WO2024094151A1

WO2024094151A1 - Multi-specific antibody and medical use thereof

Info

Publication number: WO2024094151A1
Application number: PCT/CN2023/129509
Authority: WO
Inventors: Min Ren; Yifan Zhang; Tsungyi Lin; Qing Li; Larry Lo
Original assignee: Hansoh Bio Llc; Shanghai Hansoh Biomedical Co., Ltd.; Changzhou Hansoh Pharmaceutical Co., Ltd.
Priority date: 2022-11-03
Filing date: 2023-11-03
Publication date: 2024-05-10

Abstract

Provided is a multi-specific antibody, comprising a first binding domain capable of specifically binding to receptor tyrosine kinase-like orphan receptor I, and a second binding domain capable of specifically binding to cluster of differentiation 3, and a pharmaceutical use thereof. In addition, provided is a preparation and application of a multi-specific antibody.

Description

MULTI-SPECIFIC ANTIBODY AND MEDICAL USE THEREOF

FIELD OF THE INVENTION

The present disclosure relates to the multi-specificity antibody, which may simultaneously bind to CD3 and another tumor-associated antigen, such as ROR1, and bind and activate CD3-positive T cells while binding ROR1-expressing tumor cells, thereby promoting T cells specifically killing tumor cells.

BACKGROUND

ROR (Receptor Tyrosine Kinase-Like Orphan Receptor) is a transmembrane protein of RTK (Receptor Tyrosine Kinase) family, and there are ROR1 and ROR2, which are type-I transmembrane receptor tyrosine kinases. The extracellular region of ROR1 and ROR2 contains an immunoglobulin (Ig) domain, a cysteine-rich domain (CRD) , also called a Frizzled (Fz) domain, and a Kringle (Kr) domain. All three domains are involved in protein-protein interactions. Intracellularly, ROR1 and ROR2 possess a tyrosine kinase (TK) domain and a proline-rich domain (PRD) straddled by two serine/threonine-rich domains (Borcherding et al., 2014, Protein Cell, 5: 496; Rebagay et al., 2012, Prontiers in oncology, 2: 1) .

ROR1 has been proposed as a target for cancer treatment. For example, WO2005100605, WO2007051077, WO2008103849 and WO2012097313 described antibodies against ROR1 and their use as therapeutics for targeting tumors, including solid tumors such as breast cancer, and hematological tumors such as chronic lymphocytic leukemia (CLL) . Cirmtuzumab, generated by mapping the epitope bound by the anti-ROR1 antibody D10 of WO2012097313, is a humanised monoclonal antibody in clinical trials for various cancers including chronic lymphocytic leukemia (CLL) . Cirmtuzumab can internalize into cells, and has been evaluated for use as the targeting moiety in anti-ROR1 antibody drug conjugates (ADCs) . A cirmtuzumab-based, MMAE-containing ADC, VLS-101, has been developed for the treatment of patients with ROR1-positive malignancies.

Cluster of differentiation 3 (CD3) is a multimeric protein complex expressed on T cells in association with the T cell receptor complex (TCR) , and is required for T cell activation. Antibodies against CD3 have been shown to cluster CD3 on T cells, causing T cell activation. Accordingly, anti-CD3 antibodies have been proposed for therapeutic purposes involving the activation of T cells. For example, bispecific antibodies that are capable of binding CD3 and a target antigen have been proposed for therapeutic uses involving targeting T cell immune responses to tissues and cells expressing the target antigen. Recent approval of the CD19 × CD3 bispecific T-cell engager (BiTE) , blinatumomab, has validated this approach.

Bispecific T-cell engagers (BiTEs) are a new class of immunotherapeutic molecules intended for the treatment of cancer. These molecules enhance the patient's immune response to tumors by retargeting T cells to tumor cells. BiTEs are constructed of two single-chain variable fragments (scFv) connected in tandem by a flexible linker. One scFv binds to a T-cell-specific molecule, usually CD3, whereas the second scFv binds to a tumor-associated antigen. This structure and specificity allows a BiTE to physically link a T cell to a tumor cell, ultimately stimulating T-cell activation, tumor killing and cytokine production. BiTEs have been developed, which target several tumor-associated antigens, for a variety of both hematological and solid tumors.

First-generation bispecific T-cell engagers (BiTEs) have encountered hurdles in the clinic related to cytokine release syndrome (CRS) and neurotoxicity. Next-generation molecules that drive effective tumor cell lysis while avoiding high levels of cytokine release may allow for wider use as single agents and in combination therapies. Faroudi et al. showed that, at low levels of TCR: pMHC engagement, T-cells are able to kill target cells before stimulation of cytokine release. Therefore, with more finely tuned binding characteristics and agonist activity for the CD3-engaging arm, a bispecific T-cell engagers (BiTEs) may more closely mimic the T-cell activation induced by natural TCR: pMHC engagement (Nathan D. Trinklein, et al. (2019) , mAbs, 11: 4, 639-652) .

WO2014/167022 discloses a bispecific antibody with a slowly internalized anti-ROR1 antibody, R12, as one arm and with an anti-CD3ε antibody as another arm. In light of the above, and given that ROR1 is a promising target in cancer treatment, there remains a need in the art to develop diversified anti-ROR1 molecules with different binding potency and/or binding sites or internalization properties, to develop diversified antibody formats, and to expand and/or improve therapeutic utility and suitability for manufacturing. To develop a bispecific antibody with more finely tuned binding characteristics for the CD3-engaging arm and high activity to kill target cells, also remains an urgent issue to be solved.

SUMMARY OF THE INVENTION

In recent years, there has been a growing interest in CD3-targeting BiTEs for cancer immunotherapy. However, challenges have emerged related to the efficacy and safety of this therapeutic modality. In addition to Tandem ScFv format (BiTEs) , Numerous formats and compositions have been described for bispecific antibody in the state of the art. Multiple variables impact the in vivo potency of these molecules, including PK, targeted antigen epitopes and the relative affinities of the antigen-binding components. However, despite all prior advances, a need remains for systematically evaluate the impact of many of these parameters and to select optimal bispecific binding molecules for cancer treatment.

This disclosure addresses the above needs by providing novel anti-ROR1 antibodies, anti-CD3 antibodies, and engineered bispecific proteins that bind both ROR1 and CD3.

In one aspect, the present disclosure provides a bispecific antibody that specifically binds to ROR1 and CD3, comprising: a first binding domain, designated B1, which is an anti-ROR1 antibody or antigen-binding fragment and a second binding domain, designated B2, which is an anti-CD3 antibody or antigen-binding fragment.

In some embodiments, the present disclosure a bispecific antibody, B2 comprises a set of CDRs of SEQ ID NO: 31, 32, 33, 34 and 35 as respectively HCDR1, HCDR2, HCDR3, LCDR1 and LCDR2, and LCDR3 as shown in any one of SEQ ID NOs: 36-40;

In some embodiments, the present disclosure a bispecific antibody, B2 comprises a set of CDRs of SEQ ID NO: 41, 42, 43, 44, 45 and 46 as respectively HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3;

In some embodiments, the present disclosure a bispecific antibody, (a) B1 comprises the CDRs of SEQ ID NO: 01, 02 and 03 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 04, 05 and 06 as respectively light chain CDR1, CDR2 and CDR3; (b) B1 comprises the CDRs of SEQ ID NO: 07, 08 and 09 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 10, 11 and 12 as respectively light chain CDR1, CDR2 and CDR3; (c) B1 comprises the CDRs of SEQ ID NO: 13, 14 and 15 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 16, 17 and 18 as respectively light chain CDR1, CDR2 and CDR3; (d) B1 comprises the CDRs of SEQ ID NO: 19, 08 and 09 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 16, 20 and 21 as respectively light chain CDR1, CDR2 and CDR3; (e) B1 comprises the CDRs of SEQ ID NO: 19, 22 and 09 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 23, 24 and 25 as respectively light chain CDR1, CDR2 and CDR3; (f) B1 comprises the CDRs of SEQ ID NO: 26, 27 and 28 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 29, 24 and 30 as respectively light chain CDR1, CDR2 and CDR3.

In some embodiments, the present disclosure a bispecific antibody, (a) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 36 as respectively light chain CDR1, CDR2 and CDR3; (b) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 37 as respectively light chain CDR1, CDR2 and CDR3; (c) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 38 as respectively light chain CDR1, CDR2 and CDR3; (d) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 39 as respectively light chain CDR1, CDR2 and CDR3; (e) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 40 as respectively light chain CDR1, CDR2 and CDR3.

In some embodiments, the present disclosure provides a bispecific antibody, B1 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NOs: 47, 49, 51, 53, 55 and 57, or a sequence having at least 80%, 85%, 90%, 95%or 99%identity thereto, and wherein the light chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NOs: 48, 50, 52, 54, 56 and 58, or a sequence having at least 80%, 85%, 90%, 95%or 99%identity thereto.

In some embodiments, the present disclosure provides a bispecific antibody, B2 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NOs: 59 and 71, or a sequence having at least 80%, 85%, 90%, 95%or 99% identity thereto, and wherein the light chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and 72, or a sequence having at least 80%, 85%, 90%, 95%or 99%identity thereto.

In some embodiments, the present disclosure provides a bispecific antibody, (a) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 47 and 48 respectively; (b) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 49 and 50 respectively; (c) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 51 and 52 respectively; (d) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively; (e) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 55 and 56 respectively; (f) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 57 and 58 respectively.

In some embodiments, the present disclosure provides a bispecific antibody, (a) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; (b) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 61 respectively; (c) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 62 respectively; (d) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 63 respectively; (e) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 64 respectively; (f) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 65 respectively; (g) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 66 respectively; (h) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 67 respectively; (i) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 68 respectively; (j) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 69 respectively; (k) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 70 respectively; (l) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 71 and 60 respectively; (m) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 71 and 68 respectively; (n) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively.

In some embodiments, the present disclosure provides a bispecific antibody, (a) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 57 and 58 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or (b) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or (c) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 47 and 48 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or (d) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 49 and 50 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or (e) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 51 and 52 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or (f) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 55 and 56 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or (g) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 68 respectively; or (h) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 70 respectively; or (i) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 71 and 60 respectively; or (j) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 71 and 68 respectively; or (k) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or (l) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 47 and 48 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or (m) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 49 and 50 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or (n) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 51 and 52 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or (o) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 55 and 56 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or (p) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 57 and 58 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively.

