain comprising a VH and a VL that respectively comprise the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81;

[0366] - A first antigen-binding domain comprising a VH and a VL that respectively comprise the amino acid sequences of SEQ ID NO:72 and 73, and a second antigen-binding domain comprising a VH and a VL that respectively comprise the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81;

[0367] - A first antigen-binding domain comprising a VH and a VL that respectively comprise the amino acid sequences of SEQ ID NO:72 and 73, and a second antigen-binding domain comprising a VH and a VL that respectively comprise the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79;

[0368] - A first antigen-binding domain comprising a VH and a VL that respectively comprise the amino acid sequences of SEQ ID NO:74 and 73, and a second antigen-binding domain comprising a VH and a VL that respectively comprise the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77;

[0369] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:74 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79, respectively;

[0370] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:72 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77, respectively;

[0371] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:75 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81, respectively;

[0372] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:75 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77, respectively;

[0373] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:75 and 73, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79, respectively;

[0374] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:72 and 100, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81, respectively;

[0375] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:72 and 100, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77, respectively;

[0376] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:72 and 100, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79, respectively;

[0377] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:72 and 103 respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81 respectively;

[0378] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:72 and 103 respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77 respectively;

[0379] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:72 and 103 respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79 respectively;

[0380] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:74 and 100 respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81 respectively;

[0381] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:74 and 100 respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77 respectively;

[0382] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:74 and 100 respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79 respectively;

[0383] - A first antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:74 and 103 respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81 respectively;

[0384] - A first antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:74 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77, respectively;

[0385] - A first antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:74 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79, respectively;

[0386] - A first antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:75 and 100, respectively, and a second antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81, respectively;

[0387] - A first antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:75 and 100, respectively, and a second antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77, respectively;

[0388] - A first antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:75 and 100, respectively, and a second antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79, respectively;

[0389] - A first antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:74 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81, respectively;

[0390] - A first antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:74 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77, respectively; or

[0391] - A first antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:74 and 103, respectively, and a second antigen-binding domain comprising a VH and a VL containing the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79, respectively.

[0392] In some embodiments, the bispecific binding molecules of the present disclosure comprise:

[0393] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences set forth in SEQ ID NO: 84 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NO: 70 and 71 or 80 or 81, respectively;

[0394] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences set forth in SEQ ID NO: 82 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NO: 70 and 71 or 80 or 81, respectively;

[0395] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences set forth in SEQ ID NO: 82 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NO: 68 and 69 or 78 or 79, respectively;

[0396] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences set forth in SEQ ID NO: 84 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NO: 66 and 67 or 76 or 77, respectively;

[0397] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences set forth in SEQ ID NO: 84 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NO: 68 and 69 or 78 or 79, respectively;

[0398] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences set forth in SEQ ID NO: 82 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NO: 66 and 67 or 76 or 77, respectively;

[0399] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences set forth in SEQ ID NO: 85 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NO: 70 and 71 or 80 or 81, respectively;

[0400] - A first antigen-binding domain, which comprises: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:85 and 83, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77;

[0401] - A first antigen-binding domain, which comprises: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:85 and 83, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79;

[0402] - A first antigen-binding domain, which comprises: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:82 and 101, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81;

[0403] - A first antigen-binding domain, which comprises: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:82 and 101, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77;

[0404] - A first antigen-binding domain, which comprises: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:82 and 101, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79;

[0405] - A first antigen-binding domain, which comprises: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:82 and 104, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81;

[0406] - A first antigen-binding domain, which comprises: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:82 and 104, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77;

[0407] - A first antigen-binding domain, which comprises: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:82 and 104, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79;

[0408] - The first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:84 and 101, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81;

[0409] - The first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:84 and 101, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77;

[0410] - The first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:84 and 101, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79;

[0411] - The first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:84 and 104, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81;

[0412] - The first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:84 and 104, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77;

[0413] - The first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:84 and 104, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79;

[0414] - The first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:85 and 101, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81;

[0415] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:85 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0416] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:85 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0417] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:85 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0418] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:85 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively; or

[0419] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:85 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively.

[0420] In certain embodiments, the HC sequence is modified to reduce or eliminate effector function. For example, the IgG1 portion of the HC sequence may contain L235E, "LALA" (L234A / L235A), or "LALAGA" (L234A / L235A / G237A) mutations (where all residues are numbered according to the EU numbering scheme).

[0421] In some embodiments, the bispecific binding molecules of the present disclosure comprise:

[0422] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:14 and 83, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81, respectively;

[0423] - A first antigen-binding domain, comprising: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:7 and 83, and a second antigen-binding domain, comprising: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81;

[0424] - A first antigen-binding domain, comprising: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:7 and 83, and a second antigen-binding domain, comprising: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79;

[0425] - A first antigen-binding domain, comprising: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:14 and 83, and a second antigen-binding domain, comprising: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77;

[0426] - A first antigen-binding domain, comprising: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:14 and 83, and a second antigen-binding domain, comprising: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79;

[0427] - A first antigen-binding domain, comprising: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:7 and 83, and a second antigen-binding domain, comprising: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77;

[0428] - A first antigen-binding domain, comprising: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:105 and 83, and a second antigen-binding domain, comprising: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81;

[0429] - A first antigen-binding domain, comprising: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:105 and 83, and a second antigen-binding domain, comprising: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77;

[0430] - A first antigen-binding domain, comprising: an HC and an LC respectively comprising the amino acid sequences of SEQ ID NO:105 and 83, and a second antigen-binding domain, comprising: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79;

[0431] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:7 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81, respectively;

[0432] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:7 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77, respectively;

[0433] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:7 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79, respectively;

[0434] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:7 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81, respectively;

[0435] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:7 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 and 77, respectively;

[0436] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:7 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 and 79, respectively;

[0437] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO:14 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 and 81, respectively;

[0438] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 14 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 66 and 67 or 76 and 77, respectively;

[0439] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 14 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 68 and 69 or 78 and 79, respectively;

[0440] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 14 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 70 and 71 or 80 and 81, respectively;

[0441] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 14 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 66 and 67 or 76 and 77, respectively;

[0442] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 14 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 68 and 69 or 78 and 79, respectively;

[0443] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 15 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 70 and 71 or 80 and 81, respectively;

[0444] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 15 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 66 and 67 or 76 and 77, respectively;

[0445] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 15 and 101, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 68 and 69 or 78 and 79, respectively;

[0446] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 105 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 70 and 71 or 80 and 81, respectively;

[0447] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 105 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 66 and 67 or 76 and 77, respectively; or

[0448] - A first antigen-binding domain comprising an HC and an LC comprising the amino acid sequences of SEQ ID NO: 105 and 104, respectively, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 68 and 69 or 78 and 79, respectively.

[0449] In some embodiments, the bispecific binding molecule of the present disclosure comprises:

[0450] - A first antigen-binding domain comprising an HC comprising the amino acid sequence of SEQ ID NO: 87, an HC comprising the amino acid sequence of SEQ ID NO: 107, and an LC comprising the amino acid sequence of SEQ ID NO: 83, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 70 and 71 or 80 or 81, respectively;

[0451] - A first antigen-binding domain comprising an HC comprising the amino acid sequence of SEQ ID NO: 86, an HC comprising the amino acid sequence of SEQ ID NO: 106, and an LC comprising the amino acid sequence of SEQ ID NO: 83, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 70 and 71 or 80 or 81, respectively;

[0452] - A first antigen-binding domain comprising an HC comprising the amino acid sequence of SEQ ID NO: 86, an HC comprising the amino acid sequence of SEQ ID NO: 106, and an LC comprising the amino acid sequence of SEQ ID NO: 83, and a second antigen-binding domain comprising a VH and a VL comprising the amino acid sequences of SEQ ID NO: 68 and 69 or 78 or 79, respectively;

[0453] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0454] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0455] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0456] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0457] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0458] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0459] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0460] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0461] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0462] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0463] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0464] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:106, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0465] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain, which comprises: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0466] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain, which comprises: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0467] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain, which comprises: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0468] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain, which comprises: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0469] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain, which comprises: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0470] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:107, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0471] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0472] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0473] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0474] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0475] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively; or

[0476] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:108, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively.

[0477] In some embodiments, the bispecific binding molecule of the present disclosure comprises:

[0478] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0479] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0480] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0481] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0482] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79;

[0483] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77;

[0484] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79;

[0485] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81;

[0486] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77;

[0487] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:83, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79;

[0488] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0489] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0490] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0491] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0492] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0493] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:86, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0494] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81;

[0495] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77;

[0496] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79;

[0497] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81;

[0498] - The first antigen-binding domain, which comprises: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain, which comprises: a VH and a VL respectively comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77;

[0499] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:87, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0500] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0501] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively;

[0502] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:101, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively;

[0503] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:70 and 71 or 80 or 81, respectively;

[0504] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:66 and 67 or 76 or 77, respectively; or

[0505] - A first antigen-binding domain comprising: an HC comprising the amino acid sequence of SEQ ID NO:88, an HC comprising the amino acid sequence of SEQ ID NO:109, and an LC comprising the amino acid sequence of SEQ ID NO:104, and a second antigen-binding domain comprising: a VH and a VL comprising the amino acid sequences of SEQ ID NO:68 and 69 or 78 or 79, respectively.

[0506] In some embodiments, the bispecific binding molecule of the present disclosure comprises:

[0507] - The amino acid sequences of SEQ ID NO:1 and 16;

[0508] - The amino acid sequences of SEQ ID NO:2 and 16;

[0509] - The amino acid sequences of SEQ ID NO:3 and 16;

[0510] - The amino acid sequences of SEQ ID NO:4 and 16;

[0511] - The amino acid sequences of SEQ ID NO:5 and 16;

[0512] - The amino acid sequences of SEQ ID NO:6 and 16;

[0513] - The amino acid sequences of SEQ ID NO:7 and 17;

[0514] - The amino acid sequences of SEQ ID NO:7 and 18;

[0515] - The amino acid sequences of SEQ ID NO:7 and 19;

[0516] - The amino acid sequences of SEQ ID NO:7 and 20;

[0517] - The amino acid sequences of SEQ ID NO:7 and 21;

[0518] - The amino acid sequences of SEQ ID NO:7 and 22;

[0519] - The amino acid sequences of SEQ ID NO:8, 9 and 16;

[0520] - The amino acid sequences of SEQ ID NO:8, 10 and 16;

[0521] - The amino acid sequences of SEQ ID NO:8, 11 and 16;

[0522] - The amino acid sequences of SEQ ID NO:12, 11 and 16;

[0523] -The amino acid sequences of SEQ ID NO: 12, 13, and 16;

[0524] -The amino acid sequences of SEQ ID NO: 14 and 19;

[0525] -The amino acid sequences of SEQ ID NO: 8, 15, and 16; or

[0526] -The amino acid sequences of SEQ ID NO: 7 and 23.

[0527] In some embodiments, the bispecific binding molecule of the present disclosure comprises one or two of the first antigen-binding domains as described above. In some embodiments, the bispecific binding molecule of the present disclosure comprises one or two of the second antigen-binding domains as described above.

[0528] In some embodiments, the valency of the first antigen-binding domain of the bispecific binding molecule of the present disclosure is 1 or 2. In some embodiments, the valency of the second antigen-binding domain of the bispecific binding molecule of the present disclosure is 1 or 2. Any combination of the valencies of the first and second antigen-binding domains is contemplated; for example, the first and second antigen-binding domains have valencies of 2 and 2, respectively; 1 and 1, respectively; 2 and 1, respectively; or 1 and 2, respectively.

[0529] In some embodiments, the bispecific binding molecule of the present disclosure may comprise a human IgG, IgM, IgE, IgA, or IgD constant region. In certain embodiments, the human constant region is an IgG isotype, such as IgG subclasses IgG1, IgG2a, or IgG2b, IgG3, or IgG4. In some embodiments, the bispecific binding molecule of the present disclosure may comprise a human δ constant region. The class of the bispecific binding molecule described herein may be altered or switched to another class or subclass. For example, a constant region that was initially IgM may be class-switched to IgG. Additionally, class-switching can be used to convert one IgG subclass to another, such as from IgG1 to IgG2. The κ light chain constant region can be altered to, for example, a λ light chain constant region.

[0530] In certain embodiments, the bispecific binding molecule of the present disclosure may comprise a human IgG constant region. In specific embodiments, the IgG constant region may include mutations that reduce or eliminate effector functions (see, e.g., Wang et al., Protein Cell 9(1):63 - 73 (2018)). For example, the bispecific binding molecule of the present disclosure may comprise a human IgG1 constant region having the mutations L235E, the "LALA" mutation (L234A / L235A), or the "LALAGA" mutation (L234A / L235A / G237A), all numbered according to the EU numbering scheme).

