ANTI-ROR1 ANTIBODY, BISPECIFIC ANTIBODY COMPRISING SAME, AND USES THEREOF

Abstract

Provided are: a novel anti-ROR1 antibody; a bispecific antibody comprising the anti-ROR1 antibody; and uses thereof.

Claims

1. An anti-ROR1 antibody or an antigen-binding fragment thereof, comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

2. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region of SEQ ID NO: 7 or SEQ ID NO: 127, and a light chain variable region of SEQ ID NO: 8 or SEQ ID NO: 126.

3. A nucleic acid molecule encoding the anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A pharmaceutical composition for prevention or treatment of cancer, comprising the anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 or 2 and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition according to claim 4, wherein the cancer expresses ROR1.

6. A composition for detecting ROR1, comprising the anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. A composition for diagnosing cancer, comprising the anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 or 2.

8. The composition according to claim 7, wherein the cancer expresses ROR1.

9. An anti-ROR1/anti-4-1BB bispecific antibody, comprising: an anti-ROR1 antibody or an antigen-binding fragment thereof; and an anti-4-1BB antibody or an antigen-binding fragment thereof, wherein the anti-ROR1 antibody or antigen-binding fragment thereof comprises HCDR1 comprising the amino sequence of SEQ ID NO: 1, HCDR2 comprising the amino sequence of SEQ ID NO: 2, HCDR3 comprising the amino sequence of SEQ ID NO: 3, LCDR1 comprising the amino sequence of SEQ ID NO: 4, LCDR2 comprising the amino sequence of SEQ ID NO: 5, and LCDR3 comprising the amino sequence of SEQ ID NO: 6.

10. The anti-ROR1/anti-4-1BB bispecific antibody according to claim 9, wherein the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 7 or 127, and a light chain variable region of SEQ ID NO: 8 or 126.

11. The anti-ROR1/anti-4-1BB bispecific antibody according to claim 9 or 10, wherein the anti-4-1BB antibody or antigen-binding fragment thereof comprises: HCDR1 comprising an amino acid sequence of SEQ ID NO: 13, 14, or 15; HCDR2 comprising an amino acid sequence of SEQ ID NO: 16, 17, or 18; HCDR3 comprising an amino acid sequence of SEQ ID NO: 19, 20, 21, 22, or 23; LCDR1 comprising an amino acid sequence of SEQ ID NO: 24 or 25; LCDR2 comprising an amino acid sequence of SEQ ID NO: 26 or 27; and LCDR3 comprising an amino acid sequence of SEQ ID NO: 28 or 29.

12. The anti-ROR1/anti-4-1BB bispecific antibody according to any one of claims 9 to 11, wherein the anti-4-1BB antibody or antigen-binding fragment thereof, (1) a heavy chain complementarity-determining region selected from the group consisting of: (a) HCDR1 comprising the amino acid of SEQ ID NO: 13, HCDR2 comprising the amino acid of SEQ ID NO: 16, and HCDR3 comprising the amino acid of SEQ ID NO: 19, (b) HCDR1 comprising the amino acid of SEQ ID NO: 13, HCDR2 comprising the amino acid of SEQ ID NO: 16, and HCDR3 comprising the amino acid of SEQ ID NO: 20, (c) HCDR1 comprising the amino acid of SEQ ID NO: 13, HCDR2 comprising the amino acid of SEQ ID NO: 16, and HCDR3 comprising the amino acid of SEQ ID NO: 21, (d) HCDR1 comprising the amino acid of SEQ ID NO: 14, HCDR2 comprising the amino acid of SEQ ID NO: 17, and HCDR3 comprising the amino acid of SEQ ID NO: 22, and (e) HCDR1 comprising the amino acid of SEQ ID NO: 15, HCDR2 comprising the amino acid of SEQ ID NO: 18, and HCDR3 comprising the amino acid of SEQ ID NO: 23; and (2) a light chain complementarity-determining region selected from the group consisting of: (a) LCDR1 comprising the amino acid of SEQ ID NO: 24, LCDR2 comprising the amino acid of SEQ ID NO: 26, and LCDR3 comprising the amino acid of SEQ ID NO: 28, and (b) LCDR1 comprising the amino acid of SEQ ID NO: 24, LCDR2 comprising the amino acid of SEQ ID NO: 26, and LCDR3 comprising the amino acid of SEQ ID NO: 28.

13. The anti-ROR1/anti-4-1BB bispecific antibody according to any one of claims 9 to 12, wherein the anti-4-1BB antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, or 47.

14. A nucleic acid molecule, encoding the anti-ROR1/anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to any one of claims 9 to 13.

15. A pharmaceutical composition for prevention or treatment of cancer, comprising the anti-ROR1/anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to any one of claims 9 to 13 and a pharmaceutically acceptable excipient.