In some embodiments, the present disclosure provides a bispecific antibody, (a) B1 and/or B2 is an intact IgG antibody; and/or (b) B1 and/or B2 is a Fab-like fragment, optionally a Fab fragment, a Fab' fragment or a F (ab') 2 fragment; and/or (c) B1 and/or B2 is a Fv fragment, optionally a single chain Fv (scFv) fragment, or a disulphide-bonded Fv fragment.

In some embodiments, the present disclosure provides a bispecific antibody, (a) B1 is an intact IgG antibody and B2 is a scFv fragment; and/or (b) B1 is a Fab fragment and B2 is a scFv fragment; and/or (c) B1 and B2 is a scFv fragment.

In some embodiments, the present disclosure provides a bispecific antibody, (a) B1 has a valency of 2 and B2 has a valency of 2; or (b) B1 has a valency of 2 and B2 has a valency of 1; or (c) B1 has a valency of 1 and B2 has a valency of 1.

In some embodiments, the present disclosure provides a bispecific antibody is selected from the groups consisting of: (a) IgG-scFv antibodies, wherein B1 is an intact IgG and B2 is an scFv linked to B1 at the N-terminus of a light chain and/or at the C-terminus of a light chain and/or at the N-terminus of a heavy chain and/or at the C-terminus of a heavy chain of the IgG, B2 has a valency of 2; (b) central scFv (Fab2-scFv-Fc) antibodies, wherein B1 is a Fab fragment with a valency of 2 and B2 is an scFv with a valency of 1, the one B1 is attached to the N-terminus of one Fc domain to form the first monomer, and B2 is linked to the C-terminus of another B1 and the N-terminus of another Fc domain to form the second monomer; and/or (c) dual scFv (scFv2-Fc) antibodies, wherein B1 is scFv with a valency of 1, B2 is scFv with a valency of 1, B1 is linked to the N-terminus of one Fc domain to form the first monomer and B2 is linked to the N-terminus of another Fc domain to form the second monomer.

In some embodiments, the scFv is in the VH-VL orientation.

In some embodiments, the scFv is in the VL-VH orientation.

In some embodiments, the scFv is linked to the B1 or Fc domain either directly or via a linker fragment; and/or the VH of scFv is linked to the VL of scFv either directly or via a linker fragment.

In some embodiments, the linker fragment comprise (GGGGS) n, (GGGS) n or (GKPGS) n, n being a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

In some embodiments, the present disclosure provides a bispecific antibody is selected from the groups consisting of: (a) IgG-scFv antibodies, comprising two heavy chains and two light chains, and its form a homodimer, wherein, the heavy chain comprises, from amino terminus to carboxyl terminus, VH (B1) -CH; the light chain comprises, from amino to carboxyl terminus, VL (B1) -CL-linker-VH (B2) -linker-VL (B2) ; (b) central scFv (Fab2-scFv-Fc) antibodies, comprising a first heavy chain, a second heavy chain and two common light chains, and its form a heterodimer, wherein, the first heavy chain comprises, from amino terminus to carboxyl terminus, VH (B1) -CH1-Fc (version 1) ; the second heavy chain comprises, from amino to carboxyl terminus, VH (B1) -CH1-linker-VH (B2) -linker-VL (B2) -linker-Fc (version 2) ; the common light chain comprises, from amino to carboxyl terminus, VL (B1) -CL; the VH (B1) -CH1 pairs with VL (B1) -CL to form a Fab; or (c) dual scFv (scFv2-Fc) antibodies, comprising a first heavy chain and a second heavy chain, and its form a heterodimer, wherein, the first heavy chain comprises, from amino terminus to carboxyl terminus, VL (B2) -linker-VH (B2) -Fc (version 1) ; the second heavy chain comprises, from amino to carboxyl terminus, VH (B1) -linker-VL (B1) -Fc (version 2) .

In some embodiments, the present disclosure provides a bispecific antibody wherein the heavy chain of the B1 and/or B2 comprises heavy chain constant regions derived from a human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably heavy chain constant regions derived from human IgG1 or IgG4 or a variant thereof, most preferably the heavy chain constant regions comprises amino acid sequences set forth in SEQ ID NOs: 75, 76, 77, 79, 80, 81 or 82; wherein the light chain of the B1 and/or B2 comprises light chain constant regions derived from human kappa (κ) or lambda (λ) chain, or a variant thereof, preferably light chain constant regions derived from human kappa (κ) chain, most preferably light chain constant regions as shown in SEQ ID NO: 78.

In some embodiments, the present disclosure provides a bispecific antibody wherein the B1 and/or B2 comprises a first Fc domain and a second Fc domain; optionally the first Fc domain have a set of amino acid substitutions selected from S354C or/and T366W, and second Fc domain have a set of amino acid substitutions selected from Y349C, T366S, L368A or/and Y407V to achieve heterodimerization; preferably, the first Fc domain of amino acid sequences set forth in SEQ ID NOs: 75 or 82 , the second Fc domain of amino acid sequences set forth in SEQ ID NOs: 76 or 81.

In some embodiments, the present disclosure provides a bispecific antibody is selected from the groups consisting of: (a) the heavy chain as shown in SEQ ID NO: 83 and the light chain as shown in SEQ ID NO: 84; or the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 87; or the heavy chain as shown in SEQ ID NO: 89 and the light chain as shown in SEQ ID NO: 90; or the heavy chain as shown in SEQ ID NO: 91 and the light chain as shown in SEQ ID NO: 92; or the heavy chain as shown in SEQ ID NO: 93 and the light chain as shown in SEQ ID NO: 94; or the heavy chain as shown in SEQ ID NO: 95 and the light chain as shown in SEQ ID NO: 96; or the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 109; or the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 110; or the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 111; or the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 112; or the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 113; or the heavy chain as shown in SEQ ID NO: 83 and the light chain as shown in SEQ ID NO: 114; or the heavy chain as shown in SEQ ID NO: 89 and the light chain as shown in SEQ ID NO: 115; or the heavy chain as shown in SEQ ID NO: 91 and the light chain as shown in SEQ ID NO: 116; or the heavy chain as shown in SEQ ID NO: 93 and the light chain as shown in SEQ ID NO: 117; or the heavy chain as shown in SEQ ID NO: 95 and the light chain as shown in SEQ ID NO: 118; (b) the first heavy chain as shown in SEQ ID NO: 97, second heavy chain as shown in SEQ ID NO: 98 and two common light chains as shown in SEQ ID NO: 99; or the first heavy chain as shown in SEQ ID NO: 100, second heavy chain as shown in SEQ ID NO: 101 and two common light chains as shown in SEQ ID NO: 102; or the first heavy chain as shown in SEQ ID NO: 100, second heavy chain as shown in SEQ ID NO: 103 and two common light chains as shown in SEQ ID NO: 102; or the first heavy chain as shown in SEQ ID NO: 104, second heavy chain as shown in SEQ ID NO: 105 and two common light chains as shown in SEQ ID NO: 106; (c) the first heavy chain as shown in SEQ ID NO: 107 and the second heavy chain as shown in SEQ ID NO: 108; or the first heavy chain as shown in SEQ ID NO: 119 and the second heavy chain as shown in SEQ ID NO: 108.

In some embodiments, the present disclosure provides an isolated nucleic acid molecule encoding the bispecific antibody above mention; optionally operably linked to a promoter.

In some embodiments, the present disclosure provides an expression vector comprising the isolated nucleic acid molecule above mentioned.

In some embodiments, the present disclosure provides an isolated host cell transformed with the nucleic acid molecule or the vector, wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.

In some embodiments, the present disclosure provides a pharmaceutical composition, which comprises bispecific antibody above mentioned, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

In some embodiments, the present disclosure provides a method of treatment or prevention of a ROR1-expressing cancer, comprising a step of administering a therapeutically effective amount of the bispecific antibody above mentioned, or the pharmaceutical composition, to a subject in need of treatment or prevention of the ROR1-expressing cancer.

In some embodiments, the present disclosure provides a method, wherein the ROR1-expressing cancer is chronic lymphocytic leukemia (CLL) , mantle cell lymphoma (MCL) , B-cell acute lymphoblastic leukemia (B-ALL) , marginal zone lymphoma (MZL) , neuroblastoma, multiple myeloma, renal cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, head and neck cancer, bladder cancer, esophageal cancer.

Advantages of the present invention: The bispecific antibody can specifically bind to tumor antigen ROR1 in sub-nanomolar or single digital nM range, and the CD3 binding portion has medium or low binding affinity to T cells in double digital nM range, which show the robust target-dependent T cell activation to avoid systemic activation and exhaustion of T cells and tumor cells killing in vitro potency. Also, it favors binding to the tumor target ROR1 on malignant cells over CD3 on T cells in circulation and avoids CD3 sink, thus increasing drug exposure in the tumor. The bispecific antibody cause minimal level of inflammatory cytokines release, thus the therapeutic window will be increased.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Diagrams of T cell engaging bispecific antibody formats. Illustration of format M “2+2 IgG-scFv" (A) , format Q “2+1 IgG like with enhanced knob-in-hole" (B) , and format S “1+1 scFv-Fc with enhanced knob-in hole (C) . Anti-ROR1 Fab arm, anti-CD3 scFv and Fc region were shown as black, dark grey and light grey rectangles respectively.

Figure 2. Hybridoma supernatants binding to different target cells were measured by flow cytometry analysis. The mean fluorescence intensity (MFI) of different hybridoma clones binding were shown as indicated bar graphs, HT29 (A) , HCC1187 (B) and T47D (C) .

Figure 3. Representative binding histograms from flow cytometry analysis of 13A1 hybridoma clone. The solid line represents the binding to Jurkat E6.1 cell and the dash line represents the binding to J. RT-T3.5. cell. F2B was used as positive control for CD3 binding and the secondary antibody-only was used as negative control.

Figure 4. Cyno T cell binding of 13A1 hybridoma supernatants were analyzed by flow cytometry and shown as histograms. SP34 was used as positive control and clone 27A5 was used as negative control.