[0531] The class (isotype) and subclass of the bispecific binding molecules of the present disclosure can be determined by any method known in the art. Generally, the class and subclass of an antibody can be determined using antibodies specific for a particular antibody class and subclass. Such antibodies are commercially available. The class and subclass can be determined by ELISA, Western blotting, and other techniques. Alternatively, the class and subclass of an antibody can be determined by sequencing all or part of the constant region of the heavy and / or light chains of the antibody, comparing its amino acid sequence with the known amino acid sequences of the various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibody.

[0532] In some embodiments, the bispecific binding molecules of the present disclosure are homodimers. In some embodiments, the bispecific binding molecules of the present disclosure are heterotrimers (light chain and heavy chain heterodimer combinations), wherein the heavy chain heterodimer is, for example, in the form described in Brinkmann and Kontermann, MABS 9:182-212 (2017). For example, "knobs-into-holes", HA-TF, ZW1, CH3 charge pair, EW-RVT, LUZ-Y, chain exchange domain (SEEDbody), Biclonic, DuoBody, BEAT, 7.8.60, 20.8.34, Triomab / Quadroma, or CrossMAb strategies can be used to promote the heterodimerization (e.g., over-homodimerization) of polypeptides containing the Fc region of immunoglobulins in the structure of the bispecific binding molecules of the present disclosure. In certain embodiments, the "knobs-into-holes" method can be used, wherein the "knob" variant of a domain is obtained by replacing an amino acid with a small side chain (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine) with another amino acid with a larger side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan). The "hole" variant of a domain is obtained by replacing an amino acid with a larger side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan) with another amino acid with a smaller side chain (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine). In certain embodiments, the knob and / or hole mutations are in the CH3 domain.

[0533] In some embodiments, antigen-binding fragments of anti-ROR1 antibodies and / or antigen-binding fragments of anti-CD3 antibodies can be used to prepare the bispecific binding molecules of the present disclosure. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab’-SH, F(ab')2; recombinant IgG (rIgG) fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); and single-domain antibodies (e.g., sdAb, sdFv, nanobody). In certain embodiments, the fragment is a single-chain antibody fragment that includes a variable heavy chain region and / or a variable light chain region, such as scFv.

[0534] In a particular embodiment, the first antigen-binding domain, the second antigen-binding domain, or both are single-chain antibodies (scFv) that include a variable heavy chain domain of an antibody (VH) linked to a variable light chain domain (VL), wherein the fusion protein retains the same antigen specificity as the antibody. VH and VL can be linked by a peptide linker. In some embodiments, the second antigen-binding domain is an scFv that specifically binds to CD3.

[0535] To generate a single-chain antibody (scFv), DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, such as encoding the amino acid sequence (Gly4Ser)4 (SEQ ID NO:96), such that the VH and VL sequences can be expressed as a continuous single-chain protein, and the VL and VL domains are linked by a flexible linker. If only VH and VL are used, the single-chain antibody can be monovalent; if two VH and VL are used, it is bivalent; or if more than two VH and VL are used, it is multivalent.

[0536] In some embodiments, the amino acid sequence of the first antigen-binding domain is fused to the amino acid sequence of the second antigen-binding domain, such as by a peptide linker, to form a fusion polypeptide. In certain embodiments, the second antigen-binding domain is an scFv and is fused to the heavy chain or light chain amino acid sequence of the first antigen-binding domain. For example, the second antigen-binding domain can be fused to:

[0537] - the amino terminus of the heavy chain of the first antigen-binding domain;

[0538] - the amino terminus of the light chain of the first antigen-binding domain;

[0539] - the carboxyl terminus of the heavy chain of the first antigen-binding domain; and / or

[0540] - the carboxyl terminus of the light chain of the first antigen-binding domain.

[0541] In some embodiments, the bispecific binding molecules of the present disclosure are configured as shown in any of Schematics A-E in Figure 1 as follows.

[0542] In certain embodiments, the length of the peptide linker conjugating the first and second antigen-binding domains is between 5-30, 5-25, 5-15, 10-30, 10-20, or 10-15 amino acids. In certain embodiments, the peptide linker comprises amino acids that allow the peptide linker to dissolve, such as serine and threonine. In certain embodiments, the linker can be charged (see, e.g., U.S. Patent 9,856,327). In certain embodiments, the peptide linker comprises amino acids that allow the peptide linker to be flexible, such as glycine, or amino acids that allow the peptide linker to be rigid. In certain embodiments, the peptide linker conjugating the first and second antigen-binding domains is (Gly4-Ser)X, where X can be 1, 2, 3, or 4 (SEQ ID NO:114). In a particular embodiment, the sequence of the peptide linker is GGGGSGGGGS (SEQ ID NO:93).

[0543] In some embodiments, the second antigen-binding domain as described herein comprises one or more mutations (e.g., replacing an amino acid residue with cysteine) to stabilize disulfide bond binding, such as to prevent or reduce aggregation of the bispecific binding molecule.

[0544] In some embodiments, the bispecific molecule of the present disclosure has a K D (as determined by ELISA) for immobilized ROR1 of 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 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, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, 0.005 nM, or 0.001 nM or lower (e.g., 0.5 nM or lower).

[0545] In some embodiments, the bispecific molecule of the present disclosure has a K D (as determined by ELISA) for soluble b-ROR1 of 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 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, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, 0.005 nM, or 0.001 nM or lower (e.g., 0.4 nM or lower).

[0546] In some embodiments, the K of the bispecific molecule of the present disclosure for CD3 D (as measured by ELISA) is 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 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, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, 0.005 nM, or 0.001 nM or lower (e.g., 5 nM or lower) of the K for CD3 D .

[0547] In some embodiments, the bispecific binding molecule of the present disclosure induces LDH release in ROR1-transfected MEC cells exposed to PMBC at 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL, or 0.01 ng / mL or lower.

[0548] In some embodiments, the bispecific binding molecule of the present disclosure induces LDH release in JeKo-1 cells exposed to PMBC at 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL, or 0.01 ng / mL or lower.

[0549] In some embodiments, the bispecific binding molecule of the present disclosure induces LDH release in Mino cells exposed to PMBC at 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL, or 0.01 ng / mL or lower.

[0550] In some embodiments, the bispecific binding molecule of the present disclosure induces LDH release in MDA-MB-468 cells exposed to PMBC at 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL, or 0.01 ng / mL or lower.

[0551] In some embodiments, the bispecific binding molecule of the present disclosure induces ROR1-dependent killing of ROR1-transfected MEC cells, JeKo-1 cells, Mino cells, MDA-MB-468 cells, or any combination thereof by PBMCs in vitro.

[0552] In some embodiments, the bispecific binding molecule of the present disclosure upregulates CD69 on the surface of T cells co-incubated with ROR1-transfected MEC cells at 50 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL, or 0.01 ng / mL, or lower, as measured by flow cytometry.

[0553] In some embodiments, the bispecific binding molecule of the present disclosure upregulates CD69 on the surface of T cells co-incubated with JeKo-1 cells at 50 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL or 0.01 ng / mL, or lower, as determined by flow cytometry.

[0554] In some embodiments, the bispecific binding molecule of the present disclosure upregulates CD69 on the surface of T cells co-incubated with Mino cells at 50 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL or 0.01 ng / mL, or lower, as determined by flow cytometry.

[0555] In some embodiments, the bispecific binding molecule of the present disclosure upregulates CD69 on the surface of T cells co-incubated with MDA-MB-468 cells at 50 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL or 0.01 ng / mL, or lower, as determined by flow cytometry.

[0556] In some embodiments, the bispecific binding molecule of the present disclosure induces the release of IFN-γ, TNF-α, IL-10, IL-6, IL-4, and IL-2 from T cells co-cultured with JeKo-1 or ROR1-transfected MEC cells at 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2 ng / mL, 1 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL, or 0.01 ng / mL or lower.

[0557] In some embodiments, the bispecific binding molecule of the present disclosure has at least one of the following characteristics:

[0558] a) The K of immobilized ROR1 measured by ELISA D is 0.5 mM or lower;

[0559] b) The K of soluble b-ROR1 measured by ELISA D is 0.4 nM or lower;

[0560] c) It is confirmed that there is a reduction in internalization in ROR1-transfected MEC cells and / or Jurkat cells compared to an antibody comprising the heavy and light chain amino acid sequences of SEQ ID NOs: 82 and 83, respectively;

[0561] d) Induces LDH release in ROR1-transfected MEC cells exposed to PBMC at 1 μg / mL or lower;

[0562] e) Induces LDH release in JeKo-1 cells exposed to PBMC at 1 μg / mL or lower;

[0563] f) Induces LDH release in Mino cells exposed to PBMC at 1 μg / mL or lower;

[0564] g) Induces LDH release in MDA-MB-468 cells exposed to PBMC at 1 μg / mL or lower;

[0565] h) Upregulates CD69 on the surface of T cells co-cultured with ROR1-transfected MEC cells at 1 μg / mL or lower as measured by flow cytometry;

[0566] i) Upregulating CD69 on the surface of T cells co-cultured with ROR1-transfected JeKo-1 cells at 1 μg / mL or lower, as determined by flow cytometry;

[0567] j) Upregulating CD69 on the surface of T cells co-cultured with ROR1-transfected Mino cells at 1 μg / mL or lower, as determined by flow cytometry;

[0568] k) Upregulating CD69 on the surface of T cells co-cultured with ROR1-transfected MDA-MB-468 cells at 1 μg / mL or lower, as determined by flow cytometry; and

[0569] l) Inducing the release of IFN-γ, IFN-α, IL-10, IL-6, IL-4, and IL-2 from T cells co-cultured with JeKo-1 or ROR1-transfected MEC cells at 1 μg / mL or lower.

[0570] In certain embodiments, the bispecific binding molecules of the present disclosure have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the described properties. In specific embodiments, the bispecific binding molecules of the present disclosure have all of the described properties.

[0571] In some embodiments, the bispecific binding molecules of the present disclosure induce T cell cytotoxicity against ROR-1 positive cells.

[0572] Nucleic acid molecules and vectors

[0573] The present disclosure also provides nucleic acid molecules and sequences encoding the bispecific binding molecules described herein. In some embodiments, different nucleic acid molecules encode different polypeptides that form the bispecific binding molecule. In other embodiments, the same nucleic acid molecule encodes more than one or all of the polypeptides that form the bispecific binding molecule.

[0574] Unless otherwise indicated, references to nucleotide sequences include their complements. Thus, reference to a nucleic acid having a particular sequence is to be understood as referring to its complementary strand as well as its complementary sequence. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of at least 10 bases in length, ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. The term includes single- and double-stranded forms.

[0575] In any of the above embodiments, the nucleic acid molecule can be isolated. A nucleic acid molecule referred to herein as "isolated" or "purified" is one that is (1) separated from the nucleic acids of the genomic DNA or cellular RNA from which it originated; and / or (2) not present in nature.

[0576] In some embodiments, the nucleic acid molecules of the present disclosure comprise nucleotide sequences encoding:

[0577] of the bispecific binding molecule of the present disclosure

[0578] - the H-CDR1-3 and / or L-CDR1-3 of the first antigen-binding domain;

[0579] - the H-CDR1-3 and / or L-CDR1-3 of the second antigen-binding domain;

[0580] - the VH and / or VL of the first antigen-binding domain;

[0581] - the VH and / or VL of the second antigen-binding domain; and / or

[0582] - the HC and / or LC of the first antigen-binding domain.

[0583] In some embodiments, the nucleic acid molecule of the present disclosure comprises a nucleotide sequence encoding the H-CDR1-3 and L-CDR1-3 of the first antigen-binding domain of the bispecific binding molecule of the present disclosure, and further comprises a nucleotide sequence encoding the H-CDR1-3 and L-CDR1-3 of the second antigen-binding domain. In some embodiments, the nucleic acid molecule of the present disclosure comprises a nucleotide sequence encoding the VH and VL of the first antigen-binding domain of the bispecific binding molecule of the present disclosure, and further comprises a nucleotide sequence encoding the VH and VL of the second antigen-binding domain. In some embodiments, the nucleic acid molecule of the present disclosure comprises a nucleotide sequence encoding the HC and LC of the first antigen-binding domain of the bispecific binding molecule of the present disclosure, and further comprises an amino acid sequence encoding the VH and VL of the second antigen-binding domain.

[0584] In some embodiments, the nucleic acid molecule of the present disclosure comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 24-46.