16. The pharmaceutical composition according to claim 15, wherein the cancer expresses ROR1.

17. An anti-ROR1/anti-B7-H3 bispecific antibody, comprising: an anti-ROR1 antibody or an antigen-binding fragment thereof; and an anti-B7-H3 antibody or an antigen-binding fragment thereof, wherein the anti-ROR1 antibody or antigen-binding fragment thereof comprises HCDR1 comprising the amino sequence of SEQ ID NO: 1, HCDR2 comprising the amino sequence of SEQ ID NO: 2, HCDR3 comprising the amino sequence of SEQ ID NO: 3, LCDR1 comprising the amino sequence of SEQ ID NO: 4, LCDR2 comprising the amino sequence of SEQ ID NO: 5, and LCDR3 comprising the amino sequence of SEQ ID NO: 6.

18. The anti-ROR1/anti-B7-H3 bispecific antibody according to claim 17, wherein the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 7 or 127, and a light chain variable region of SEQ ID NO: 8 or 126.

19. The anti-ROR1/anti-B7-H3 bispecific antibody according to claim 17 or 18, wherein the anti-B7-H3 antibody or antigen-binding fragment thereof comprises: HCDR1 comprising an amino acid sequence selected from SEQ ID NOS: 48 to 51; HCDR2 comprising an amino acid sequence selected from SEQ ID NOS: 52 to 57; HCDR3 comprising an amino acid sequence selected from SEQ ID NOS: 58 to 62; LCDR1 comprising an amino acid sequence selected from SEQ ID NOS: 63 to 67; LCDR2 comprising an amino acid sequence selected from SEQ ID NOS: 68 to 73; and LCDR3 comprising an amino acid sequence selected from SEQ ID NOS: 74 to 79.

20. The anti-ROR1/anti-B7-H3 bispecific antibody according to any one of claims 16 to 19, wherein the anti-B7-H3 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain complementarity-determining region selected from the group consisting of: (a) HCDR1 comprising the amino acid of SEQ ID NO: 48, HCDR2 comprising the amino acid of SEQ ID NO: 52, and HCDR3 comprising the amino acid of SEQ ID NO: 58, (b) HCDR1 comprising the amino acid of SEQ ID NO: 49, HCDR2 comprising the amino acid of SEQ ID NO: 53, and HCDR3 comprising the amino acid of SEQ ID NO: 59, (c) HCDR1 comprising the amino acid of SEQ ID NO: 50, HCDR2 comprising the amino acid of SEQ ID NO: 54, and HCDR3 comprising the amino acid of SEQ ID NO: 60, (d) HCDR1 comprising the amino acid of SEQ ID NO: 48, HCDR2 comprising the amino acid of SEQ ID NO: 55, and HCDR3 comprising the amino acid of SEQ ID NO: 61, and (e) HCDR1 comprising the amino acid of SEQ ID NO: 51, HCDR2 comprising the amino acid of SEQ ID NO: 56, and HCDR3 comprising the amino acid of SEQ ID NO: 62, and (f) HCDR1 comprising the amino acid of SEQ ID NO: 50, HCDR2 comprising the amino acid of SEQ ID NO: 57, and HCDR3 comprising the amino acid of SEQ ID NO: 60; and (ii) a light chain complementarity-determining region selected from the group consisting of: (a) LCDR1 comprising the amino acid of SEQ ID NO: 63, LCDR2 comprising the amino acid of SEQ ID NO: 68, and LCDR3 comprising the amino acid of SEQ ID NO: 74, (b) LCDR1 comprising the amino acid of SEQ ID NO: 64, LCDR2 comprising the amino acid of SEQ ID NO: 69, and LCDR3 comprising the amino acid of SEQ ID NO: 75; (c) LCDR1 comprising the amino acid of SEQ ID NO: 65, LCDR2 comprising the amino acid of SEQ ID NO: 70, and LCDR3 comprising the amino acid of SEQ ID NO: 76; (d) LCDR1 comprising the amino acid of SEQ ID NO: 66, LCDR2 comprising the amino acid of SEQ ID NO: 71, and LCDR3 comprising the amino acid of SEQ ID NO: 77; (e) LCDR1 comprising the amino acid of SEQ ID NO: 67, LCDR2 comprising the amino acid of SEQ ID NO: 72, and LCDR3 comprising the amino acid of SEQ ID NO: 78; and (f) LCDR1 comprising the amino acid of SEQ ID NO: 65, LCDR2 comprising the amino acid of SEQ ID NO: 73, and LCDR3 comprising the amino acid of SEQ ID NO: 76.

21. The anti-ROR1/anti-B7-H3 bispecific antibody according to any one of claims 16 to 20, wherein the anti-B7-H3 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 80 to 91; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 92 to 103.

22. A nucleic acid molecule, encoding the anti-ROR1/anti-B7-H3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 16 to 21.

23. A pharmaceutical composition for prevention or treatment of cancer, comprising the anti-ROR1/anti-B7-H3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 16 to 21, and a pharmaceutically acceptable excipient.

24. The pharmaceutical composition according to claim 23, wherein the cancer expresses either or both of ROR1 and B7-H3.

25. An antibody-drug conjugate, comprising the anti-ROR1/anti-B7-H3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 16 to 21 and a cytotoxic drug linked thereto.