Figure 5. Graphs showing the T cell activation activity of selected hybridoma clone supernatants using NFAT reporter assay. F2B and OKT3 are anti-CD3 mAbs that were used as positive controls.

Figure 6. Binding curves of CD3xROR1 bispecific antibodies to both tumor and T cell lines measured by flow cytometery analysis and shown as the mean fluorescence intensity (MFI) value at indicated antibody concentration. The M format bispecific antibodies binding to Jurkat E6.1 cells and Jeko-1 cells are shown in (A) and (B) respectively, and similarly Q format bindings to these two cells lines are shown in (C) and (D) .

Figure 7. Graphs showing the T cell activation by indicated bispecific antibodies using NFAT reporter assay. Activities of M format bispecific antibodies were shown in (A) . Activities of Q format bispecific antibodies were shown in (B) .

Figure 8. Tumor antigen-dependent activation of human primary T cells mediated by CD3xROR1 bispecific antibodies. Human PBMCs were incubated with ROR1⁺ tumor cell line Jeko-1 (A and B) or ROR1^-cell THP1 (C and D) at an E-to-T ratio of 10: 1 with a serial diluation of indicated bispecific antibodiesfor 48 hours. Percentages of activated CD4 or CD8 T cells (CD69⁺CD4⁺ and CD69⁺CD4⁺) of total CD4 or CD8 T cells were shown.

Figure 9. Bispecific antibody-mediated PBMCs cytotoxicity toward tumor cells. Human PBMCs from one healthy donor were co-cultured with tumor cells at a ratio of 10: 1 with a serial dilution of bispecific antibody for two days then analyzed by flow cytometry. The live tumor cell percentages were shown as bispecific antibody dose-response curves on ROR1 ⁺ cell line Jeko-1 (A) , or ROR1 ^-cell line, K562 (B) . EC₅₀ and EC₉₀ values from different independent experiments with multiple donors were pooled and shown as in C and D respectively. Each dot represent data from an individual donor PBMCs.

Figure 10. Graphs showing the killing of solid tumor cells (Hs746T and HCC827) using Cell Titer Glo assay and obtained with controls and candidate bi-specific antibodies. M31 is the negative control, R11v9 and NVG111 are the positive controls.

Figure 11. Graphs showing the killing of Jeko-1 cells and release of various cytokines (IFNγ, IL-6, IL-2, TNFα) from a single donor PBMCs, after treatment with candidate bi-specific antibodies: M3 (panel A) , M5 (panel B) , Q3 (panel C) , Q5 (panel D) , or positive ctrl R11v9 (panel E) .

Figure 12. Graph showing the release of TNFα from PBMCs, after treatment with 10 nM of control and candidate bi-specific antibodies. Data were pooled from independent assays, and each dot represents data from one individual donor.

Figure 13 Bispecific antibody mediated cyno PBMCs activation and cytotoxicity toward Jeko-1 cell. cyno PBMCs were co-cultured with Jeko-1 cell for 48 hours at an E-to-T ratio of 10: 1 and live Jeko-1 was quantified as percentage of tumor-only control (A) . CD69⁺ population was also quantified as percentage of CD4⁺ and CD8⁺ T cell population (B, C) .

Figure 14. Pharmacokinetic (PK) property of CD3 bispecific antibodies. BALB/C mice were given a single dose (5mg/kg) bispecific antibody via i.v. injection. Serum concentration of bispecific antibody was determined by ELISA and plotted over time as indicated.

Figure 15. Bispecific antibodies inhibit tumor growth in vivo. NSG mice were inoculated Jeko-1 cells via s. c. injection, then engrafted with expanded human T cells and treated with BsAbs as indicated dosages. Tumor growth curves were shown in (A) , and excised tumor weight at Day11 were shown in (B) .

Figure 16. Bispecific antibodies inhibit tumor growth in a disseminated Jeko-1 CDX model with human PBMCs. NSG mice were inoculated Jeko-1-luc cells via i.v. injection, then engrafted with human PBMCs from health donor. The tumor bearing mice were treated with BsAbs as indicated. The bioluminescence images of the mice at Day 14 were shown in panel (A) and quantified bioluminescence signals were shown in panel B.

DETAILED DESCRIPTION

In the present invention, unless otherwise defined, the scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In addition, the laboratory operations of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are the routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

DEFINITIONS

Before the present invention is detailed below, it is to be understood that the present invention is not limited to the particular methodologies, protocols and reagents described herein, as those may vary. It is also to be understood that the terminology used herein is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims.

For interpretation of the specification, the following definitions will be applied and wherever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

A “bispecific antibody” is an antibody with binding activity for two different antigens (or different epitopes of the same antigen) . The bispecific antibodies of the present disclosure are specific to two different antigens, namely ROR1 as the first antigen and CD3 as the second antigen. The bispecific antibodies of the present disclosure comprise the first binding domain, designated B1, which is an anti-ROR1 antibody or antigen-binding fragment and a second binding domain, designated B2, which is an anti-CD3 antibody or antigen-binding fragment.

One homodimer bispecific antibody that finds particular use in the present invention is the IgG-scFv format shown in Figure 1A. In this embodiment, the format relies on the use of the C-terminal attachment of scFv to the IgG light chains, thus forming a second antigen binding domain, wherein the Fab portions of the IgG binds ROR1 and the two "extra" scFv domains bind CD3.

One heterodimeric bispecific antibody that finds particular use in the present invention is the central scFv format shown in Figure 1B. In this embodiment, the format relies on the use of an inserted scFv domain thus forming a second antigen binding domain, wherein the Fab portions of the two monomers bind ROR1 and the "extra" scFv domain binds CD3. The scFv domain is inserted between the Fc domain and the Fv-CHl region of one monomer, thus providing a second antigen binding domain.

The antibodies described herein also provide dual scFv formats in Figure 1C as are known in the art. In this embodiment, the ROR1 and CD3 heterodimeric bispecific antibody is made up of two scFv-Fc monomers.

As used herein, the term "antibody" as used herein refers to a monoclonal antibody or a fragment thereof. An antibody consists of two pairs of a "light chain" (LC) and a "heavy chain" (HC) (such light chain (LC) /heavy chain pairs are abbreviated herein as LC/HC) . The light chains and heavy chains of such antibodies are polypeptides consisting of several domains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Each light chain comprises a light chain variable domain VL and a light chain constant domain CL. The variable domains VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The "constant domains" of the heavy chain and of the light chain are not involved directly in binding of an antibody to a target, but exhibit various effector functions.

The term "antibody" includes e.g. mouse antibodies, human antibodies, chimeric antibodies, humanized antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as their characteristic properties are retained. Especially preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies.

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 a portion thereof) . It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” include (i) a Fab fragment: a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab') 2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) capable of specifically binding to an antigen. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain variable fragments (scFv) ) . Also within the present disclosure are antigen-binding molecules comprising a VH and/or a VL. In the case of a VH, the molecule may also comprise one or more of a CH1, hinge, CH2, or CH3 region. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites.

As used herein, the term "comprising" in regard to the bispecific antibody as used herein means that the bispecific antibody comprises as ROR1 and CD3 binders only those binders mentioned. Therefore a bispecific antibody according the disclosure comprising a bivalent anti-ROR1 antibody specifically binding to ROR1, and a bivalent antibody specifically binding to CD3 has in regard to ROR1 and CD3 binding only two binding valence for ROR1 and only two valence for CD3 and is therefore tetravalent. A bispecific antibody according the invention comprising a bivalent anti-ROR1 antibody specifically binding to ROR1, and a monovalent antibody specifically binding to CD3 has in regard to ROR1 binding two binding valences and in regard to CD3 binding one valence and is therefore trivalent.

As used herein, the term "peptide linker" as used within the invention may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity. Useful linkers include glycine-serine polymers, including for example (GS) n, (GSGGS) n, (GGGGS) n, and (GGGS) n, where n is an integer of at least one (and generally from 3 to 4) , glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the scFv can also comprises a positively charged scFv linker, including (GKPGS) 4.

As used herein, "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL) , and a VH domain and the first constant domain (CH1) of a heavy chain. Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1) , and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL) . This crossover Fab molecule is also referred to as crossFab (VLVH) . On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL) , and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1) . This crossover Fab molecule is also referred to as crossFab (CLCH1) . The term "Fab fragment" also includes parts or all of the hinge region, like Fab' fragment. As used herein, "F (ab) 2 fragment" refers to a bivalent monospecific antibody fragment without an Fc part. Preferably a F (ab) 2 fragment is linked at the C-terminus by disulphide bond (s) in the hinge region and usually such a "F (ab) 2 fragment" is a F (ab') 2 fragment.

As used herein, A "Fc part of an antibody" is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies. The antibodies according to the invention, which comprise an Fc part, contain as Fc part, preferably a Fc part derived from human origin and preferably all other parts of the human constant regions.

In some embodiments, a bispecific binding molecule of the present disclosure is a homodimer. In some embodiments, a bispecific binding molecule of the present disclosure is a heterotrimer (light chain combined with heavy chain heterodimer) , wherein the heavy chain heterodimer is, e.g., in a format described in Brinkmann and Kontermann, MABS 9: 182-212 (2017) . For example, a “knobs-into-holes, ” HA-TF, ZW1, CH3 charge pair, EW-RVT, LUZ-Y, Strand Exchange Engineered Domain body (SEEDbody) , Biclonic, DuoBody, BEAT, 7.8.60, 20.8.34, Triomab/Quadroma, or CrossMAb strategy may be used to promote heterodimerization (e.g., over homodimerization) of the polypeptide comprising the Fc region of the immunoglobulin in the architecture of a bispecific binding molecule of the present disclosure. In certain embodiments, a "knobs-into-holes" approach may be used, wherein a "knob" variant of a domain is obtained by replacing an amino acid with a small side chain (for example, alanine, asparagine, aspartic acid, glycine, serine, threonine or valine) with another amino acid with a larger side chain (for example, arginine, phenylalanine, tyrosine, or tryptophan) . A "hole" variant of a domain is obtained by replacing an amino acid with a large side chain (for example, arginine, phenylalanine, tyrosine, or tryptophan) with another amino acid with a smaller side chain (for example, alanine, asparagine, aspartic acid, glycine, serine, threonine or valine) . In certain embodiments, the knob and/or hole mutations are in the CH3 domain.