[0585] In certain embodiments, the nucleic acid molecule of the present disclosure comprises:

[0586] - the nucleotide sequences of SEQ ID NO: 24 and 39;

[0587] - the nucleotide sequences of SEQ ID NO: 25 and 39;

[0588] - the nucleotide sequences of SEQ ID NO: 26 and 39;

[0589] - the nucleotide sequences of SEQ ID NO: 27 and 39;

[0590] - the nucleotide sequences of SEQ ID NO: 28 and 39;

[0591] -The nucleotide sequences of SEQ ID NO:29 and 39;

[0592] -The nucleotide sequences of SEQ ID NO:30 and 40;

[0593] -The nucleotide sequences of SEQ ID NO:30 and 41;

[0594] -The nucleotide sequences of SEQ ID NO:30 and 42;

[0595] -The nucleotide sequences of SEQ ID NO:30 and 43;

[0596] -The nucleotide sequences of SEQ ID NO:30 and 44;

[0597] -The nucleotide sequences of SEQ ID NO:30 and 45;

[0598] -The nucleotide sequences of SEQ ID NO:31, 32 and 39;

[0599] -The nucleotide sequences of SEQ ID NO:31, 33 and 39;

[0600] -The nucleotide sequences of SEQ ID NO:31, 34 and 39;

[0601] -The nucleotide sequences of SEQ ID NO:35, 34 and 39;

[0602] -The nucleotide sequences of SEQ ID NO:35, 36 and 39;

[0603] -The nucleotide sequences of SEQ ID NO:37 and 42;

[0604] -The nucleotide sequences of SEQ ID NO:31, 38 and 39; or

[0605] -The nucleotide sequences of SEQ ID NO:30 and 46.

[0606] In another aspect, the present disclosure provides vectors suitable for expressing one or more polypeptides that form a bispecific binding molecule as described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is intended to be ligated. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded DNA fragment to which additional DNA fragments can be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA fragments can be ligated into the viral genome. In some embodiments, the vector is capable of autonomous replication in the host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vector (e.g., a non-episomal mammalian vector) can integrate into the genome of the host cell after introduction into the host cell and thereby replicate with the host genome. In addition, certain vectors are capable of directing the expression of a gene to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0607] In some embodiments, the nucleic acid molecule of the present disclosure may comprise a nucleotide sequence encoding an amino acid sequence from a first antigen-binding domain, the nucleotide sequence being ligated in-frame to a nucleotide sequence encoding an amino acid sequence from a second antigen-binding domain.

[0608] In some embodiments, the bispecific binding molecule of the present disclosure is expressed by inserting DNA encoding the polypeptide components of the bispecific binding molecule into an expression vector such that the DNA is operably linked to the necessary expression control sequences such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The coding sequence can be ligated into the vector such that the transcriptional and translational control sequences in vivo perform their intended function of regulating the transcription and translation of the coding sequence. Expression vectors and expression control sequences compatible with the expression host cell used can be selected. Nucleotide sequences encoding different polypeptide components of the bispecific binding molecule of the present disclosure can be inserted into the same or different vectors and can be operably linked to the same or different expression control sequences (e.g., promoters). In one embodiment, two or more coding sequences are inserted into the same expression vector and can be operably linked to the same expression control sequence (e.g., a common promoter) to split the same expression control sequence (e.g., a promoter), or different expression control sequences (e.g., promoters). The coding sequence can be inserted into the expression vector by standard methods (e.g., ligating antibody gene fragments and complementary restriction sites on the vector, or ligating blunt ends if no restriction sites are present).

[0609] Host cells and methods for producing bispecific binding molecules

[0610] Another aspect of the present disclosure relates to methods of generating the bispecific binding molecules of the present disclosure. In some embodiments, methods for generating bispecific binding molecules as defined herein include providing a recombinant host cell capable of expressing the bispecific binding molecule (e.g., a host cell as described herein), culturing the host cell under conditions suitable for expression of the bispecific binding molecule, and isolating the resulting bispecific binding molecule.

[0611] Mammalian cell lines that can be used as hosts are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-203T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatoma cells (e.g., Hep G2), A549 cells, and many other cell lines. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells, and yeast cell lines. The cell line can be selected based on its expression level.

[0612] In some embodiments, the host cells of the present disclosure comprise nucleotide sequences encoding:

[0613] - H-CDR1-3 and / or L-CDR1-3 of the first antigen-binding domain of the bispecific molecule of the present disclosure;

[0614] - H-CDR1-3 and / or L-CDR1-3 of the second antigen-binding domain;

[0615] - VH and / or VL of the first antigen-binding domain;

[0616] - VH and / or VL of the second antigen-binding domain; and / or

[0617] - HC and / or LC of the first antigen-binding domain.

[0618] In some embodiments, the host cells of the present disclosure comprise nucleotide sequences encoding the H-CDR1-3 and L-CDR1-3 of the first antigen-binding domain of the bispecific binding molecules of the present disclosure, and further comprise nucleotide sequences encoding the H-CDR1-3 and L-CDR1-3 of the second antigen-binding domain. In some embodiments, the host cells of the present disclosure comprise nucleotide sequences encoding the VH and VL of the first antigen-binding domain of the bispecific binding molecules of the present disclosure, and further comprise nucleotide sequences encoding the VH and VL of the second antigen-binding domain. In some embodiments, the host cells of the present disclosure comprise nucleotide sequences encoding the HC and LC of the first antigen-binding domain of the bispecific binding molecules of the present disclosure, and further comprise nucleotide sequences encoding the VH and VL of the second antigen-binding domain.

[0619] In some embodiments, the host cells of the present disclosure comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 24-46.

[0620] In certain embodiments, the host cells of the present disclosure comprise:

[0621] - the nucleotide sequences of SEQ ID NO: 24 and 39;

[0622] - the nucleotide sequences of SEQ ID NO: 25 and 39;

[0623] - the nucleotide sequences of SEQ ID NO: 26 and 39;

[0624] - the nucleotide sequences of SEQ ID NO: 27 and 39;

[0625] - the nucleotide sequences of SEQ ID NO: 28 and 39;

[0626] - the nucleotide sequences of SEQ ID NO: 29 and 39;

[0627] - the nucleotide sequences of SEQ ID NO: 30 and 40;

[0628] - the nucleotide sequences of SEQ ID NO: 30 and 41;

[0629] - the nucleotide sequences of SEQ ID NO: 30 and 42;

[0630] - the nucleotide sequences of SEQ ID NO: 30 and 43;

[0631] - the nucleotide sequences of SEQ ID NO: 30 and 44;

[0632] - the nucleotide sequences of SEQ ID NO: 30 and 45;

[0633] -The nucleotide sequences of SEQ ID NO: 31, 32, and 39;

[0634] -The nucleotide sequences of SEQ ID NO: 31, 33, and 39;

[0635] -The nucleotide sequences of SEQ ID NO: 31, 34, and 39;

[0636] -The nucleotide sequences of SEQ ID NO: 35, 34, and 39;

[0637] -The nucleotide sequences of SEQ ID NO: 35, 36, and 39;

[0638] -The nucleotide sequences of SEQ ID NO: 37 and 42;

[0639] -The nucleotide sequences of SEQ ID NO: 31, 38, and 39; or

[0640] -The nucleotide sequences of SEQ ID NO: 30 and 46.

[0641] Pharmaceutical composition

[0642] Another aspect of the present disclosure is a pharmaceutical composition comprising the bispecific binding molecule of the present disclosure as an active ingredient (or as the sole active ingredient). The pharmaceutical composition may comprise any bispecific binding molecule as described herein. In some embodiments, the composition is intended to ameliorate, prevent, and / or treat the cancers described herein (e.g., ROR1-positive cancers).

[0643] Generally, the bispecific binding molecules of the present disclosure are suitable for administration as a formulation in combination with one or more pharmaceutically acceptable excipients, as described below, for example.

[0644] As used herein, the term "excipient" is used to describe any ingredient other than the compounds of the present disclosure. The choice of excipient depends to a large extent on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include an isotonic agent such as sugar, a polyol such as mannitol, sorbitol, or sodium chloride in the composition. Other examples of pharmaceutically acceptable substances are wetting agents or minor auxiliary substances, such as wetting agents or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the bispecific binding molecule.

[0645] The pharmaceutical compositions of the present disclosure and methods for their preparation will be apparent to those skilled in the art. Such compositions and methods of preparation can be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical compositions are preferably prepared under GMP (Good Manufacturing Practice) conditions.

[0646] The pharmaceutical compositions of the present disclosure can be prepared, packaged, or sold in bulk, as a single unit dose, or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of the active ingredient. The amount of the active ingredient is usually equal to the dose of the active ingredient to be administered to a subject or a convenient fraction of such dose, for example, one-half or one-third of such dose.

[0647] Any method accepted in the art for administering peptides, proteins, or antibodies can be suitably used for the bispecific binding molecules of the present disclosure.

[0648] The pharmaceutical compositions of the present disclosure are generally suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical disruption of the tissue of a subject and administration of the pharmaceutical composition through the disruption in the tissue, and thus generally results in administration into the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, using the pharmaceutical composition by injection of the composition, by surgical incision to administer the composition, by percutaneous non-surgical wound to administer the composition, etc. In particular, parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, and intrasynovial injection or infusion; and renal dialysis infusion techniques. Regional perfusion is also contemplated. Specific embodiments include intravenous and subcutaneous routes.

[0649] Formulations of pharmaceutical compositions suitable for parenteral administration (e.g., intravenous administration) typically comprise an active ingredient in combination with a pharmaceutically acceptable carrier (e.g., sterile water or sterile isotonic saline). Such formulations can be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage form, e.g., in an ampoule or a multi-dose container containing a preservative. Parenteral formulations include, but are not limited to, suspensions, solutions, emulsions of oily or aqueous carriers, pastes, etc. Such formulations can also contain one or more additional ingredients, including but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a parenteral formulation, the active ingredient is provided in a dry (e.g., powder or granule) form, which is used to reconstitute the composition with a suitable carrier (e.g., sterile pyrogen-free water) prior to parenteral administration. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably with a pH ranging from 3 to 9), but for certain applications, they may be more suitably formulated as sterile non-aqueous solutions or in a dry form for use in combination with a suitable carrier such as sterile, pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, such as aqueous solutions of propylene glycol or glucose. Such dosage forms can be suitably buffered if desired. Other useful parenteral formulations include those containing the active ingredient in microcrystalline form or in liposomal formulations. Parenteral formulations can be formulated for immediate release and / or sustained release. Sustained release formulations include delayed release, extended release, pulsatile release, controlled release, targeted release, and programmed release.

[0650] Therapeutic use of bispecific binding molecules

[0651] In some embodiments, the bispecific binding molecules of the present disclosure are used to treat cancer, such as ROR1-positive cancer, in a patient. The patient can be a mammal, such as a human. ROR1 has been shown to be expressed in multiple types of tumors, including lymphomas and solid tumors. A high proportion of human cancers express ROR1. For example, Zhang et al. showed that 54% of ovarian cancers, 57% of colon cancers, 77% of lung cancers, 90% of lymphomas, 89% of skin cancers, 83% of pancreatic cancers, 73% of testicular cancers, 43% of bladder cancers, 96% of uterine cancers, 90% of prostate cancers, and 83% of adrenal cancers they examined had moderate to strong staining with the anti-ROR1 antibody 4A5 (Zhang et al., Am J Pathol. 181(6):1903-10(2012)). Daneshmanesh et al. similarly found that ROR1 was almost ubiquitously expressed in chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL), and was expressed to varying degrees in other lymphomas, such as mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL) / marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and myeloma (Daneshmanesh et al., Leuk Lymphoma 54(4):843-50(2013)). Multiple groups have demonstrated that ROR1 is expressed in a subset of B-cell acute lymphoblastic leukemia (ALL) (see Dave et al., PLoS One 7:e52655(2012)). ROR1 is also expressed in a significant proportion of hepatocellular carcinoma (HCC) or non-small cell lung cancer (NSCLC) pathologies (U.S. Patent Publication 2018 / 0369406). In addition, it has been shown that ROR1 expression is increased in aggressive cancers and is associated with poor prognosis; thus, the bispecific binding molecules of the present disclosure are particularly suitable for treating aggressive or advanced cancers.

[0652] "Treat / treating / treatment" refers to a method of alleviating or eliminating a biological disorder and / or at least one accompanying symptom. As used herein, "alleviating" a disease, disorder, or condition refers to reducing the severity and / or frequency of occurrence of the disease, disorder, or condition. In addition, the reference to "treatment" herein includes references to curative, palliative, and prophylactic treatment. Treatment of cancer includes inhibiting cancer growth (including causing partial or complete cancer regression), inhibiting cancer progression or metastasis, preventing cancer recurrence or residual disease, and / or prolonging the survival of a patient.

[0653] A bispecific binding molecule can be administered to a patient suffering from cancer as described herein in a therapeutically effective amount. A "therapeutically effective amount" refers to the amount of a therapeutic agent administered that will, to some extent, alleviate one or more symptoms of the disorder being treated. A therapeutically effective amount of an anti-cancer therapeutic agent can, for example, result in tumor shrinkage, increased survival, elimination of cancer cells, slowed disease progression, metastasis reversal, or other clinical endpoints desired by a healthcare professional.