As used herein, the "variable domain" (variable domain of a light chain (VL) , variable region of a heavy chain (VH) ) as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the target. The domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs) . The framework regions adopt a β-sheet conformation and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the target binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.

As used herein, the terms "monoclonal antibody (mAb) " or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.

As used herein, the term "human antibody" , as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374) . Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon target challenge. Human antibodies can also be produced in phage display libraries.

As used herein, the term "recombinant human antibody" , as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

A useful comparison of CDR numbering is as below:

Note ¹: some of these definitions (particularly for Chothia loops) vary depending on the individual publication examined; Note²: any of the numbering schemes can be used for these CDR defintions, except the contact definition uses the Chothia or Martin (enhanced Chothia) definition; Note³: the end of the Chothia HCDR1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. This is because the Kabat numbering scheme places the insertions at H35A and H35B.

As used herein, the term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of a target that is bound by an antibody.

As used herein, the term "nucleic acid molecule" as used herein refers to a DNA molecule and a RNA molecule. The nucleic acid molecule may be single stranded or double stranded but is preferably a double stranded DNA. A nucleic acid is “effectively linked” when it is placed into functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.

The preparation method of the nucleic acid is a conventional preparation method in the art. Preferably, it comprises the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by the method of artificial full-length sequence synthesis.

Those skilled in the art know that the base sequence encoding the amino acid sequence of the protein can be replaced, deleted, changed, inserted or added appropriately to provide a polynucleotide homolog. The homolog of the polynucleotide of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the activity of the antibody.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked. In one embodiment, the vector is a “plasmid” that refers to a circular double stranded DNA loop into which additional DNA segment can be ligated. In another embodiment, the vector is a viral vector, wherein an additional DNA segment can be ligated into viral genome. The vectors disclosed herein are capable of self-replicating in a host cell into which they have been introduced (for example, a bacterial vector having a bacterial replication origin and an episomal mammalian vector) or can be integrated into the genome of a host cell upon introduction into host cell, thereby is replicated along with the host genome (e.g., a non-episomal mammalian vector) .

The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors, thus being constructed. The expression vector is one of a variety of conventional vectors in the art, as long as it can carry the above-mentioned nucleic acid molecule. The vector preferably includes: various plasmids, cosmids, phage or virus vectors and the like.

As used herein, the term "transfectoma" , as used herein, includes recombinant eukaryotic host cell expressing the antibody, such as CHO cells, NS/0 cells, HEK293 cells, plant cells, or fungi, including yeast cells.

The sequence of the DNA molecule for the antibody or a fragment thereof according to the present invention can be obtained by conventional techniques, for example, methods such as PCR amplification or genomic library screening. In addition, the sequences encoding light chain and heavy chain can be fused together, to form a single-chain antibody.

Once a relevant sequence is obtained, the relevant sequence can be obtained in bulk using a recombination method. This is usually carried out by cloning the sequence into a vector, transforming a cell with the vector, and then separating the relevant sequence from the proliferated host cell by conventional methods.

In addition, a relevant sequence can be synthesized artificially, especially when the fragment is short in length. Usually, several small fragments are synthesized first, and then are linked together to obtain a fragment with a long sequence.

At present, it is possible to obtain a DNA sequence encoding the antibody of the present invention (or fragments thereof, or derivatives thereof) completely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or, for example, vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention by chemical synthesis.

In general, under conditions suitable for expression of the antibody according to the present invention, the host cell obtained is cultured. Then, the antibody of the present invention is purified by using conventional immunoglobulin purification steps, for example, the conventional separation and purification means well known to those skilled in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.

The antibody obtained can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) ) . The binding affinity of a monoclonal antibody can be determined by, for example, the Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980) ) .

The antibody according to the present invention can be expressed in a cell or on the cell membrane, or is secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods according to its physical, chemical, and other properties. These methods are well known to those skilled in the art. The examples of these methods comprise, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation) , centrifugation, cell lysis by osmosis, ultrasonic treatment, supercentrifugation, molecular sieve chromatography (gel chromatography) , adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) , and any other liquid chromatography, and the combination thereof.

As used herein, the term "variant" of a polypeptide such as for example, an antigen-binding fragment, a protein or an antibody is a polypeptide in which one or more amino acid residues are inserted, deleted, added and/or substituted, as compared to another polypeptide sequence, and includes a fusion polypeptide. In addition, a protein variant includes one modified by protein enzyme cutting, phosphorylation or other posttranslational modification, but maintaining biological activity of the antibody disclosed herein, for example, binding to ROR1 and specificity. The variant may be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%identical to the sequence of the antibody or its antigen-binding fragment disclosed herein. The percent identity (%) or homology may be calculated with reference to the following description.

As used herein, the term "K_D" (M) , as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. "K_D" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M) . K_D values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the K_D of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a system, or by using bio-layer interferometry technology, such as an Octet RED96 system.

As used herein, the term "affinity" is the strength of interaction between an antibody or its antigen-binding fragment and an antigen, and it is determined by properties of the antigen such as size, shape and/or charge of antigen, and CDR sequences of the antibody or antigen-binding fragment. The methods for determining the affinity are known in the art, and the followings can be referred.

As used herein, the term "EC₅₀" refers to the concentration for 50%of maximal effect, i.e. the concentration that can cause 50%of the maximal effect.

As used herein, the term "Pharmaceutical composition" , as used herein, is intended to refer to a mixture containing one or more of the compounds or a physiological/pharmaceutically acceptable salt or prodrug thereof described herein with other chemical components, such as physiological /pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and exerts the biological activity.

As used herein, the term "Administration" and "treatment" , when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact with an exogenous pharmaceutical, therapeutic, diagnostic reagent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contacting the cell with a reagent, as well as contacting a fluid with a reagent, wherein the fluid is in contact with the cell. "Administration" and "treatment" also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition or by another cell. “Treatment, ” when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

EXAMPLE

The invention is further illustrated by the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The experimental methods without detailed conditions in the following examples are generally in accordance with the conditions described in the conventional conditions such as Sambrook. J et al. "Guide to Molecular Cloning Laboratory" (translated by Huang Peitang et al., Beijing: Science Press, 2002) , or in accordance with the conditions recommended by the manufacturer (for example, product manuals) . Percentages and parts are by weight unless otherwise stated. The experimental materials and reagents used in the following examples are commercially available unless otherwise specified.

The room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30℃.

EXAMPLE 1: Immunization and Screening of Antibody

Preparation of immunogen

Design of ROR1 antigen: A combination of recombinant protein antigen huROR1-Fc (R&D systems, catalog number 9490-RO) , engineered huROR1-HEK293 cell, and HT-29 cells (ATCC, catalog number HTB38) were used to immunize mice. For the engineered huROR1-HEK293 cell line, HEK293 cells were transduced by lentivirus containing a gene sequence encoded for human ROR1 ectodomain and a puromycin resistance gene. A single clone from the transduced HEK293 cells with highest huROR1 expression was selected.

Design of CD3 antigen: A combination of Jurkat E6.1, DNA and recombinant protein antigens were used to immunize mice. The recombinant ectodomain of human CD3E and CD3D were expressed and purified as heterodimer and were commercially available at AcroBiosystems (catalog numbers CDD-H52Wa, CDD-H52W1, CDD-C52W4, CDD-C52W9) . Either Fc, His Tag&Fc, Flag Tag or His Tag&Tag Free were used.

Immunization

Anti-ROR1 antibodies:

Antibodies were obtained by immunizing genetically modified mouse encoding human immunoglobulin heavy and kappa light chain variable regions with a combination of recombinant protein antigen huROR1-Fc (R&D systems, catalog number 9490-RO) , engineered huROR1-HEK293 cell, and HT-29 cells (ATCC, catalog number HTB38) . The antibody immune response was monitored by a ROR1-specific immunoassay. When a desired immune response was achieved splenocytes were harvested from each mouse and fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for ROR1 specificity.

Anti-CD3 antibodies

antibodies were obtained by immunizing genetically modified mouse encoding human immunoglobulin heavy and kappa light chain variable regions with a combination of human CD3 antigen, cynomolgus CD3 antigen, human CD3 DNA, and/or Jurkat E6.1 cells (Sigma-Aldrich, Catalog No. 88042803) which naturally express human CD3. The antibody immune response was monitored by a CD3-specific immunoassay. When a desired immune response was achieved splenocytes were harvested from each mouse and fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for CD3 specificity.

Spleen cell fusion

The spleen lymphocytes and myeloma cells Sp2/0 (CRL-158) were fused to obtain hybridoma cells by electrofusion or PEG fusion. PEG fusion was performed using ClonacellTM HY technology (STEMCELL technologies) , following manufacturer’s instructions. The primary cell: mouse myeloma cell line ratio was 1: 1 for electrofusion, and 10: 1 for PEG fusion.

Screening of hybridoma clones specifically binding to human ROR1 protein by ELISA

ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . ELISA plates were coated with 1μg/ml of human ROR1-Fc (9490-R0-050) , human ROR2-Fc (8609-RO-050) or BSA overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50μl ROR1 hybridoma supernatant was added in duplicate wells and incubated for 1 hour. Excess unbound antibodies were washed off and 50μl of 1: 30000 diluted secondary antibody Goat anti-mouse IgG Fc-HRP (ab5870) was added to each well for another 1 hour. Plates were washed before the addition 50 μL of chemiluminescence agents (color A and color B) according to manufacturer's protocol. The reactions were stopped using 25μL of 2N sulfuric acid. Optical density at 450nm of samples was measured by a microplate reader (PerkinElmer) . All tested clones bound selectively to human ROR1 but not to human ROR2 nor BSA, demonstrating ROR1 specificity.

Table 1. Binding characterization of hybridoma clones to human ROR1 and human ROR2 by ELISA assay (OD₄₅₀)

Screening of hybridoma clones specifically binding to ROR1 expressing cancer cells by Flow cytometry

Hybridoma supernatants were subjected to binding tests on ROR1⁺ cell line HT29 (ATCC, HTB-38) , HCC1187 (ATCC, CRL-2322) , and ROR1^-ROR2⁺ cell line T47D (ATCC, HTB-133) using flow cytometry analysis. Briefly, 50 μL of cells in cell staining buffer (2×10⁶ cells/mL) was mixed with 50 μL undiluted hybridoma supernatants. The mixture was incubated on ice for 20 min and then washed with ice cold staining buffer twice. The cells were subsequently stained with 50 μL of PE labelled secondary antibody (1: 400 dilution, BioLegend, Cat#405307) for 20 min. After washing by staining buffer and fixing by 4%PFA, cells were analyzed by flow cytometry. A purified anti-human ROR1 antibody was used as positive control (BioLegend, Cat#357802) . A purified mouse IgG1 antibody was used as isotype control (Biolegend, Cat#400102) . Figure 2 shows examples of selected cell binding signals measured by flow cytometry. The clones 8B10, 15G4, 29D4, 31G8, 37F9, 38H10 were identified with enhanced binding profile to HT29 cells and HCC1187 cells compared to T47D cells.

Screening of hybridoma clones specifically binding to human/cynomolgus CD3 protein by ELISA

ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . ELISA plates were coated with 5ug/ml of human CD3δε (AcroBio CDD-H52W1) and cyno CD3δε (AcroBio, CDD-C52W4) overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50ul CD3 hybridoma supernatant was added in duplicate wells and incubated for 1 hour. Excess unbound antibodies were washed off and 50ul of 1: 1000 diluted secondary antibody Goat anti-mouse IgG Fc-HRP (Jackson ImmunoResearch 115-035-071) was added to each well for another 1 hour. Plates were washed before the addition of chemiluminescence agents (color A and color B) according to manufacturer's protocol. Optical density at 450nm of samples were measured by microplate reader (PerkinElmer) .

Table 2. The binding activity results of ELISA assay

The results show that the supernatant of hybridoma clone 13A1 antibody of the present disclosure have high affinity to the human and cynomolgus CD3.

Jurkat Binding Screening

Hybridoma supernatants were subjected to binding tests on human T cell lines (CD3⁺ Jurkat cell line clone E6.1 and CD3^-clone J. RT-T3.5 (ATCC) using flow cytometry analysis. The primary staining was performed using undiluted hybridoma supernatants, followed by fluorescence labeled secondary antibody (PE Goat anti-mouse IgG, Biolegend Cat#405307) . Figure 3 shows examples of selected flow cytometry histograms. The clone 13A1 was identified with enhanced binding profile to Jurkat E6.1 compared to J. RT-T3.5 cells.

Cyno-T cell (HSC-F) Binding

Selected clones from Jurkat cell binding screening were further tested for cynomolgus monkey T cell binding (HSC-F) using flow cytometry analysis. Cynomolgus monkey T cell line (HSC-F) was obtained from Non-Human Primate Reagent Resource (NHPRR) . The staining was performed in a similar fashion as described above. Purified anti-CD3 antibodies F2B and SP34 are the positive control. Representative results are shown in Figure 4.

T cell activation (NFAT Reporter Assay)

T cell activation activity of hybridoma supernatants or recombinant anti-CD3 antibodies were measured using NFAT reporter assay, using Jurkat-Lucia^TM NFAT cells (InvivoGen jktl-nfat) . U-bottom 96-well plates were dry coated with capturing antibody (goat anti-mouse IgG Fcγfragment specific, Jackson ImmunoResearch 115-005-071) , at 4μg/ml in PBS and 50 μl/well. Hybridoma supernatants diluted in culture medium were added to the pre-coated plate at 50 μl/well for overnight. The Jurkat-Lucia^TM NFAT cells were added to each well at 400,000 cells per well in 200ul culture medium. The cells were maintained at 37℃ with 5%CO₂ for 24 hours, then the culture supernatants were collected. Luciferase activity in culture supernatant was measured using QUANTI-Luc assay solution (InvivoGen) according to manufacturer’s instruction. Luminescence signal was recorded by VICTOR Nivo Multimode Microplate Reader (PerkinElmer) . Purified anti-CD3 antibodies F2B and OKT3 were used as positive controls. Supernatant from another hybridoma clone was used as a negative control. Clone 13A1 was selected for their ability to active human T cells indicated by the in vitro reporter assay as shown in Figure 5.

EXAMPLE 2 Sequencing of Positive Hybridoma Clones

The process of cloning sequences from positive hybridomas are as follows. The logarithmic growth phase hybridoma cells were collected, RNA was extracted, and reverse transcription was performed then followed by VDJ region amplification. Amplified cDNA library from each clone were subjected to next-generation sequencing. The amino acid sequences of the heavy and light chain variable region DNA sequences corresponding to the antibodies 8B10, 15G4, 29D4, 31G8, 37F9, and 38H10 and 13A1 were obtained. Upon the manufacturability assessment evaluation, several mutations were made in the FR region, and the amino acid sequence of heavy chain variable and light chain variable regions and CDR sequence of each antibody are as the following tables. The amino acid residues of the CDRs in VH/VL are numbered and annotated according to the Kabat &Wu definition system.

Table 3. CDR sequence of heavy chain variable domain for ROR1 hybridoma clones

Table 4. CDR sequence of light chain variable domain for ROR1 hybridoma clones

Table 5. CDR sequence of heavy chain variable domain for CD3 hybridoma clones

Table 6. CDR sequence of light chain variable domain for CD3 hybridoma clones

Table 7. Sequences of heavy chain and light chain variable domains for ROR1 hybridoma clones

Table 8. Sequences of heavy chain and light chain variable domains for CD3 hybridoma clones

Example 3 Construction, Expression and Purification of Bispecific Antibody

To identify the structure that provides the optimal configuration, specifically the most active relative spatial placement of the ROR1 and CD3 binding components, a variety of constructs were designed as summarized in FIG. 1.

ScFv Generation:

To create a single chain antibody (scFv) , the VH and VL of CD3 are operatively linked to a flexible linker (SEQ ID NO: 120, 122) or a positively charged linker (SEQ ID NO: 121) , and in some format the scFv is fused to the ROR1 antigen-binding fragment via another peptide linker (SEQ ID NO: 124, 125) to fuse to ROR1 CH1 and Fc, or SEQ ID NO: 123 to fuse to ROR1 CL, to form a fusion polypeptide.

Table 9. Sequences of the peptide linker

For certain constructs, use of antibody Fc with LALA mutation sequences enabled the evaluation of bispecific constructs that are bivalent for both ROR1 and CD3 (FIG. 1A) . For other constructs, the use of knobs-into-holes mutations together with silencing mutations in the Fc region enabled the expression of bispecific binding molecules with different valencies for ROR1 and CD3. For example, one construct is bivalent for ROR1 and monovalent for CD3 (FIG. 1B) while another construct is monovalent for both ROR1 and CD3 (FIG. 1C) .

Finally, ROR1 paratopes with altered affinities and valencies were tested in combination with the CD3 paratopes to evaluate the effect of CD3 and ROR1 paratopes with differing relative affinities or avidities. Based on these design parameters, multiple bispecific constructs were expressed, purified and characterized.

Table 10. Summary of bispecific construct engineering

The antigen-binding fragment (B1) that specifically binds to ROR1 and the antigen-binding fragment (B2) that specifically binds to CD3 are linked in different ways to obtain a bispecific antibody containing the following sequence, the constant light chain (CL) of the bispecific antibody according to the invention is human kappa (κ) type:

The IgG-scFv antibodies are constructed as follows (ordered from amino terminus to carboxyl terminus) (the structure is shown in Figure 1A) :

the heavy chain: VH (B1) -CH (IgG4) ;

the light chain: VL (B1) -CL-linker-VH (B2) -linker-VL (B2) ;

The central scFv antibodies are constructed as follows (ordered from amino terminus to carboxyl terminus) (the structure is shown in Figure 1B) :

the first heavy chain: VH (B1) -CH1-Fc (IgG1 Fc hole) ;

the second heavy chain: VH (B1) -CH1-linker-VH (B2) -linker-VL (B2) -linker-partial Fc (IgG1 partial knob) ;

the light chain: VL (B1) -CL;

The dual scFv antibodies are constructed as follows (ordered from amino terminus to carboxyl terminus) (the structure is shown in Figure 1C) :

the first heavy chain: VL (B2) -linker-VH (B2) -Fc (IgG1 knob N297A) ;

the second heavy chain: VH (B1) -linker-VL (B1) -Fc (IgG1 hole N297A) .

Table 11. Sequences of IgG constant regions and Fc domains

Molecular cloning of recombinant antibodies:

The cDNA sequences that encode VH and VL regions of designed bispecific antibodies were directly synthesized as DNA fragments with 5’-end in-frame leader sequence (MGWSCIILFLVATATGVHS) . These DNA fragments were cloned into mammalian expression vectors (with backbone sequence similar to pcDNA3.1) , using NEBuilder DNA Assembly Cloning Kit (New England Biolabs) . The constant regions of both M and Q format of bispecific antibodies adopt hIgG4 sequences, with engineered mutations (S228P, F234A, L235A) in the M format, and knob-in-hole mutations in the Q format.

Expression and purification of recombinant antibodies:

The heavy chain expression plasmid and light chain plasmids were co-transfected into Expi293F cells (ThermoFisher, #A14527) using the ExpiFectamine 293 Transfection Kit (ThermoFisher, A14524) , or into ExpiCHO-S cells (ThermoFisher #A29127) using the ExpiFectamine CHO Transfection Kit (ThermoFisher, A29129) . Based on the manufacturer’s instructions, plasmid DNA concentration reached 1.0 ug per ml of suspended cells, with LC: HC vector ratio 3: 2 for the M format of bispecific antibodies, or LC: HC1: HC2 vector ratio 2: 1: 1 for the Q format of bispecific antibodies. The transfected cells were cultured 5 to 7 days on an orbital shaker at 37 ℃, 8%CO₂. Conditioned medium was collected and antibodies were purified using HiTrap MabSelect SuRe column (Cytiva, #17549112) on AKTA Pure 25 machine (Cytiva) . Eluted antibodies were neutralized with Tris Buffer (pH 9.0) and subjected to PBS buffer exchange. The concentrated proteins were further purified using SEC-superdex 200 column (Cytiva, #28989335) on AKTA Pure 25 machine. The peak fractions were pooled and concentrated. The final products were quantified by UV absorption, and quality was determined by SDS-PAGE and HPLC.