[0654] In some embodiments, the cancers treatable by the bispecific binding molecules described herein are cancers that express ROR1 (i.e., ROR1-positive). Any suitable method for determining by gene or protein expression, such as by histology, flow cytometry, RT-PCR, or RNA-Seq, can identify a cancer as ROR1-expressing. The cancer cells used for the assay can be obtained by tumor biopsy or by collecting circulating tumor cells. In certain embodiments, if an antibody-based assay such as flow cytometry or immunohistochemistry is used, a cancer that expresses ROR1 is any cancer that has cells showing higher reactivity to an anti-ROR1 antibody than an isotype control antibody. In certain embodiments, if an RNA-based assay is used, a cancer that expresses ROR1 is one that exhibits elevated ROR1 RNA levels compared to negative control cells or cancers that do not express ROR1.

[0655] In certain embodiments, the bispecific binding molecules of the present disclosure are used to treat hematological malignancies (such as leukemia and / or lymphoma). In certain embodiments, the bispecific binding molecules of the present disclosure are used to treat solid tumors. The cancers to be treated can be selected from, for example, lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, marginal cell B-cell lymphoma, Burkitt lymphoma, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, non-Hodgkin lymphoma that has undergone Richter transformation, T-cell non-Hodgkin lymphoma, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, T-cell leukemia, sarcoma, osteosarcoma, Ewing sarcoma, renal cell carcinoma, hepatocellular carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell carcinoma, glioblastoma, melanoma, myeloma, multiple myeloma, gastric cancer, brain cancer, lung cancer, non-small cell lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer. In certain embodiments, the cancers are selected from mantle cell lymphoma, breast cancer, lung cancer, osteosarcoma, and Ewing sarcoma.

[0656] In some embodiments, the cancer to be treated can be a cancer refractory to other therapeutic agents (such as triple-negative breast cancer). The cancer can be, for example, in an early, intermediate, advanced, or metastatic stage.

[0657] In some embodiments, the patient to be treated with the bispecific binding molecule of the present disclosure has received prior cancer treatment. In other aspects, the patient has not received prior cancer treatment.

[0658] The bispecific binding molecule of the present disclosure can be administered without additional therapeutic treatment, i.e., as a monotherapy. Alternatively, treatment with the bispecific binding molecule of the present disclosure can include at least one additional therapeutic treatment (combination therapy). In some embodiments, the bispecific binding molecule can be co-administered or formulated with another drug / drug product for cancer treatment. The additional treatment can include, for example, chemotherapy, anti-tumor agents, or anti-angiogenic agents, and / or radiotherapy.

[0659] In certain embodiments, the bispecific binding molecules of the present disclosure are used in combination with additional therapeutic or bioactive molecules (e.g., for treating cancers described herein). Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, prodrugs, carbohydrates, imaging agents, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, antibiotics, antifungals, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroid agents, microbe-derived toxins, etc. In some embodiments, the additional therapeutic agent is a vascular endothelial growth factor (VEGF) inhibitor, Bruton's tyrosine kinase (BTK) inhibitor, mammalian target of rapamycin (mTOR) inhibitor, phosphoinositide 3-kinase (PI3K) inhibitor, Janus kinase / signal transducer and activator of transcription (Jak / STAT) signaling inhibitor, B-cell lymphoma 2 (Bcl-2) inhibitor, spleen tyrosine kinase (SYK) inhibitor, microtubule inhibitor, epidermal growth factor receptor (EGFR) inhibitor, poly ADP ribose polymerase (PARP) inhibitor, anaplastic lymphoma kinase (ALK) inhibitor, DNA repair inhibitor, DNA cross-linking agent, nucleoside analog, or immunomodulator. In some embodiments, the additional therapeutic agent is an antibody, such as rituximab (anti-CD20) or bevacizumab (anti-VEGF); a Bruton tyrosine kinase inhibitor such as acalabrutinib or ibrutinib; an mTOR inhibitor such as idelalisib or buparlisib; a Jak / STAT signaling inhibitor such as ruxolitinib; a Bcl-2 inhibitor such as ABT-199 / venetoclax, Bcl-2i-1 or Bcl-2i-2; a SYK inhibitor such as fostamatinib; a microtubule inhibitor such as paclitaxel or vincristine; an EGFR inhibitor such as erlotinib; a PARP inhibitor such as olaparib; an ALK inhibitor such as crizotinib; a DNA repair inhibitor such as carboplatin; a DNA cross-linking agent such as oxaliplatin / cisplatin; a nucleoside analog such as gemcitabine; or an immunomodulatory drug (IMiD) such as lenalidomide or pomalidomide. In certain embodiments, the additional therapeutic agent is ibrutinib, acalabrutinib, venetoclax, Bcl-2i-1, Bcl-2i-2, everolimus, sapanisertib, idelalisib, pacritinib, buparlisib, BEZ235, ruxolitinib, fostamatinib, rituximab, CHO-PIXALIDEN, pomalidomide, paclitaxel, vincristine, erlotinib, crizotinib, carboplatin, oxaliplatin / cisplatin, bevacizumab or gemcitabine.

[0660] In certain embodiments, the bispecific binding molecules of the invention are used in combination with immune checkpoint modulators that enhance the patient's immune system (e.g., for treating cancers described herein). For example, the conjugate is used in combination with an immune checkpoint inhibitor, such as an antibody or antibody derivative, antisense oligonucleotide, small interfering RNA, aptamer, or peptide, targeting programmed death ligand 1 (PD-L1, also often B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell co-stimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagenous structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof.

[0661] In certain embodiments, the bispecific binding molecules of the present disclosure are administered to a patient in combination with T cells (e.g., autologous or allogeneic T cells of the patient). In certain embodiments, where the patient is human, the T cells are also human. In some embodiments, the bispecific binding molecule binds to the T cells prior to administration to the patient.

[0662] It should be understood that the bispecific binding molecules of the present disclosure can be used in the treatment methods described herein, can be used for the treatments described herein, and / or can be used to prepare a medicament for the treatments as described herein. The present disclosure also provides kits and articles of manufacture comprising the bispecific binding molecules of the present disclosure as described herein.

[0663] Articles of manufacture and kits

[0664] The present disclosure also provides articles, such as kits, that include one or more containers (e.g., single-use or multi-use containers) that contain a pharmaceutical composition of the bispecific binding molecules of the present disclosure, optionally additional bioactive molecules (e.g., another therapeutic agent), and instructions for use. The bispecific binding molecules and the optional additional bioactive molecules can be separately packaged in suitable packages, such as vials or ampoules made of non-reactive glass or plastic. In certain embodiments, the vials or ampoules contain lyophilized powder containing the bispecific binding molecules and / or additional bioactive molecules. In certain embodiments, the vials or ampoules contain a concentrated solution (e.g., 2x, 5x, 10x, or more) of the bispecific binding molecules or bioactive molecules. In certain embodiments, the article, such as a kit, includes a medical device (e.g., syringe and needle) for administering the bispecific binding molecules and / or bioactive molecules; and / or a suitable diluent (e.g., sterile water and saline). The present disclosure also includes methods for treating with the articles.

[0665] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. In general, the terms and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceuticals and medicinal chemistry, and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly accomplished in the art or as described herein.

[0666] Furthermore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. In this specification and the examples, the words "having" and "comprising", "" or variants such as "has", "have", "having", "comprises", "comprise", "comprising", "comprises" shall be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0667] All publications and other references mentioned herein are incorporated by reference in their entirety. Although many documents are cited herein, such citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0668] To better understand the present invention, the following examples are presented. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0669] Examples

[0670] The following examples illustrate representative embodiments of the present invention and are not meant to be limiting in any way.

[0671] Example 1: Design and expression of anti-ROR1 / anti-CD3 bispecific binding molecules

[0672] Multiple variables affect the in vivo potency of bispecific binding molecules, including PK, epitopes, the relative affinities of the binding components, and the valence and spatial conformation of the paratopes. Currently, there is no reliable method to a priori design a single molecule that optimizes all of these parameters. Accordingly, multiple anti-ROR1 / anti-CD3 bispecific constructs were designed, fabricated, and tested to systematically evaluate the impact of many of these parameters.

[0673] To identify the structure that provides the optimal conformation, in particular the most active relative spatial positions of the ROR1 and CD3 binding components, multiple constructs were designed, as Figure 1 summarized. Although multiple bispecific formats have been described (see, e.g., Brinkmann and Kontermann, MABS 9:182-212 (2017)), current efforts focus on formats that include the antibody Fc component to achieve favorable PK and use well-established protein A-based antibody purification methods.

[0674] The ROR1 binding component used in the bispecific binding molecule has been demonstrated to be highly selective for human ROR1 and has been shown to be safe in human clinical trials (see, e.g., Choi et al., Cell Stem Cell 22:951-959 (2018)). In addition, using anti-CD3 scFv sequences based on the SP34 and OKT3 antibodies, bispecific binding molecules were tested with CD3 binding components specific for two different CD3 epitopes. CD3 antibodies have been classified according to the different characteristics of their epitopes (Tunnacliffe et al., International Immunology 1:546-550 (1989)). Using this system, SP34 was classified as group I, while OKT3 was classified as group II.

[0675] For some constructs, the use of the wild-type antibody Fc sequence enabled the evaluation of bispecific constructs that are bivalent for both ROR1 and CD3 ( Figure 1 , A, B, and E). For other constructs, the use of knobs-into-holes mutations in the Fc region enabled the expression of bispecific binding molecules with different valences for ROR1 and CD3. For example, one construct is bivalent for ROR1 and monovalent for CD3 ( Figure 1 , D), while another construct is monovalent for both ROR1 and CD3 ( Figure 1 , C).

[0676] Finally, ROR1 epitopes with altered affinities and valences were tested in combination with different CD3 epitopes to evaluate the effects of CD3 and ROR1 epitopes with different relative affinities / avidities.

[0677] Based on these design parameters, multiple bispecific constructs were expressed, purified, and characterized.

[0678] Materials and Methods

[0679] The codon-optimized DNA encoding the bispecific construct with the leader sequence of SEQ ID NO: 110 was cloned into the pcDNA3.4 vector and then introduced into Expi293 cells by transient transfection. The cells were grown in 40 mL of medium containing Expi293F expression medium (ThermoFisher cat.# A1435103). Then, the antibody was purified in one step from the culture supernatant using HiTrap MabSelect SuRe (GE Healthcare cat.# 11-0034-93). The purified antibody was stored in PBC, pH 7.2, and the purity was characterized by SDS-PAGE. The purity of the construct consisting of three different polypeptide chains was further characterized by SEC using a TSKgel G3000SWxl 7.8 mm x 380 mm column. The mobile phase was 0.1 M phosphate buffer, 0.1 M Na2SO4, pH 6.7, and the column was run at a flow rate of 0.7 mL / min.

[0680] Results

[0681] The sequences of the anti-ROR1 / anti-CD3 bispecific binding molecules are summarized in Table 1 (protein) and Table 2 (DNA).

[0682] Table 1. Summary of polypeptide sequences of bispecific constructs

[0683]

[0684]

[0685] Table 2. Summary of DNA sequences of bispecific constructs

[0686]

[0687]

[0688] Most constructs were well-expressed (17 / 20 expressed > 1 mg; 14 / 20 expressed > 2 mg) and were substantially purified in a single step in most cases using protein A chromatography (Table 3), as determined by SDS-PAGE.

[0689] Table 3. Summary of expression and purification of bispecific constructs

[0690] Construct Yield (mg) Concentration (mg / mL) Purity (%) 1 2.72 0.34 90 2 5.32 0.76 95 3 1.36 0.68 95 4 3.52 0.44 60 5 5.49 0.61 55 6 0.38 0.19 90 7 7.84 3.92 95 8 5.50 5.50 95 9 1.93 1.29 95 10 3.28 0.41 65 11 3.78 0.63 70 12 0.19 0.12 65 13 8.47 1.21 73 14 9.08 4.54 95 15 9.20 1.15 65 16 7.42 1.06 80 17 4.60 0.92 79 18 0.89 0.59 90 19 4.60 0.92 60 20 1.32 0.88 85

[0691] Constructs without a disulfide-stabilized anti-CD3 scFv generally had higher purity (> 90%) after protein A chromatography (Table 3, compare Constructs 2 and 5).

[0692] SEC analysis was performed on constructs composed of three different polypeptide chains. Generally, after a single protein A purification step, constructs composed of two different chains (wild-type Fc) had higher purity than constructs composed of three different chains (heteromeric Fc) (Table 3, compare the purity of Samples 1-3 and 7-9 with that of Samples 13-17).

[0693] Overall, these data indicate that most constructs were well-expressed and had a purity > 90% after a single protein A purification step.

[0694] Example 2: Binding of anti-ROR1 / anti-CD3 bispecific binding molecule to ROR1

[0695] The binding of bispecific binding molecules with different configurations, ROR1 affinities, and ROR1 potencies to human ROR1 was evaluated to determine the effect of these parameters on cytotoxicity. The binding of bispecific constructs 1-20 to human ROR1 was characterized using ROR1-transfected MEC cells, JeKo-1 cells, and recombinant ROR1 extracellular domain (ECD).

[0696] Materials and Methods

[0697] ROR1 binding assays were performed using cells and flow cytometry

[0698] ROR1 binding was quantified using ROR1-transfected MEC cells and JeKo-1 cells. For each condition, 2.5E5 cells were used. The cells were placed in 50 μL of PBS containing 2% FBS. Then, the cells were diluted with an equal volume of the stock solution of the 2X test bispecific binding molecule. The cells and the antibody were co-incubated on ice for 20 minutes, and then the cells were washed 3 times with 300 μL of FACS buffer and resuspended in 100 μL of anti-human Fc-PE conjugated antibody (Invitrogen cat.#12499882) and incubated on ice in the dark for 20 minutes. Subsequently, the cells were washed 3 times with 300 μL of FACS buffer and fixed with 2% paraformaldehyde at 25 °C for 10 minutes. Then the cells were washed 2 times with 300 μL of FACS buffer and analyzed on a Miltenyi MACSQuant Analyzer.

[0699] ROR1 binding assay was performed using recombinant ROR1-ECD and ELISA

[0700] Binding of the bispecific binding molecule to recombinant ROR1 was evaluated by ELISA using two different configurations. In the first configuration, the ROR1 protein was immobilized on a 96-well plate and the bispecific construct was titrated (details below). The ROR1 binding of the bispecific binding molecule was also characterized using a second configuration, in which the bispecific binding molecule was immobilized on a 96-well plate and soluble biotinylated ROR1 (b-ROR1) was titrated (details below).

[0701] Immobilized ROR1 ELISA

[0702] A 96-well Costar-3366 plate was coated overnight at 4 °C with 50 μL / well of a 2 μg / mL human TL1A) (Acro Biosystems cat.#R01-H522Y) PBS solution. The plate was rinsed once with PBS containing 0.05% Tween 20 (PBS-T), and then blocked for 1 hour at 25 °C with a 5% non-fat milk in PBS (5% M-P) solution (100 μL / well). Antibody samples were serially diluted 2-fold using 5% M-P and incubated for 1 hour at 25 °C (50 μL / well). The plate was washed 3 times with PBS-T, and 50 μL / well of an anti-human κ, HRP-conjugate (Southern Biotech cat.#2060-05) diluted 10,000-fold with 5% M-P was added and incubated at 25 °C for 1 hour. The plate was washed 3 times with PBS-T, developed with 50 μL / well of 1-Step Ultra TMB-ELISA (Thermo Scientific cat.#34028). The reaction was terminated by adding 2N H2SO4, and A450 was measured using a Spectramax plate reader.

[0703] Soluble ROR1 ELISA

[0704] For biotinylation of ROR1, 200 μg of human ROR1 (Acro Biosystems cat.#R01-H522Y) was resuspended in 2 mL of water to 100 μg / mL and then mixed with EZ-Link Sulfo-NHS-LC-biotin (Thermo Scientific cat.#21327) at a 1:5 molar ratio and incubated at 25 °C for 2 h. The reaction was terminated by adding 31 μL of 750 mM arginine hydrochloride and stored at 4 °C.

[0705] To measure the binding of the bispecific construct to soluble ROR1, 96-well Costar-3366 plates were coated overnight at 4 °C with 50 μL / well of a PBS solution of 2 μg / mL goat anti-human κ (Southern Biotech cat.#2060-01). The plates were washed once with PBS-T, blocked for 1 h at 25 °C with 100 μL / well of 5% M-P, washed once with PBS-T, and 2 μL / mL of the bispecific binding molecule was added and incubated at 25 °C for 1 h. The plates were washed 3 times with PBS-T. To detect the binding of b-ROR1, 50 μL / well of Neutravidin-HRP, diluted 1:5000 in 1% BSA-PBS, was added and incubated at 25 °C for 1 h. The plates were washed 3 times with PBS-T and then developed with 50 μL / well of 1-Step Ultra TMB-ELISA (Thermo Scientific cat.#34028). The reaction was terminated by adding 2N H2SO4 and A450 was measured using a Spectramax plate reader.

[0706] Results

[0707] Three anti-ROR1 Fab variants were characterized using ELISA-based screening and live cell binding, each variant differing from the parental antibody by a single amino acid in H-CDR2. Two variants, T32A and T32E, showed higher affinity than the parental Fab ( Figure 2 , Figure A, open circles and squares vs. solid circles), while another variant, W110Y, showed lower affinity ( Figure 2 , Figure A, triangles). The relative affinities of these variants were further characterized using live ROR1-transfected MEC cells. Similar to the ELISA-based screening, variants T32A and T32E showed higher affinity ( Figure 2 , Figure B, open circles and squares vs. solid circles), while the binding of W110Y was diminished ( Figure 2, Figure B, triangle). When using MEC cells transfected with a blank vector (not shown), no binding of any variant was detected, demonstrating the specificity of the variant for ROR1. Regardless of the assay format, the relative binding strengths of the variants were very similar. For example, the affinity of the T32A variant was 4 to 4.7 times higher than that of the parental antibody, while the affinity of the W110Y variant was 5.9 to 6.8 times lower than that of the parental antibody. These very closely related variants differed from each other by one or two amino acids, had a binding activity range for ROR1 of 27-fold, and were capable of designing anti-ROR1 / anti-CD3 bispecific binding molecules with a range of affinities for ROR1 and CD3.

[0708] Next, two additional anti-ROR1 Fab variants, each containing a different light chain CDR mutation combined with the heavy chain T32A mutation, were characterized using a modified version of the previously described ELISA-based screening. Specifically, the first wash step after binding of the Fab to immobilized ROR1 was altered by placing the plate in a large volume of PBS-T for an extended one-hour dissociation step. This allowed variants with slow dissociation rates to be distinguished from each other. Using the modified ELISA, two variants, T32A(HC)+A25P(LC) and T32A(HC)+T69R(LC), showed stronger binding than T32A ( Figure 3 ). As previously described, the T32A variant showed a greater than 5-fold improvement in binding relative to the parental (WT), while T32A(HC)+A25P(LC) and T32A(HC)+T69R(LC) showed further binding improvements relative to the parental Fab (>8- and >13-fold, respectively). These very closely related variants differed from each other by 1-3 amino acids, had a binding activity range for ROR1 of 80 to 90-fold, and were capable of designing with a range of affinities for ROR1 and CD3.

[0709] Three assays were used to measure the ROR1 binding of the bispecific binding molecules: (1) binding to live cells and detection by flow cytometry, (2) binding to immobilized ROR1, as determined by ELISA, and (3) binding to soluble biotinylated ROR1, as determined by ELISA. Regardless of the screening format employed, all constructs selectively bound ROR1 ( Figures 4 - 7 ).

[0710] Selectivity for ROR1 was demonstrated by measuring binding to ROR1-transfected MEC cells and control MEC cells. Binding of the bispecific binding molecules to ROR1-transfected cells was concentration-dependent ( Figure 4 , Figure A) and no binding to control cells was observed at any concentration ( Figure 4 , Figure B). Nonspecific human IgG was included as a control for both cell lines ("control").

[0711] Changes in the binding strength of bispecific constructs to ROR1-transfected MEC cells were observed ( Figure 4 , Figure A). For example, the ROR1 bivalent construct binds more tightly than the ROR1 monovalent construct ( Figure 4 , Figure A, comparing constructs 1-3 and 7-9 with 13-15). This difference in binding strength is most evident at lower concentrations (<100 ng / mL).

[0712] The effect of ROR1 expression levels on bispecific construct binding was evaluated by flow cytometry using JeKo-1 cells. The level of ROR1 expressed by JeKo-1 cells (∼13,000 copies / cell) is lower than that of ROR1-transfected MEC cells (∼56,000 copies / cell). All constructs bind JeKo-1 cells ( Figure 5 ). The relative binding trends observed using ROR1-transfected MEC cells were observed using JeKo-1 cells. Specifically, monovalent ROR1 constructs generally bind more weakly than bivalent constructs ( Figure 5 , Figure A, comparison of constructs 14 and 15 with other constructs; Figure 5 , Figure C, comparison of constructs 15-17 with other constructs).

[0713] Next, the binding of bispecific binding molecules to ROR1 was characterized by ELISA using the extracellular domain of recombinantly expressed ROR1. ROR1 was immobilized on microtiter plates, the bispecific binding molecules were titrated, and binding was quantified using an anti-human κ-HPR reagent. Consistent with the cell-binding data, all constructs bind ROR1 in a concentration-dependent manner ( Figure 6 ). In addition, monovalent ROR1 constructs 13-15 bind more tightly than all constructs except for two bivalent constructs. The binding activity of the bivalent bispecific binding molecules is similar to that of the parental anti-ROR1 IgG (control), indicating that attaching scFv domains to Ig molecules does not inhibit ROR1 binding. The relative affinities of the bispecific constructs tested in this format are summarized in Table 4.

[0714] The stronger binding observed with bivalent constructs compared to monovalent constructs in antigen-coated cell assays and ELISA is consistent with an avidity effect. To determine if this is the case, binding to soluble monomeric ROR1 was evaluated by immobilizing the bispecific binding molecules on microtiter plates coated with goat anti-human κ antibody and titrating biotinylated ROR1. All bispecific binding molecules are active in this format ( Figure 7), and in most cases, there was no significant difference between the monovalent and bivalent constructs. These results are consistent with the avidity effect, which depends on the assay format used. Weaker binding was observed in both variants in which the anti-CD3 scFv was modified at the amino terminus of the heavy or light chain (constructs 19 and 20, respectively), suggesting that placing the CD3 complementarity-determining regions close to the ROR1 complementarity-determining regions may inhibit ROR1 binding to some extent.

[0715] Bispecific binding molecule constructs containing a higher-affinity ROR1 binding domain (constructs 16 - 18) bound biotinylated ROR1 with higher affinity than the corresponding constructs utilizing the wild-type (low-affinity) ROR1 binding domain (constructs 9, 19, 20). The bispecific construct 18 (bivalent, higher-affinity ROR1) bound more tightly than the corresponding construct 9 (bivalent, lower-affinity ROR1). Similarly, the bispecific constructs 16 and 17 (monovalent, higher-affinity ROR1) bound more tightly than construct 15 (monovalent, lower-affinity ROR1). These data are summarized in Table 4. In addition, bispecific binding molecules with a higher-affinity ROR1 binding domain bound soluble ROR1 with higher affinity than the parental IgG (control).

[0716] Table 4. Affinity of bispecific binding molecules for ROR1

[0717]

[0718]

[0719] Example 3: Internalization of anti-ROR1 / anti-CD3 bispecific binding molecules into ROR1

[0720] Internalization and recycling of ROR1 and bispecific binding molecules that recognize ROR1 may affect therapeutic efficacy in vivo. For example, binding, internalization, and degradation of ROR1 prior to integration with T cells may reduce cytotoxicity. Anti-ROR1 / anti-CD3 bispecific binding molecules with five different configurations were characterized for binding and internalization.

[0721] While characterizing the internalization of various bispecific constructs, cell surface expression of ROR1 was detected using two different anti-ROR1 antibodies. One antibody (UC961) recognizes the same ROR1 epitope as the bispecific binding molecule, while the other (4A5) recognizes a non-overlapping ROR1 epitope.

[0722] In addition, cell surface binding of the bispecific binding molecules was evaluated after incubation at 37 °C for 24 hours to determine whether extended exposure affects the cell surface levels of the various binding molecules.

[0723] Characterize the internalization of the ROR1 bispecific binding molecule and the recycling / re-expression of ROR1 using ROR1-transfected MEC cells and flow cytometry.

[0724] Materials and Methods

[0725] Measurement of bispecific binding molecule internalization

[0726] Measure the internalization of the bispecific binding molecule using a pulse-chase method that quantifies non-internalized cell surface antibody at 0, 30, 60, 120, 180, and 240 minutes using flow cytometry. ROR1-transfected MEC cells are washed with cold PBS and resuspended at 1 x 10 7 / mL in cold incubation buffer (RPMI-Fisher / Gibco cat.# 16140071 containing 2% FBS). Then, for each bispecific construct tested, 3.5 x 10 6 cells are aliquoted into microcentrifuge tubes. An equal volume (350 μL) of the bispecific binding molecule is combined with the cells to a final concentration of 30 μg / mL. Based on previous binding studies, 30 μg / mL is expected to be saturating for the higher affinity / avidity of the divalent ROR1 constructs (constructs 1-12). The cells and antibody are incubated on ice for 20 minutes, washed 5 times with 1 mL of cold FACS buffer (PBS containing 2% FBS), and then resuspended in 1.4 mL of incubation buffer. Subsequently, the samples are incubated at 37 °C for 0, 30, 60, 120, 180, and 240 minutes, and then 200 μL of each sample is transferred to ice to stop internalization.