Table 12. Summary of bispecific construct sequences

Example 4 The CD3 ScFv Engineering Mutations

The purpose of the mutation is to obtain a group of anti-CD3 antibodies with varied affinities and T cell activation activities in addition to potentially lower immunogenicity by converting certain residues to the germline sequence.

Selection of a template and mutation design for hybridoma clone 13A1-1

Table 13. the mutations of 13A1-1 light chain and heavy chain varable regions

NOTE: For example, I14T indicates a mutation from I to T at position 14 according to Kabat numbering system. WT indicates that the sequence does not contain amino acid mutations.

Table 14. the sequence of 13A1-1 mutations CDR regions and light chain and heavy chain variable regions

Table 15. Sequences of heavy chain and light chain variable domains for CD3 hybridoma clones

The method of anti-ROR1/anti-CD3 T cell bispecific antibodies generation are described in Example 3.

Table 16. Summary of bispecific construct sequences

Example 5 Basic Antigen Binding Properties of T cell Engaging Bispecific Antibody (TCE)

The binding of antibodies to soluble CD3δε and ROR1 antigens:

The binding affinity of CD3xROR1 bispecific antibodies with recombinant CD3δε-His (Acro Biosystems Cat#CDD-H52W1) and ROR1-His (Acro Biosystems Cat#RO1-H522y) protein were determined by Octet (Octet Red 384) instrument. A kinetic experiment was performed to look at binding and dissociation to antigens. Briefly, antibodies were immobilized onto the anti-hIgG Fc Capture (AHC) Biosensors by dipping the probe into wells containing 0.75ug/ml antibody. Non-bound antibodies were washed off by dipping the probe into buffer-only wells. Antigen association was performed in the wells containing serial dilution of CD3δε-His (1500nM-23nM) and ROR1-His (600nM-0.82nM) for 10min. Bound antigens were allowed to dissociate in the buffer-only wells for 30min. Association-dissociation curves were generated, and the global fit was performed using the 1: 1 binding model. K_D values were calculated and shown in Table 17.

Table 17. K_D values of CD3xROR1 bispecific antibodies

ND = not defined

As expected, bispecific antibodies have weaker binding to CD3 than ROR1. M format has stronger binding to CD3 than Q format because it contains two CD3 binding sites compared to Q format, which only has one. All four bispecific antibodies show similar binding affinity to ROR1.

Cell binding of CD3xROR1 bispecific antibodies:

Each arm of CD3xROR1 bispecific antibodies was functionally tested for its binding to cell surface CD3 or ROR1 in a cell-based binding assay. CD3 positive T cell, Jurkat E6.1, and ROR1 positive cancer cell, Jeko-1, were used in the flow cytometry experiment. Briefly, antibodies were serially diluted from 1000nM all the way down to 0.001nM. 0.3 million cells were dispensed into each well in duplicate. 60ul antibody was added to each well and stained the cell for 1hour at 4℃ in dark. Cells were washed once with 200ul PBS. The secondary antibody, goat anti-human IgG (Jackson ImmunoResearch Cat 109-116-170) conjugated to R-phycoerythrin (PE) was diluted 1: 200 and 60ul was used for staining. Flow cytometry experiment was carried out subsequently to measure the PE signal. Purified anti-CD3 antibodies 20G6 and F2B are the positive control. Prism Graphpad was used to generate lines of best-fit (Figure 6) and EC₅₀ was calculated (Table 18 and Table 19) .

Table 18. EC₅₀ of CD3xROR1 bispecific antibody M format

Table 19. EC₅₀ of CD3xROR1 bispecific antibody in Q format

The results above show both M and Q format led to decreased binding affinity to CD3 compared to 13A1-1 parental mAb, while ROR1 binding remains intact.

Example 6 Effect of the Bispecific Antibodies on human T cell line or PBMCs Activation.

Anti-ROR1/anti-CD3 T cell bispecific antibodies generated in Example 3 and 4 were analyzed by NFAT reporter assay and flow cytometry based for their potential to induce T cell activation in a similar fashion as described in Example 1.

NFAT reporter assay:

To assess the potential of Bispecific antibodies to trigger T-cell activation, 96 well U-bottom plates were dry coated with 10 ug/ml capturing antibody (Jackson ImmunoResearch’s AffiniPure goat anti-human IgG, Fcγ specific, #109-005-170) , and then after PBS wash, were wet coated with bispecific antibodies (1: 5 serial dilution, up to 67 nM) . After PBS wash, NFAT reporter cells (InvivoGen #jktl-nfat) were added to each well at 0.4 million/well in RPMI 1640, with 10%FBS. After 24 hours of incubation at 37 ℃, 5%CO₂, reporter activity was measured using QUANTI-Luc assay (InvivoGen) following manufacture’s instruction. Luminescence was measured using Victor Nivo multimode microplate reader (PerkinElmer) , and data were analyzed by GraphPad Prism 9 software. The effect of bispecific antibodies on T cell activations were shown in Figure 7A and 7B, and EC₅₀ values were shown in Table 20.

Table20. EC₅₀ values of control and candidate M format bispecific antibodies, activating the NFAT reporter cells

Primary human T cells activation assay:

CD3xROR1 bispecific antibodies were tested for their ability to activate the human primary T cells. Human PBMCs from a health donor were co-cultured with a ROR1⁺ cell line (Jeko-1) or a ROR1^-cell line (THP1) at an effector-to-tumor ratio (E-to-T) of 10: 1 for 48 hours in the presence of antibody. Cells were washed with 1x PBS and anti CD4-APC (BioLegend Cat#317416, clone OKT4) , anti CD8-PE (Invitrogen Cat#12-0088-42 clone RPA-T8) , and anti CD69-BV506 (BioLegend Cat#310938, clone FN50) antibodies were used to stain CD4⁺CD69⁺ and CD8⁺CD69⁺T cell populations. Flow cytometery data were acquired on BD Fortessa X20. Data were analyzed by FlowJo software. Prims Graphpad was used to plot the CD69⁺ population as a percentage of total CD4 or CD8 T cell. EC₅₀ values were calculated and tabulated in Table 21 and 22.

Robust activation of human primary CD4 and CD8 T cells by bispecific antibodies were observed only in the presence of Jeko-1 cell, which is ROR1 positive (Figure 8A and 8B) , but not the ROR1 negative THP-1 cells (Figure 8C and 8D) . These results indicated the bispecific antibodies can only active T cells when tumor antigen was engaged. As expected, 13A1-1 and Mut-36 monoclonal antibody controls (mabs) , which recognize CD3 only can active T cells equally well with either cell lines, independent of tumor antigen.

Table 21. EC₅₀ values of human primary T cells activation by bispecific antibodies in the presence of Jeko-1 cells

Table 22. EC₅₀ values of human primary T cells activation by bispecific antibodies in the presence of THP-1 cells

Example 7 Redirected human PBMCs or amplified T cells Cytotoxicity of Bispecific Antibodies

ROR1xCD3 bispecific antibodies were tested for their cytotoxicity toward ROR1⁺ tumor cell lines in a co-culture experiment with human primary immune cells.

PBMCs co-culture cytotoxicity assays on suspension tumor cell lines:

Tumor cells in suspension were labeled with 5uM CellTrace Violet dye (ThermoFisher Cat#C34557) for 20min according to manufacturer’s instructions. Human PBMCs from health donors were isolated from whole blood using lymphocyte separation medium (Corning Cat#25-072-CV) according to manufacturer’s instruction. Live PBMCs and tumor cells were counted and mixed in an E-to-T ratio of 10: 1. Antibodies were serially diluted, with a final starting concentration of 10nM. R11v9 is the positive control, which is the ROR1-targeting scFv in R11v9. The R11 is disclosure in patent WO2012/075158. The cells were incubated with antibodies at 37℃ for 48 hours. At the end of the incubation, cells were stained with Invitrogen^TM LIVE/DEAD^TM Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific Cat#L10119) according to manufacturer’s instructions, and analyzed with BD Fortessa X20 flow cytometer. Live tumor cell numbers of each well are normalized to wells without PBMCs.

Bispecific antibodies M5 and Q5 can effectively mediated Jeko-1 tumor cells killing by activated PBMCs (Figure 9A) , while show minimal cytotoxicity toward ROR1^-K562 cells under the same condition (Figure 9B) . The results from multiple donors were summarized in Figure 9C and 9D.

Table 23. EC ₅₀ values of PBMC-mediated Jeko-1 killing of different ROR1 clones

Cytotoxicity On Solid Tumor Cells

Human T cells were expanded from frozen pan human CD3⁺ T cells from healthy donors (Stemexpress) . In brief, 4 x 10⁷ cells were seeded with Dynabeads-Human T-Activator CD3/CD28 (Gibco, REF 11131D) at a ratio of 2: 1 in RPMI with L-Glutamine, 10%FBS, 1%Pen-Strep, 0.2%Plasmocin and 30 U/ml IL-2 (Pepotech, Cat#200-02 -500UG) . Fresh media added everyday up to 7 to 9 days or 14 days until the harvesting. The beads were removed, and the cells were cultured for one additional day without the beads before cytotoxicity assays.

Killing of solid tumor cells were measured using Cell Titer Glo assay (Promega’s Cell Titer Glo 2.0 kit, #G9242) . Solid tumor cells were seeded on opaque-walled 96-well plates (Corning #3610) , at 5,000 cells in 75 ul volume for each well and cultured overnight. On the next day, bi-specific antibodies diluted in culture medium were added to the plate at 50 ul/well, followed by addition of activated T cells at 25,000 cells in 75 ul volume of medium for each well. The cells were maintained at 37℃ with 5%CO2 for 48 hours. T cells in each well were then washed off with 200 ul of PBS. Subsequently, 100 ul of PBS and equal volume of Cell Titer reagent were added to each well and the adherent tumor cells were fully lysed at room temperature within 10 minutes. Luciferase activity in lysis buffer was measured according to manufacturer’s instruction. Luminescence signal was recorded by VICTOR Nivo Multimode Microplate Reader (PerkinElmer) . The killing effect of bispecific antibodies on solid tumor cell line Hs746T and Hcc827 were shown in Figure 10A and 10B respectively, and IC₅₀ values were summarized in Table 24.