[0727] Samples for each construct and time point are split in half (100 μL each) and centrifuged. One half of the sample is resuspended in 100 μL of secondary antibody (goat anti-human IgG-PE, Fc-γ specific) to detect the bound bispecific construct. The other half is stained with a 100 μL mixture of UC961-PE and 4A5-Alexa Fluor 647. UC961 binds the same epitope as the bispecific construct, thereby detecting free ROR1 not bound by the bispecific binding molecule. 4A5 binds ROR1 at a different epitope and thus does not compete for binding with the bispecific binding molecule. Therefore, 4A5 detects total cell surface ROR1 (bound and free). Both stained sets are incubated in the dark in ice-cold incubation buffer for 20 minutes. The cells are washed 3 times with FACS buffer and resuspended in 100 μL of fixation buffer (PBS with 2% paraformaldehyde) for 10 minutes at 25 °C. Subsequently, the fixed cells are washed once with FACS buffer and analyzed.

[0728] Quantify the median fluorescence intensity (MFI) of each stain. Several control conditions were used. The PE and Al647 MFI of unstained control cells were measured for background fluorescence subtraction. Cells not exposed to the bispecific construct were stained with the secondary antibody PE to address non-specific staining of the secondary antibody (secondary negative control), and stained with UC961-PE to obtain a control for maximum UC961 binding. Quantification of surface bispecific binding molecules, unoccupied cell surface ROR1 epitopes, and total ROR1 was performed as follows:

[0729] Surface biAb = [(Secondary PEMFI at time point x) - (PE MFI of secondary negative control)]

[0730] [(PE MFI at time point 0 of the secondary)-(PE MFI of the secondary negative control)]

[0731] Total ROR1 = [(Al647 MFI of 4A5 at time point x) - (Al647 MFI of unstained cells)]

[0732] [(Al647 MFI of 4A5 at time point 0)-(AL647 MFI of unstained cells)]

[0733]

[0734] Results

[0735] Previous experiments using the anti-ROR1 antibody used to construct the bispecific binding molecules described herein demonstrated that: (1) the antibody was rapidly internalized, and (2) ROR1 reappeared on the cell surface unbound to the antibody. This cell surface expression was either the result of recycling of ROR1 from endosomal compartments or reflected rapid upregulation of ROR1 by de novo synthesis or trafficking from intracellular stores (see, e.g., U.S. Patent Publication 2018 / 0369406). Different bispecific constructs showed variable internalization and ROR1 recycling / re-expression patterns ( Figure 8 and Figure 9 ).

[0736] Placing the anti-CD3 scFv at the carboxyl terminus of the heavy chain (configuration A, construct 1) resulted in rapid internalization ( Figure 8 , Figure A, circles). At time point 0, the available ROR1 bispecific epitopes were saturated as no free epitopes were detected. Over the course of the experiment, ROR1 did not recycle to the cell surface or re-express at significant levels as the total ROR1 surface levels decreased ( Figure 8 , Figure A, squares), and no free ROR1 epitopes recognized by the bispecific binding molecule were detected ( Figure 9, Figure A, triangle). These data are consistent with the internalization and trafficking to lysosomes of the bispecific binding molecule-ROR1 complex. Very similar internalization and trafficking were observed with bispecific binding molecule constructs 2 and 3 (not shown).

[0737] Placement of the anti-CD3 scFv at the carboxyl terminus of the light chain (configuration B, construct 7) significantly inhibited internalization ( Figure 8 , Figure B, comparing the degree of internalization relative to Figure 8 construct 3 in Figure A). At time 0, the construct had fully saturated its epitope. The total cell surface ROR1 levels appeared relatively constant ( Figure 8 , Figure B, squares). In addition, the free ROR1 epitopes recognized by the bispecific increased in a time-dependent manner ( Figure 8 , Figure B, triangles), consistent with dissociation of the bispecific binding molecule from the cell surface or recycling / re-expression of ROR1. Very similar internalization and trafficking were observed with bispecific binding molecule constructs 8 and 9 (not shown).

[0738] The monovalent anti-ROR1 (configuration C, construct 13) was also not efficiently internalized ( Figure 8 , Figure C). Previous studies have shown that binding of the bivalent construct saturates at 30 μg / mL ( Figure 4 , Figure A and Figure 5 ). However, in this experiment, binding of the monovalent anti-ROR1 bispecific binding molecule at 30 μg / mL did not appear to saturate cell surface ROR1. At time 0, the degree of binding of construct 13 was only about 80% of the degree of binding of the corresponding saturating MFIs of constructs 1 and 7 at time 0 ( Figure 8 , Figure C, comparing circles and squares at time 0). In addition, free ROR1 epitopes recognized by construct 13 were also detected at time 0 ( Figure 8 , Figure C, triangles). In summary, the levels of the monovalent bispecific binding molecule construct 13, total cell surface ROR1, and unoccupied bispecific epitopes remained relatively constant throughout the experiment. Overall, these data are consistent with little internalization of bispecific binding molecule 13. Very similar internalization and trafficking were observed with construct 15 (not shown).

[0739] Placement of the anti-CD3 scFv at the amino terminus of one heavy chain (configuration D, construct 19) resulted in partial internalization ( Figure 9 , Figure A, circles). During the course of the experiment, ROR1 was recycled to the cell surface or re-expressed at significant levels as the total ROR1 surface levels increased ( Figure 9 , Figure A, squares) and there was an increasing, time-dependent detection of ROR1 epitopes recognized by the bispecific binding molecule ( Figure 9, Figure A, triangle). Placing the anti-CD3 scFv portion at the amino terminus of a heavy chain results in internalization and trafficking, very similar to that of a bispecific binding molecule with Configuration B (anti-CD3 scFv fused to the carboxyl terminus of a light chain).

[0740] Placing the anti-CD3 scFv at the amino terminus of the light chain (Configuration E, construct 20) results in partial internalization ( Figure 9 , Figure B, circle). During the experimental time course, ROR1 was recycled to the cell surface or re-expressed at significant levels as the total ROR1 surface level increased ( Figure 9 , Figure B, square) and there was an increased, time-dependent detection of ROR1 epitopes recognized by the bispecific binding molecule ( Figure 9 , Figure B, triangle). Placing the anti-CD3 scFv portion at the amino terminus of the light chain results in internalization and trafficking, very similar to that of a bispecific binding molecule with Configuration B (anti-CD3 scFv fused to the carboxyl terminus of a light chain).

[0741] To determine the long-term effect of ROR1 trafficking and expression on the level of bispecific binding molecule bound to the cell surface, cells were co-incubated with the bispecific binding molecule for 24 hours, washed, and the cell surface bispecific binding molecule was measured. Quantification of the cell surface bispecific binding molecule after 24 hours of incubation showed that the level of the bispecific binding molecule with anti-CD3 scFv fused to the carboxyl terminus of the heavy chain ( Figure 10 , construct 1, circle) was lower than that of the bispecific binding molecule with anti-CD3 scFv fused to the carboxyl terminus of the light chain ( Figure 10 , construct 7, square) or the bispecific binding molecule with anti-CD3 scFv fused to the amino terminus of the Fc ( Figure 10 , construct 13, triangle). The reduced cell surface level of constructs in which anti-CD3 scFv was fused to the carboxyl terminus of the heavy chain rather than other bispecific configurations indicated downregulation of surface ROR1 and was consistent with the observations in the internalization experiments ( Figure 8 ).

[0742] Example 4: CD3 Binding and Internalization of Anti-ROR1 / Anti-CD3 Bispecific Binding Molecules

[0743] The number of TCR-CD3 complexes on the surface of T cells reflects a combination of de novo protein synthesis and secretion, internalization, recycling, and degradation. Treatment with the anti-CD3 antibody OKT3 has been shown to selectively remove CD3 from the surface by internalization.

[0744] Three anti-CD3 sequences were used as scFv constructs in anti-ROR1 / anti-CD3 bispecific binding molecules. Two of the sequences were different humanized versions of OKT3 (Ab8 and Ab9), while the third sequence was a third-party humanized version of a different anti-CD3 antibody, SP34 (Ab10). As described above, OKT3 and SP34 bind to different epitopes, and anti-CD3 antibodies have been classified according to the different characteristics of their epitopes (Tunnacliffe et al., International Immunology 1:546-550 (1989)). Using this system, SP34 was classified as group I, while OKT3 was classified as group II. Cross-reactivity with cynomolgus monkey CD3 was expected based on the sequence of SP34, rather than on the sequence of OKT3.

[0745] The various CD3 complementarity-determining regions were evaluated in five different configurations ( Figure 1 , A-E), and the monovalent anti-CD3 bispecific constructs ( Figure 1 , C and D) were compared with certain bivalent anti-CD3 constructs ( Figure 1 , A, B and E). The disulfide-stabilized versions of all three scFv sequences were also evaluated in various bispecific configurations.

[0746] The internalization of the various bispecific constructs in the absence of ROR1 was evaluated. The binding and internalization of the bispecific binding molecules were characterized using Jurkat cells and flow cytometry.

[0747] Materials and Methods

[0748] CD3 Binding Assay

[0749] To quantify CD3 binding, 2.5E5 Jurkat cells were used for each condition. The cells were prepared as a 2X cell stock solution by resuspending the cells to 5E6 / mL and placing the cells in 50 μL of PBS containing 2% FBS. Then, the cells were diluted with an equal volume of a 2X stock solution of the test bispecific binding molecule. The cells and the antibody were co-incubated on ice for 20 minutes. Then, the cells were washed 3 times with 300 μL of FACS buffer and resuspended in 100 μL of goat anti-human Fc-PE conjugated antibody (Invitrogen cat.#12499882) at a 1:500 dilution and incubated on ice in the dark for 20 minutes. Then, the cells were washed 3 times with 300 μL of FACS buffer and fixed with 2% paraformaldehyde at 25 °C for 10 minutes. The cells were washed 2 times with 300 μL of FACS buffer and analyzed on a Miltenyi MACSQuant Analyzer.

[0750] CD3 Internalization Assay

[0751] To quantify the internalization of bispecific binding molecules, 2.5E5 Jurkat cells were co-incubated with 1 μg / mL of the bispecific binding molecule on ice for 20 minutes. The cells were then washed three times with 300 μL of ice-cold FACS buffer and resuspended in medium and cultured at 37 °C. At different times, the cells were removed, washed with 300 μL of ice-cold FACS buffer and processed as described above.

[0752] Results

[0753] Five different bispecific configurations were evaluated ( Figure 1 ). In all tested bispecific configurations, the sequences based on OKT3 and SP34 retained CD3 binding, as demonstrated by binding to Jurkat cells. The constructs bound to varying degrees ( Figure 11 and Figure 12 ).

[0754] For all bispecific configurations tested with OKT3 and SP34 sequences (constructs 1 - 15), the OKT3-based sequences bound with higher affinity than the SP34-based sequences. For example, constructs 1 and 2 bound more tightly than 3 ( Figure 11 , Figure A), constructs 7 and 8 bound more tightly than 9 ( Figure 11 , Figure B), and constructs 13 and 14 bound more tightly than 15 ( Figure 11 , Panel C). Nonspecific human IgG was used as a control ("Control").

[0755] Introducing cysteine residues to generate disulfide-stabilized variants of the anti-CD3 scFv did not negatively impact binding. The binding of the disulfide-stabilized variants was indistinguishable from the corresponding non-disulfide-stabilized sequences ( Figure 11 , Figure A, comparing constructs 1 - 3 and 4 - 6 respectively, and Figure 11 , Figure B, comparing constructs 7 - 9 and 10 - 12 respectively.)

[0756] The configuration of the bispecific binding molecule had a profound effect on the binding of the anti-CD3 scFv ( Figure 12 ). When the anti-CD3 scFv portion was placed at the amino terminus of the light chain ( Figure 12 , Figure B, construct 20) or the heavy chain ( Figure 12 , Figure B, construct 19), the strongest binding to Jurkat cells was observed. In contrast, when the anti-CD3 scFv was placed at the carboxyl terminus of the light chain, weaker binding was observed ( Figure 12 , Figure B, constructs 9 and 18). A single anti-CD3 scFv portion placed at the amino terminus of one heavy chain (monovalent CD3 binding; Figure 12, Figure B, construct 19) also binds as well as or better than placing two anti-CD3 scFv moieties at the carboxy terminus of the heavy chain (bivalent CD3 binding; Figure 12 , Figure B, construct 3) or the light chain (bivalent CD3 binding; Figure 12 , Figure B, constructs 9 and 18). In one experiment, non-specific human IgG was used as a control ( Figure 12 , Figure A, "control"), while in a second experiment, an irrelevant ROR1 x CD3 (U.S. Patent Publication 2017 / 0233472; SEQ ID NO: 111 - 113) bispecific construct was used as a control ( Figure 12 , Figure B, "control").

[0757] Similar trends were observed for a subset of these bispecific configurations using the OKT3 sequence. Specifically, the monovalent anti-CD3 scFv fused to the amino terminus of the Fc bound the strongest ( Figure 12 , Figure A, construct 13), followed by constructs in which the scFv was fused to the carboxy terminus of the heavy chain ( Figure 12 , Figure A, construct 1), and finally constructs in which the scFv was fused to the carboxy terminus of the light chain ( Figure 12 , Figure A, construct 7).

[0758] Notably, the configuration of the bispecific binding molecule appears to be a more important determinant of CD3 binding strength than the CD3 epitope valence. Monovalent anti-CD3 constructs fused to the amino terminus of the heavy chain ( Figure 12 , Figure B, construct 19) or the Fc ( Figure 12 , Figure A, constructs 13 and Figure 12 , Figure B, constructs 15 and 16) were found to bind as well as or better than bivalent anti-CD3 constructs in which the scFv was fused to the carboxy termini of the heavy and light chains ( Figure 12 , Figure B, constructs 3, 9, and 18).

[0759] Overall, these data demonstrate that there are different ways to modulate the strength of CD3 binding. First, when compared to each other in the same bispecific format, SP34- and OKT3-based sequences show different binding strengths. Second, the binding strength of SP34- and OKT3-based sequences is affected by the configuration of the bispecific binding molecule. Finally, monovalent or bivalent anti-CD3 bispecific binding molecules can be achieved using heteromeric Fc constructs.

[0760] T cells activated by anti-CD3 antibodies can lead to increased CD3 internalization. Once internalized, CD3 may enter the recycling pathway and be re-expressed on the cell surface, or it may be sorted to lysosomes and degraded. In the absence of tumor antigen (ROR1), the internalization and degradation of CD3 induced by bispecific binding molecules may inhibit the cytotoxic activity of bispecific binding molecules in vivo. Thus, bispecific binding molecules are characterized as internalized after binding to CD3 in the absence of ROR1 ( Figure 13 ).

[0761] Jurkat cells were incubated at 37 °C after binding bispecific binding molecules and washing at 4 °C. Subsequently, the amount of bispecific binding molecules on the cell surface was quantified at different time points. Since bispecific binding molecules initially bind with different intensities ( Figure 11 and Figure 12 ), internalization was reported as the percentage of binding observed at time 0. The level of surface bispecific binding molecules quantified in this way reflects a combination of internalization and dissociation. Nevertheless, for the reasons outlined below, the main factor affecting the surface level appears to be the internalization rather than the dissociation of bispecific binding molecules.

[0762] For all tested bispecific constructs, the level of surface bispecific binding molecules decreased in a time-dependent manner, although the extent of loss varied. Rapid internalization (>50%) was observed within the first 30 minutes for eight of the nine constructs tested, followed by slow but continuous internalization over the next 3.5 hours. Construct 9, consisting of an SP34-based sequence fused to the carboxyl terminus of the light chain, showed the least internalization during the experiment, with approximately 50% of bispecific binding molecules still detectable on the cell surface after 4 hours. In addition, constructs 1 and 3, consisting of an anti-CD3 scFv portion fused to the carboxyl terminus of the heavy chain, showed moderate internalization, with approximately 20% still detectable on the cell surface after 4 hours of incubation.

[0763] As previously mentioned, the observed decrease in cell surface bispecific binding molecule staining over time may reflect dissociation of bispecific binding molecules from the surface rather than internalization. However, the two bispecific constructs that showed the least signal loss over time, constructs 3 and 9, are among the weakest binding constructs characterized ( Figure 11 , Figures A and B), while the binding of construct 13, which showed very strong binding at time 0 ( Figure 11 , Figure C), was weakly stained at 4 hours (8% of the initial signal). Overall, these data suggest that certain configurations consisting of an SP34-based anti-CD3 scFv portion may internalize to a lesser extent than those consisting of an OKT3-based scFv portion.

[0764] Example 5: Redirected T cell cytotoxicity of anti-ROR1 / anti-CD3 bispecific binding molecules in vitro

[0765] Twenty anti-ROR1 / anti-CD3 bispecific binding molecules were tested to evaluate the effects of multiple parameters on antibody potency in a T cell-mediated cytotoxicity assay. The variables tested included anti-CD3 epitope and titer, anti-ROR1 affinity and titer, and various configurations of the CD3 and ROR1 binding components.

[0766] The killing activity of the constructs was tested against a panel of blood and solid tumor cancer cells with different ROR1 expression levels. In addition to characterizing the killing activity of the bispecific binding molecules, the activity of the T cells was evaluated using CD69 as a marker. The cytokines released by the activated T cells were also characterized.

[0767] Materials and Methods

[0768] Cytotoxicity assay of human PBMCs

[0769] For the cytotoxicity assay, target cells were resuspended in RPMI (without phenol red), 10% FBS containing penicillin-streptomycin to 4E5 cells / mL. Subsequently, 2E4 cells / well (50 μL / well) were transferred to a 96-well plate and incubated in a 37 °C incubator for 1 h. Round-bottom plates were used for suspension cells and flat-bottom plates were used for adherent cell lines. Human PBMCs were isolated as described below. PBMCs were resuspended to 2E6 cells / mL and then 2E5 cells / well (100 μL / well) were added to the wells containing the target cells. Unless otherwise specified, the ratio of PBMCs (effector cells) to tumor cells (targets) used was 10:1 (E:T ratio). The target cells were mixed with PBMCs and incubated in a 37 °C incubator for 1 h. The bispecific binding molecule was diluted to an appropriate concentration in the medium, 50 μL was added to the mixture of target cells and PBMCs, and then incubated in a 37 °C incubator for 24 h. Each experiment included the following controls: (1) media only (Media), (2) media adjusted for lysis buffer (Adj), (3) PBMCs only (PBMC), (4) target cells only for spontaneous release of LDH (SR), (5) target cells only for maximum release of LDH (MR), and (6) target cells and PBMCs without the bispecific binding molecule (AICC).

[0770] Isolation of PBMCs

[0771] The lower chamber of a SepMate-50 (StemCell Technologies) conical tube was filled with 15 mL of Ficoll. Blood taken from an unidentified normal donor was placed in a heparinized blood collection tube and mixed with sterile PBS at a 1:1 ratio at 25 °C. The diluted blood was layered on top of the upper chamber of the SepMate-50 tube and centrifuged at 1200 x g for 15 minutes at 25 °C with the brake on. The diluted blood was layered on top of the upper chamber of the SepMate-50 tube and centrifuged at 1200 x g for 15 minutes at 25 °C with the brake on. The cells were then washed 3 times with cold 5% FBS PBS solution by centrifuging at 500 x g for 3 minutes. Finally, the cells were resuspended in 10 mL of medium (RPMI without phenol red, 10% FBS containing penicillin-streptomycin), and aliquots were taken for cell counting. The cells were then used for plating the cytotoxicity assay.

[0772] Quantification of LDH release

[0773] To quantify the LDH release after 24 hours of culture, 20 μL of 10X lysis buffer was added to the Adj and MR wells, and the contents of each well were transferred to a V-bottom 96-well plate and centrifuged at 500 x g for 5 minutes. Subsequently, 170 μL of the supernatant was transferred to a separate 96-well plate for further analysis. In a fresh clear-bottom 96-well plate, 50 μL of the supernatant was mixed with 50 μL of CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega cat.# G1780), and the plate was incubated at 25 °C in the dark for 30 minutes. Finally, 50 μL of stop solution was added before reading the absorbance at 490 nm. % LDH release was calculated according to the manufacturer's instructions.

[0774] Assessment of T cell activation

[0775] Upregulation of CD69 is used to characterize early activation of T cells. CD69 was quantified using flow cytometry. Briefly, the pelleted cells were resuspended in 100 μL of PBS with 2% PFA and incubated at 25 °C for 10 minutes for fixation. The cells were washed twice with 300 μL of PBS, resuspended in 150 μL of PBS, and stored at 4 °C in the dark for later use. Subsequently, the cells were collected by centrifugation and resuspended in 100 μL of staining mixture, and incubated at 4 °C in the dark for 20 minutes. The staining mixture consisted of Pacific Blue anti-human CD8 clone SK1 (BioLegend cat.#344717) diluted 1:75 in PBS, PE / Cy7 anti-human CD69 clone FN50 (BioLegend cat.#310911) diluted 1:500, and AlexaFluor647 mouse anti-human ROR1 clone 4A5 diluted 1:500. Then the cells were washed three times with 300 μL of cold PBS and resuspended in 150 μL of PBS with 2% FBS. The samples were analyzed on a Mitenyi MACSQuant Analyzer.

[0776] Quantifying cytokines released from PBMCs in redirected T cell assays

[0777] To measure cytokine release, IFN-γ, IL-2, IL-4, IL-6, IL-10, and TNF-α were measured simultaneously using the MSD-R V-PLEX cytokine panel 1 human kit. The assay was performed according to the manufacturer's instructions. Briefly, the V-plex Cytokine Panel 1 plate was washed three times with 150 μL / well of wash buffer (PBS containing 0.05% Tween-20). Next, 50 μL of diluted samples and calibrators were added to each well. The plate was sealed with a sticky plate sealer and incubated overnight on an oscillator at 4 °C and 500 rpm. The next day, the plate was washed three times with 150 μL / well of wash buffer and incubated with 25 μL of detection antibody solution on a shaker at room temperature for 2 hours. The plate was washed three more times with the wash buffer. Finally, 150 uL of read buffer T was added before analysis using the MSD instrument.

[0778] Results

[0779] Although with different relative potencies, antigen-dependent killing of ROR1-transfected MEC cells was observed in most of the bispecific constructs tested. Representative titration curves are shown in Figure 14 , Figure A (using constructs of configurations A, B, and C) and Figure 15, in Figure A (using constructs of configurations A, B, C, D, and E). The titration curves of constructs 2, 8, and 14 (not shown) were similar to those of constructs 1, 7, and 13, respectively.

[0780] As expected, when different PBMC donors were used, the absolute potency of the bispecific binding molecule varied to some extent. Nevertheless, the relative potencies of the different constructs did not fluctuate significantly. For example, bispecific binding molecule construct 9 was consistently the most active one, and in ROR1-transfected MEC target cells, the EC50 for one donor showed <0.1 ng / mL ( Figure 14 , Figure A) and an EC50 of 1.1 ng / mL for the second donor ( Figure 15 , Figure A). <

[0781] Constructs 1-3, 7-9, and 13-15 all showed selective killing at 1 μg / mL (5 mM for constructs 1-3 and 7-9, 8 nM for constructs 13-15 ( Figure 16 , black bars). The antigen-dependence of the killing was demonstrated by the lack of LDH release in samples without target cells ( Figure 16 , "PBMC") or antibody ( Figure 16 , "bispecific antibody-free") or in samples of non-transfected MEC cells used as target cells ( Figure 16 , gray bars). In contrast, the control anti-CD19 / anti-CD3 bispecific binding molecule (Creative Biolabs cat.#BSAB-L002) induced cytotoxicity in both ROR1-transfected and non-transfected MEC cells ( Figure 16 , "CD19 x CD3"). Bispecific constructs 16-20 were not tested in this experiment.

[0782] Activation of T cells in the cultures was evaluated by examining the upregulation of CD69, an early marker of T cell activation. Consistent with the cytotoxicity data, activation was observed at 1 μg / mL for all constructs except #17 and #19 ( Figure 14 , Figure B; Figure 15 , Figure B) For the more potent bispecific binding molecules, activation was observed at lower concentrations ( Figure 14 , Figure B, comparing 7 with 3; Figure 15 , Figure B, comparing 18 with 3).

[0783] T cell activation, as evaluated by increased CD69 expression, generally reflected the cytotoxic activity of the bispecific binding molecule. However, there were examples where CD69 activation was not directly correlated with cytotoxicity ( Figure 19 , comparing 7 and 9; Figure 20 , comparing 7 with 8 and 9).

[0784] T cell activation is antigen - dependent ( Figure 17 ). T cells co - cultured with ROR1 - / CD19 + MEC cells were in the presence of a control CD19 x CD3 bispecific binding molecule ( Figure 17 , Figure A, left) but not in the presence of an ROR1 bispecific binding molecule ( Figure 18 , Figure A, right). However, when T cells were co - cultured with ROR1 - transfected MEC cells (ROR1 + / CD19 + ), the T cells were activated in the presence of the ROR1 bispecific binding molecule ( Figure 17 , Figure B, right). These data indicate that ROR1 is required for the activation of T cells by the bispecific constructs characterized in this study.

[0785] Next, the ROR1 - dependent killing of different human target cells by the bispecific binding molecules was examined. ROR1 - dependent killing of JeKo - 1 cells (a mantle cell lymphoma cell line) was observed with all bispecific binding molecules of configurations A, B, C, and E, although with different potencies. Representative titration curves are shown in Figure 18 , Figure A (constructs 1 - 15) and Figure 19 , Figure A (constructs 16 - 20). Consistent with the observations made with ROR1 - transfected MEC cells, the activation of T cells generally reflected the relative cytotoxicity of the bispecific binding molecules ( Figure 18 , Figure B; Figure 19 , Figure B).

[0786] A second mantle cell lymphoma cell line, Mino cells, was also tested as a target cell line. All three non - disulfide - stabilized constructs with configuration B showed effective activity against Mino cells ( Figure 20 , Figure A), with the SP34 - based CD3 sequence (construct 9) being the most effective. Interestingly, construct 9 activated fewer T cells (determined by upregulation of CD69) than the OKT3 - based constructs ( Figure 20 , Figure B).