Table 24. IC₅₀ values of bispecific antibodies killing selected solid tumor cells

Example 8 Quantitation of Cytokine Release from Activated T cells upon Engagement of Bispecific Antibody

Anti-ROR1/anti-CD3 T cell bispecific antibodies generated in Example 3 and 4 are analyzed for their potential to induce T-cell mediated cytokine production.

Suspension tumor cells were co-cultured with PBMC cells (EtoT=10: 1) in U-bottom 96-well plates, at 37℃ with 5%CO2 for 48 hours. After spinning down the cell pellets, supernatant from each well were transferred to V-bottom 96-well plates. Release of cytokines (including TNFα, IFN γ) from activated T cells were measured using LEGENDplex Muliplex Assay (Biolegend’s LEGNEDplex kit, HU Essential Immune Response Panel 13-plex, #740930) according to manufacture's instructions. Data were acquired and analyzed on iQue3 (Sartorius) Individual cytokine level of each sample was interpolated from standard curve using Prism (Graphpad) software.

Results showed minimal levels of inflammatory cytokines (IL-2, IFNγ, IL-6, TNFα) released upon bispecific antibodies mediated T cell activations, with better therapeutic window than Ref Ab, as shown in Figure 11 and Figure 12.

Example 9 Effect of bispecific antibodies on cynomolgus monkey primary PBMCs activation and cytotoxicity toward tumor cells

CD3xROR1 bispecific antibodies were also tested for their ability to activate primary cynomolgus monkey T cells. Cyno PBMCs (iQ Bioscience Cat#IQB-MnPB102) were co-cultured with Jeko-1 at an E-to-T ratio of 10: 1 for 48 hours in the presence of antibody.

Anti-CD4-APC (BioLegend Cat#317416, clone OKT4) , anti CD8-PE (Invitrogen Cat#12-0088-42 clone RPA-T8) , and anti CD69-BV506 (BioLegend Cat#310938, clone FN50) antibodies were used to stain CD4⁺ CD69⁺ and CD8⁺ CD69⁺ T cell populations. Flow cytometry experiment was performed to count the CD69 positive population of T cells. Prims Graphpad was used to plot the CD69⁺ population as a percentage of total CD4 or CD8 T cells.

Bispecific antibodies can mediate robust tumor cells killing by cyno PBMCs (Figure 13A) , and activated primary cyno T cells in the presence of tumor antigens (Figure 13B and 13C) . EC₅₀ values were calculated and tabulated in Table 25 and 26.

Table 25. EC₅₀ values of tumor cells killing by cyno PBMCs

Table 26. EC₅₀ values of cyno T cells in the presence of tumor antigens

Example 10 In vivo Mouse PK study of Bispecific Antibody

To determine the serum half-life of CD3 bispecific antibodies in mice, a single dose of M5, M5-Mut36 or Q5v2 at 5mg/kg were intravenously administered into BALB/c mice. Serum was collected at 1hr, 4hr, 24hr, 48hr, 72hr, 120hr, 168hr, 336hr, and 504hr post injection, each time point from 3 mice. ELISA assay was performed to determine the concentration of antibody at each time point.

Briefly, high-binding ELISA plates were coated with 1ug/ml ROR1-His (Acro Biosystems Cat#RO1-H522y) overnight at 4℃. Plates were washed 3x with 120ul PBST buffer and blocked in 1x BSA solution for 1hour. Serial dilution of standards was performed, with known concentration of antibody ranging from 800ng/ml to less than 1 ng/ml. Quality control (QC) samples at 350ng/ml, 150ng/ml, 40ng/ml, and 10ng/ml were included to ensure standard curves is capable of accurately determine the concentration at this range. Appropriate pre-bleed mouse serum was included in both standards and QC samples to ensure matrix effects were accounted for. Unknown samples were diluted 1: 1000 and/or 1: 100. Plates were washed in PBST after blocking and 30ul of standards, QC and unknown samples were incubated on the plate for 1 hour. The secondary antibody goat anti-human IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Cat#109-035-008) was diluted 10,000-fold and 30ul was used for staining after the primary antibody. Plates were developed for 10 minutes in chemiluminescence reagent and subsequently read in i3x plate reader (Molecular Devices) . Serum concentration of each time point was calculated using Softmax Pro 7 software (Molecular Devices) , shown in Figure 14. PK solver was used to fit the data point using non-compartmental analysis. PK parameters were calculated and summarized in Table 27.

Table 27. Summary of mouse PK parameters of M5, M5_Mut36 and Q5v2

The result shows the serum half life of M5, M5_Mut36 and Q5v2 in mice are ～2, 6 and 3 days respectively.

Example 11 Evaluation of Therapeutic Efficacy of Bispecific Antibody in JeKo-1 Xenograft M-NSG Mouse Model

For 6-to 8-week-old NOD-scid-IL2Rgnull (NSG) mice (The Jackson Laboratory) were injected via s. c. in the flank with a 2x10⁶ Jeko-1 tumor cells at Day-20. The animals were randomized into 5 treatment groups on day 0 with 5 mice per group and the mean tumor volume is ～150mm³, then each mouse was i.v. injected (tail vein) with 10 x 10⁶ primary T cells expanded from health donor PBMCs and bispecific antibodies (10ug/mouse or 1ug/mouse) or PBS.

The mice received a total of three doses of expanded primary T cells every 8 days and a total of six doses of bsAbs or PBS solution alone every 4 days. Body weight and tumor volume were measured twice weekly. Tumor volume was measured by using calipers and calculated by the following formula: volume = (length x width²) /2. The animal were euthanized when the tumor size exceeded 2000mm³, and tumors were excised and weighted. The animal were euthanized when the tumor size exceeded 2000mm³, and tumors were excised and weighted.

Compared with vehicle control group, M5_Mut36 treated groups show slower tumor growth (Figure 15A) and lower tumor weight (Figure 15B) . These results suggested that M5_Mut36 can activate engrafted human T cells and inhibit tumor growth in vivo.

The mice also received a total of three or four doses of expanded primary T cells every 4 days and a total of six doses of bsAbs or PBS solution alone every 4 days. Body weight and tumor volume were measured every 2 days.

Compared with vehicle control group, all treatment groups show tumor inhibition, M5-Mut36 exhibit better efficacy. TGI value of M5-Mut36 is between 80%-100%, greater than positive control 30-60%.

The bispecific antibodies were also evaluated in a disseminated tumor model. In brief, On day 1-, 6-to 8-week-old female NCG mice were injected intravenously (tail vein) with 2 x 10⁶ human PBMCs from health donor. One day later, 2 x 10⁶ Jeko-1-Luc cells were i.v. injected (tail vein) into each mouse. The treatments were initiated on day 6, and bispecific antibodies or PBS were administered intravenously (tail vein) with 6 mice per treatment group. Each mice received a total 6 dosages of BsAb at 1ug/mouse or 10ug/mouse every 4 days. Tumor growth was monitored weekly by bioluminescent imaging 5 min after i. p. injections with 150 mg/kg D-luciferin. Luciferase activity was analyzed by using Living Image Software, and the photon flux was analyzed within regions of interest that encompassed the entire body of each individual mouse. The weight of the mice was measured every 4 days.

All bispecific antibodies exhibited robust anti-tumor activity, which evidenced by significant lower tumor burden measured by bioluminescent in all treatment groups compared with vehichle control group (Figure 16A) . High dosage treatment groups (10ug) show more prolonged tumor growth inhibition than low dosage groups (1ug) (Figure 16B) . These results demonstrated that ROR1xCD3 bispecific antibodies, M5, M5-Mut36 and Q5v2, have in vivo anti-tumor efficacy.

Claims

A bispecific antibody comprising a first binding domain, designated B1, which is capable of specifically binding to ROR1, and a second binding domain, designated B2, which is capable of specifically binding to CD3, the first and/or second binding domains are selected from the group consisting of: antibodies or antigen binding fragments thereof.
The bispecific antibody according to claim 1, wherein B2 comprises a set of CDRs of SEQ ID NO: 31, 32, 33, 34 and 35 as respectively HCDR1, HCDR2, HCDR3, LCDR1 and LCDR2, and LCDR3 as shown in any one of SEQ ID NOs: 36-40;

and wherein:

(a) B1 comprises the CDRs of SEQ ID NO: 01, 02 and 03 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 04, 05 and 06 as respectively light chain CDR1, CDR2 and CDR3;

(b) B1 comprises the CDRs of SEQ ID NO: 07, 08 and 09 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 10, 11 and 12 as respectively light chain CDR1, CDR2 and CDR3;

(c) B1 comprises the CDRs of SEQ ID NO: 13, 14 and 15 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 16, 17 and 18 as respectively light chain CDR1, CDR2 and CDR3;

(d) B1 comprises the CDRs of SEQ ID NO: 19, 08 and 09 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 16, 20 and 21 as respectively light chain CDR1, CDR2 and CDR3;

(e) B1 comprises the CDRs of SEQ ID NO: 19, 22 and 09 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 23, 24 and 25 as respectively light chain CDR1, CDR2 and CDR3;

(f) B1 comprises the CDRs of SEQ ID NO: 26, 27 and 28 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 29, 24 and 30 as respectively light chain CDR1, CDR2 and CDR3.
The bispecific antibody of according to claim 2, wherein:

(a) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 36 as respectively light chain CDR1, CDR2 and CDR3;

(b) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 37 as respectively light chain CDR1, CDR2 and CDR3;

(c) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 38 as respectively light chain CDR1, CDR2 and CDR3;

(d) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 39 as respectively light chain CDR1, CDR2 and CDR3;

(e) B2 comprises the CDRs of SEQ ID NO: 31, 32 and 33 as respectively heavy chain CDR1, CDR2 and CDR3 and the CDRs of SEQ ID NO: 34, 35 and 40 as respectively light chain CDR1, CDR2 and CDR3.
The bispecific antibody of according to any one of claims 1-3, wherein: B1 comprises a heavy chain variable domain and a light chain variable domain,

wherein the heavy chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NOs: 47, 49, 51, 53, 55 and 57, or a sequence having at least 80%, 85%, 90%, 95%or 99%identity thereto, and wherein the light chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NOs: 48, 50, 52, 54, 56 and 58, or a sequence having at least 80%, 85%, 90%, 95%or 99%identity thereto.
The bispecific antibody of according to any one of claims 1-4, wherein: B2 comprises a heavy chain variable domain and a light chain variable domain,

wherein the heavy chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NOs: 59 and 71, or a sequence having at least 80%, 85%, 90%, 95%or 99%identity thereto, and wherein the light chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and 72, or a sequence having at least 80%, 85%, 90%, 95%or 99%identity thereto.
The bispecific antibody according to claim 4, wherein:

(a) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 47 and 48 respectively;

(b) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 49 and 50 respectively;

(c) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 51 and 52 respectively;

(d) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively;

(e) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 55 and 56 respectively;

(f) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 57 and 58 respectively.
The bispecific antibody according to claim 5, wherein:

(a) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively;

(b) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 61 respectively;

(c) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 62 respectively;

(d) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 63 respectively;

(e) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 64 respectively;

(f) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 65 respectively;

(g) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 66 respectively;

(h) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 67 respectively;

(i) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 68 respectively;

(j) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 69 respectively;

(k) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 70 respectively;

(l) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 71 and 60 respectively;

(m) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 71 and 68 respectively;

(n) B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively.
The bispecific antibody of according to any one of claims 1-7, wherein:

(a) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 57 and 58 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or

(b) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or

(c) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 47 and 48 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or

(d) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 49 and 50 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or

(e) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 51 and 52 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or

(f) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 55 and 56 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 60 respectively; or

(g) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 68 respectively; or

(h) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 70 respectively; or

(i) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 71 and 60 respectively; or

(j) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 71 and 68 respectively; or

(k) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 53 and 54 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or

(l) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 47 and 48 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or

(m) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 49 and 50 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or

(n) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 51 and 52 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or

(o) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 55 and 56 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively; or

(p) B1 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 57 and 58 respectively and B2 comprises a set of heavy and light chain variable region of amino acid sequences set forth in SEQ ID NOs: 59 and 72 respectively.
The bispecific antibody according to claim 1, wherein:

(a) B1 and/or B2 is an intact IgG antibody;

(b) B1 and/or B2 is a Fab-like fragment, optionally a Fab fragment, a Fab' fragment or a F (ab') 2 fragment; and/or

(c) B1 and/or B2 is a Fv fragment, optionally a single chain Fv (scFv) fragment, or a disulphide-bonded Fv fragment.
The bispecific antibody according to claim 9, wherein:

(a) B1 is an intact IgG antibody and B2 is a scFv fragment;

(b) B1 is a Fab fragment and B2 is a scFv fragment; and/or

(c) B1 and B2 is a scFv fragment.
The bispecific antibody according to claim 9 or claim 10, wherein:

(a) B1 has a valency of 2 and B2 has a valency of 2;

(b) B1 has a valency of 2 and B2 has a valency of 1; or

(c) B1 has a valency of 1 and B2 has a valency of 1.
The bispecific antibody according to any one of claims 9-11, wherein the bispecific antibody is selected from the groups consisting of:

(a) IgG-scFv antibodies, wherein B1 is an intact IgG and B2 is an scFv linked to B1 at the N-terminus of a light chain and/or at the C-terminus of a light chain and/or at the N-terminus of a heavy chain and/or at the C-terminus of a heavy chain of the IgG, B2 has a valency of 2;

(b) central scFv antibodies, namely Fab2-scFv-Fc, wherein B1 is a Fab fragment with a valency of 2 and B2 is an scFv with a valency of 1, the one B1 is attached to the N-terminus of one Fc domain to form the first monomer, and B2 is linked to the C-terminus of another B1 and the N-terminus of another Fc domain to form the second monomer; and/or

(c) dual scFv antibodies, namely scFv2-Fc, wherein B1 is scFv with a valency of 1, B2 is scFv with a valency of 1, B1 is linked to the N-terminus of one Fc domain to form the first monomer and B2 is linked to the N-terminus of another Fc domain to form the second monomer.
The bispecific antibody according to claim 12, wherein

the scFv is linked to the B1 or Fc domain either directly or via a linker fragment; and/or the VH of scFv is linked to the VL of scFv either directly or via a linker fragment.
The bispecific antibody according to claim 13, wherein

the linker fragment comprise (GGGGS) n , (GGGS) n or (GKPGS) n, n being a positive integer;

preferably, n is 1, 2, 3, 4, 5 or 6.
The bispecific antibody according to any one of claims 9-14, wherein the bispecific antibody is selected from the groups consisting of:

(a) IgG-scFv antibodies, comprising two heavy chains and two light chains, and its form a homodimer,

wherein, the heavy chain comprises, from amino terminus to carboxyl terminus, VH (B1) -CH; the light chain comprises, from amino to carboxyl terminus, VL (B1) -CL-linker-VH (B2) -linker-VL (B2) ;

(b) central scFv (Fab2-scFv-Fc) antibodies, comprising a first heavy chain, a second heavy chain and two common light chains, and its form a heterodimer,

wherein, the first heavy chain comprises, from amino terminus to carboxyl terminus, VH (B1) -CH1-Fc; the second heavy chain comprises, from amino to carboxyl terminus, VH (B1) -CH1-linker-VH (B2) -linker-VL (B2) -linker-Fc; the common light chain comprises, from amino to carboxyl terminus, VL (B1) -CL, the VH (B1) -CH1 pairs with VL (B1) -CL to form a Fab; or

(c) dual scFv (scFv2-Fc) antibodies, comprising a first heavy chain and a second heavy chain, and its form a heterodimer,

wherein, the first heavy chain comprises, from amino terminus to carboxyl terminus, VL (B2) -linker-VH (B2) -Fc; the second heavy chain comprises, from amino to carboxyl terminus, VH (B1) -linker-VL (B1) -Fc.
The bispecific antibody according to claim 8, wherein

the B1 and/or B2 comprises heavy chain constant regions derived from a human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably heavy chain constant regions derived from human IgG1 or IgG4 or a variant thereof, most preferably the heavy chain constant regions comprises amino acid sequences set forth in SEQ ID NOs: 75, 76, 77, 79, 80, 81 or 82; wherein

the B1 and/or B2 comprises light chain constant regions derived from human kappa (κ) or lambda (λ) chain, or a variant thereof, preferably light chain constant regions derived from human kappa (κ) chain, most preferably light chain constant regions as shown in SEQ ID NO: 78.
The bispecific antibody according to claim 8, wherein the B1 and/or B2 comprises a first Fc domain and a second Fc domain;

preferably, the first Fc domain of amino acid sequences set forth in SEQ ID NOs: 75 or 82 , the second Fc domain of amino acid sequences set forth in SEQ ID NOs: 76 or 81.
The bispecific antibody according to claim 8, wherein the bispecific antibody is selected from the groups consisting of:

(a)

the heavy chain as shown in SEQ ID NO: 83 and the light chain as shown in SEQ ID NO: 84; or

the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 87; or

the heavy chain as shown in SEQ ID NO: 89 and the light chain as shown in SEQ ID NO: 90; or

the heavy chain as shown in SEQ ID NO: 91 and the light chain as shown in SEQ ID NO: 92; or

the heavy chain as shown in SEQ ID NO: 93 and the light chain as shown in SEQ ID NO: 94; or

the heavy chain as shown in SEQ ID NO: 95 and the light chain as shown in SEQ ID NO: 96; or

the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 109; or

the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 110; or

the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 111; or

the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 112; or

the heavy chain as shown in SEQ ID NO: 86 and the light chain as shown in SEQ ID NO: 113; or

the heavy chain as shown in SEQ ID NO: 83 and the light chain as shown in SEQ ID NO: 114; or

the heavy chain as shown in SEQ ID NO: 89 and the light chain as shown in SEQ ID NO: 115; or

the heavy chain as shown in SEQ ID NO: 91 and the light chain as shown in SEQ ID NO: 116; or

the heavy chain as shown in SEQ ID NO: 93 and the light chain as shown in SEQ ID NO: 117; or

the heavy chain as shown in SEQ ID NO: 95 and the light chain as shown in SEQ ID NO: 118;

(b)

the first heavy chain as shown in SEQ ID NO: 97, second heavy chain as shown in SEQ ID NO: 98 and two common light chains as shown in SEQ ID NO: 99; or

the first heavy chain as shown in SEQ ID NO: 100, second heavy chain as shown in SEQ ID NO: 101 and two common light chains as shown in SEQ ID NO: 102; or

the first heavy chain as shown in SEQ ID NO: 100, second heavy chain as shown in SEQ ID NO: 103 and two common light chains as shown in SEQ ID NO: 102; or

the first heavy chain as shown in SEQ ID NO: 104, second heavy chain as shown in SEQ ID NO: 105 and two common light chains as shown in SEQ ID NO: 106;

(c)

the first heavy chain as shown in SEQ ID NO: 107 and the second heavy chain as shown in SEQ ID NO: 108; or

the first heavy chain as shown in SEQ ID NO: 119 and the second heavy chain as shown in SEQ ID NO: 108.
An isolated nucleic acid molecule encoding the bispecific antibody of claims 1 to 18; optionally operably linked to a promoter.
An expression vector comprising the isolated nucleic acid molecule of claim 19.
An isolated host cell comprising the nucleic acid molecule of claim 19, or the vector of claim 20, wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
A pharmaceutical composition, which comprises bispecific antibody according to any one of claims 1-18, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
A method of treatment or prevention of a ROR1-expressing cancer, comprising a step of administering a therapeutically effective amount of the bispecific antibody according to any one of claims 1-18, or the pharmaceutical composition of claim 22, to a subject in need of treatment or prevention of the ROR1-expressing cancer.
The method according to claim 23, wherein the ROR1-expressing cancer is chronic lymphocytic leukemia, mantle cell lymphoma, B-cell acute lymphoblastic leukemia, marginal zone lymphoma, neuroblastoma, multiple myeloma, renal cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, head and neck cancer, bladder cancer, esophageal cancer.