[0787] Similarly, ROR1 - dependent killing of the breast tumor cell line MDA - MB - 468 with different potencies was observed for bispecific configurations A, B, C, and E. Representative titration curves are shown in Figure 21 , Figure A (constructs 1 - 15) and Figure 22 , Figure A (constructs 16 - 20). Consistent with the observations made with ROR1 - transfected MEC cells and JeKo - 1 cells, the activation of T cells generally reflected the relative cytotoxicity of the bispecific binding molecules ( Figure 21 , Figure B; Figure 22 , Figure B).

[0788] Characteristics of cytokine release induced after T cell activation in redirected killing of JeKo-1 cells, ROR1-transfected MEC cells, and mock-transfected (ROR1-) MEC cells. Representative dose-response plots of constructs 7, 9, 18, 20, and control ROR1 x CD3 bispecific binding molecules are shown (U.S. Patent Publication 2017 / 0233472).( Figure 23 (TNF-α), Figure 24 (IFN-α), Figure 25 (IL-2), Figure 26 (IL-4), Figure 27 (IL-6), Figure 28 (IL-10)). Dose-dependent cytokine production (TNF-α, IFN-α, IL-2, IL-4, IL-6, and IL-10) was observed in JeKo-1 cells (Figure A) and ROR1-transfected MEC cells (Figure B), but not in mock-transfected (ROR1-) MEC cells (Figure C), for all tested constructs. ROR1-transfected MEC cells expressed higher levels of ROR1 than JeKo-1 cells (Table 5) and were more sensitive to the tested bispecific binding molecules than JeKo-1 cells, demonstrating the importance of target expression levels. Cytokine secretion was dependent on ROR1 expression. Incubation of 1 μg / mL of the constructs with mock-transfected (ROR1-) MEC cells (Figure C) did not induce secretion of any cytokine significantly above background levels (no bispecific antibody control). In contrast, incubation of the ROR1 x CD3 bispecific binding molecule (“Control 1”) with mock-transfected (ROR1-) MEC cells resulted in some secretion of the inflammatory cytokine IFN-α( Figure 24 , Figure C). Control 1 induced a higher overall level of cytokine secretion when tested with JeKo cells, except for IL-6. In contrast, all constructs, including Control 1, induced similar levels of cytokine secretion when tested with MEC cells transfected with higher ROR1 expression.

[0789] A summary of the activities of some bispecific binding molecules against various target cells is shown in Table 5.

[0790] Table 5 Summary of cell lines tested with bispecific constructs

[0791]

[0792] Overall, redirected T cell killing of various target cell lines by anti-ROR1 / anti-CD3 bispecific binding molecules with a range of properties and configurations identified certain trends.

[0793] Based on the LCK cytotoxicity assay using the user's PBMC as effector cells and various target cell lines, constructs B, E, and C tend to be more effective than constructs A and D. The relative potency based on the configuration can reflect the relative spatial orientation of the CD3 and ROR1 binding arms. Potency can also reflect the effect of various constructs on the trafficking of bispecific binding molecules. For example, the bispecific binding molecules of constructs B and C do not internalize as rapidly or to the same extent as those of construct A.

[0794] Although the OKT3-based sequences appear to have a higher affinity for CD3 than the SP34-based sequences, bispecific binding molecules with bivalent SP34-based sequences are generally more effective. The enhanced activity can reflect the affinity, or alternatively or in addition, the unique epitope recognized by SP34. Interestingly, when evaluating bispecific binding molecules with monovalent CD3 binding, the trend is different. In some cases, the OKT3-based sequences are more effective than the SP34-based sequences ( Figure 14 , compare constructs 13 and 15).

[0795] No direct correlation was observed between the ROR1 expression level of the target cells and cytotoxicity (Table 5). Nevertheless, higher affinity variants of the anti-ROR1 antibody are more effective than the parental WT sequence. This may be more important in cases where the ROR1 expression level is low.

[0796] The results of these experiments suggest that while the CD3 and ROR1 binding components can be optimized and characterized individually, the most active bispecific binding molecules can only be identified through empirical testing of different pairings of binding components using different bispecific configurations. The experiments demonstrated that constructs 9 and 18 (configuration B) were consistently among the most effective anti-ROR1 / anti-CD3 bispecific binding molecules. This is particularly notable because CD3 binding experiments showed that these constructs were among the weakest binders to Jurkat cells in the absence of ROR1. This is expected to be a useful feature as activation and internalization of CD3 on T cells will be minimal before ROR1 is involved in the tumor microenvironment.

[0797] Table 6. Antigen-binding domains

[0798]

[0799]

Claims

1. A bispecific binding molecule comprising a first antigen-binding domain that specifically binds to the extracellular domain of human ROR1 and a second antigen-binding domain that specifically binds to the extracellular domain of human CD3; wherein the first antigen-binding domain comprises: a) heavy chain (HC)-CDR1-3 and light chain (LC)-CDR1-3 consisting of the amino acid sequences of SEQ ID NO:97, 61, 62, 63, 64, and 65, respectively; or b) HC-CDR1-3 and LC-CDR1-3 consisting of the amino acid sequences of SEQ ID NO:60, 61, 62, 63, 64, and 65, respectively; and wherein the second antigen-binding domain comprises: heavy chain (HC)-CDR1-3 and light chain (LC)-CDR1-3 consisting of the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58, and 59, respectively.

2. The bispecific binding molecule according to claim 1, wherein the first antigen-binding domain comprises: a) VH comprising the amino acid sequence of SEQ ID NO:74 and VL comprising the amino acid sequence of SEQ ID NO:73; b) VH comprising the amino acid sequence of SEQ ID NO:72 and VL comprising the amino acid sequence of SEQ ID NO:73; c) heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:84 and light chain (LC) comprising the amino acid sequence of SEQ ID NO:83; d) HC comprising the amino acid sequence of SEQ ID NO:82 and LC comprising the amino acid sequence of SEQ ID NO:83; e) HC comprising the amino acid sequence of SEQ ID NO:87 and LC comprising the amino acid sequence of SEQ ID NO:83; or f) HC comprising the amino acid sequence of SEQ ID NO:86 and LC comprising the amino acid sequence of SEQ ID NO:

83.

3. The bispecific binding molecule according to claim 1, wherein the second antigen-binding domain comprises: a) VH comprising the amino acid sequence of SEQ ID NO:70 and VL comprising the amino acid sequence of SEQ ID NO:71; or b) VH comprising the amino acid sequence of SEQ ID NO:80 and VL comprising the amino acid sequence of SEQ ID NO:

81.

4. The bispecific binding molecule according to claim 1, which comprises: a) a first antigen-binding domain comprising heavy chain (HC)-CDR1-3 and light chain (LC)-CDR1-3 consisting of the amino acid sequences of SEQ ID NO:97, 61, 62, 63, 64, and 65, respectively, and a second antigen-binding domain comprising HC-CDR1-3 and LC-CDR1-3 consisting of the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58, and 59, respectively; or b) A first antigen-binding domain, which comprises: HC-CDR1-3 and LC-CDR1-3 respectively consisting of the amino acid sequences of SEQ ID NO:60, 61, 62, 63, 64 and 65, and a second antigen-binding domain, which comprises: HC-CDR1-3 and LC-CDR1-3 respectively consisting of the amino acid sequences of SEQ ID NO:54, 55, 56, 57, 58 and 59; Alternatively, it comprises: a) A first antigen-binding domain, which comprises: a heavy chain variable domain (VH) and a light chain variable domain (VL) respectively comprising the amino acid sequences of SEQ ID NO:74 and 73, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71; or b) A first antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:72 and 73, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71; Alternatively, it comprises: a) A first antigen-binding domain, which comprises: a heavy chain (HC) and a light chain (LC) respectively comprising the amino acid sequences of SEQ ID NO:74 and 73, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:80 and 81; or b) A first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:72 and 73, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:80 and 81; Alternatively, it comprises: a) A first antigen-binding domain, which comprises: a heavy chain (HC) and a light chain (LC) respectively comprising the amino acid sequences of SEQ ID NO:84 and 83, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71; or b) A first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:82 and 83, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:70 and 71; Alternatively, it comprises: a) A first antigen-binding domain, which comprises: a heavy chain (HC) and a light chain (LC) respectively comprising the amino acid sequences of SEQ ID NO:84 and 83, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:80 and 81; b) A first antigen-binding domain, which comprises: HC and LC respectively comprising the amino acid sequences of SEQ ID NO:82 and 83, and a second antigen-binding domain, which comprises: VH and VL respectively comprising the amino acid sequences of SEQ ID NO:80 and 81.

5. A bispecific binding molecule that specifically binds to human ROR1 and human CD3, which comprises the amino acid sequences of SEQ ID NO: 14 and 19; or which comprises the amino acid sequences of SEQ ID NO: 7 and 19; or which comprises the amino acid sequences of SEQ ID NO: 7 and 23.

6. The bispecific binding molecule according to any one of claims 1-5, wherein: a) the valency of the first antigen-binding domain is 2, and the valency of the second antigen-binding domain is 2; or b) the valency of the first antigen-binding domain is 1, and the valency of the second antigen-binding domain is 1; or c) the valency of the first antigen-binding domain is 2, and the valency of the second antigen-binding domain is 1.

7. The bispecific binding molecule according to any one of claims 1-5, which comprises a human IgG1 constant region.

8. The bispecific binding molecule according to any one of claims 1-5, which comprises an IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A, wherein the residues are numbered according to the EU system.

9. The bispecific binding molecule according to any one of claims 1-5, wherein the second antigen-binding domain is a scFv.

10. The bispecific binding molecule according to any one of claims 1-5, wherein the heavy chain or light chain amino acid sequence of the second antigen-binding domain is fused to the heavy chain or light chain amino acid sequence of the first antigen-binding domain via a peptide linker.

11. The bispecific binding molecule according to claim 10, wherein the peptide linker between the heavy chain or light chain amino acid sequence of the second antigen-binding domain and the heavy chain or light chain amino acid sequence of the first antigen-binding domain has the amino acid sequence of GGGGSGGGGS shown in SEQ ID NO:

93.

12. The bispecific binding molecule according to claim 10, wherein the second antigen-binding domain is fused to: a) the carboxyl terminus of the light chain of the first antigen-binding domain; or b) the amino terminus of the light chain of the first antigen-binding domain.

13. A pharmaceutical composition, which comprises the bispecific binding molecule according to any one of claims 1-12 and a pharmaceutically acceptable excipient.

14. An isolated nucleic acid molecule, which comprises nucleotide sequences encoding the heavy chain variable domain (VH) and light chain variable domain (VL) of the first antigen-binding domain of the bispecific binding molecule according to any one of claims 1-12, and further comprises nucleotide sequences encoding the VH and VL of the second antigen-binding domain of the bispecific binding molecule according to any one of claims 1-12.

15. The isolated nucleic acid molecule according to claim 14, which comprises a) the nucleotide sequences of SEQ ID NO: 37 and 42; b) the nucleotide sequences of SEQ ID NO: 30 and 42; or c) the nucleotide sequences of SEQ ID NO: 30 and 46.

16. A vector comprising the isolated nucleic acid molecule according to claim 14 or 15.

17. A host cell comprising nucleotide sequences encoding a heavy chain variable domain (VH) and a light chain variable domain (VL) of a first antigen-binding domain of a bispecific binding molecule according to any one of claims 1-12, and further comprising nucleotide sequences encoding a VH and a VL of a second antigen-binding domain of the bispecific binding molecule according to any one of claims 1-12.

18. The host cell according to claim 17, wherein the host cell comprises the following nucleotide sequences: a) SEQ ID NO: 37 and 42; b) SEQ ID NO: 30 and 42; or c) SEQ ID NO: 30 and 46.

19. A method for producing a bispecific binding molecule according to any one of claims 1-12, comprising: providing the host cell according to claim 17 or 18, culturing the host cell under conditions suitable for the expression of the bispecific binding molecule, and isolating the resulting bispecific binding molecule.

20. The bispecific binding molecule according to any one of claims 1-5, characterized in that, It is used for treating cancer in a patient.

21. Use of a bispecific binding molecule encoded by the nucleotide sequences of SEQ ID NO: 30 and 42 in the preparation of a medicament for treating mantle cell lymphoma, breast cancer, lung cancer, osteosarcoma and Ewing's sarcoma in a patient.

22. The use according to claim 21, wherein the patient is treated with an additional therapeutic agent.

23. The use according to claim 22, wherein the additional therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, B-cell lymphoma 2 (Bcl-2) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and phosphatidylinositol 3-kinase (PI3K) inhibitors.

24. The use according to claim 22, wherein the additional therapeutic agent is selected from ibrutinib, acalabrutinib, venetoclax, everolimus, sapanisertib and idelalisib.

25. A kit comprising a bispecific binding molecule according to any one of claims 1-12.

26. The kit according to claim 25, wherein It is used for treating cancer.

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