26. The antibody-drug conjugate according to claim 25, wherein the antibody-drug conjugate has a s structure of antibody-linker-drug.

27. The antibody-drug conjugate according to claim 25 or 26, wherein the cytotoxic drug is an anticancer agent.

28. A pharmaceutical composition for prevention or treatment of cancer, comprising the antibody-drug conjugate of any one of claims 25 to 27 and a pharmaceutically acceptable excipient.

29. The pharmaceutical composition according to claim 28, wherein the cancer expresses either or both of ROR1 and B7-H3.

Description

DESCRIPTION OF DRAWINGS

[0308] FIGS. 1a to 1c are graphs showing the binding affinity of an anti-ROR1 antibody to ROR1 according to an embodiment.

[0309] FIGS. 2a and 2b are graphs showing the binding affinity of an anti-ROR1/anti-4-1BB bispecific antibody to 4-1BB and/or ROR1 according to an embodiment.

[0310] FIG. 2c is a graph showing the binding affinity of an anti-ROR1/anti-4-1BB bispecific antibody to cells expressing 4-1BB on their surface according to an embodiment.

[0311] FIG. 2d is a graph showing the binding affinity of an anti-ROR1/anti-4-1BB bispecific antibody to cells expressing ROR1 on their surface according to an embodiment.

[0312] FIGS. 3a to 3d are graphs showing the serum stability of an anti-ROR1/anti-4-1BB bispecific antibody according to an embodiment (a and b: in human serum; c and d: in monkey serum).

[0313] FIG. 4a is a graph showing 4-1BB signaling activation by an anti-ROR1/anti-4-1BB bispecific antibody in the presence of ROR1 according to an embodiment.

[0314] FIG. 4b is a graph showing ROR1-dependent 4-1BB signaling activation by an anti-ROR1/anti-4-1BB bispecific antibody according to an embodiment.

[0315] FIG. 4c is a graph showing the correlation between ROR1-dependent 4-1BB signaling activation and ROR1 expression by an anti-ROR1/anti-4-1BB bispecific antibody according to an embodiment.

[0316] FIG. 4d is a graph showing FcRI-dependent 4-1BB signaling activation by an anti-ROR1/anti-4-1BB bispecific antibody according to an embodiment.

[0317] FIG. 4d is a graph showing FcRI-dependent 4-1BB signaling activation by an anti-ROR1/anti-4-1BB bispecific antibody according to an embodiment.

[0318] FIG. 5a is a graph demonstrating the activation of PBMCs and the induction of cytokine (interferon-gamma) release by an anti-ROR1/anti-4-1BB bispecific antibody in CHO-K1 cells overexpressing ROR1 according to an embodiment.

[0319] FIG. 5b are graphs illustrating the activation of PBMCs and the induction of cytokine (interferon-gamma) release by an anti-ROR1/anti-4-1BB bispecific antibody in gastric cancer cell lines expressing ROR1 according to an embodiment.

[0320] FIG. 5c are graphs illustrating the activation of PBMCs and the induction of cytokine (interferon-gamma) release by an anti-ROR1/anti-4-1BB bispecific antibody in gastric cancer cell lines expressing ROR1 according to an embodiment.

[0321] FIGS. 6a and 6b are graphs showing the anti-tumor effect (in vivo) of an anti-ROR1/anti-4-1BB bispecific antibody according to an embodiment.

[0322] FIG. 6c is a graph depicting the long-lasting anti-tumor memory response by an anti-ROR1/anti-4-1BB bispecific antibody according to an embodiment.

[0323] FIG. 6d is a graph presenting the results of measuring immune modulation activity of an anti-ROR1/anti-4-1BB bispecific antibody in tumor, blood, and liver according to an embodiment.

[0324] FIG. 6e is a graph showing the results of measuring immune modulation activity of an anti-ROR1/anti-4-1BB bispecific antibody in PBMCs not expressing ROR1 according to an embodiment.

[0325] FIGS. 7a-7d are graphs showing the binding affinity of an anti-ROR1/anti-B7-H3 bispecific antibody to ROR1 or B7-H3 according to an embodiment.

[0326] FIG. 8 is a graph demonstrating the binding affinity of an anti-ROR1/anti-B7-H3 bispecific antibody to cells expressing ROR1 and B7-H3 on their surface according to an embodiment.

[0327] FIG. 9 is a graph illustrating the degree of cellular internalization of an anti-ROR1/anti-B7-H3 bispecific antibody according to an embodiment.

[0328] FIGS. 10a to 10c are graphs resenting the results of measuring the purity of an antibody-drug conjugate (ADC) comprising an anti-ROR1/anti-B7-H3 bispecific antibody using size-exclusion high-performance liquid chromatography (SE-HPLC) according to an embodiment. The peak values (min.) shown in FIGS. 10a to 10c are as follows: 10a: 12.441, 14.677; 10b: 13.564, 16.170; 10c: 13.752, 16.314.

[0329] FIG. 11 is a graph showing the results of measuring the drug-to-antibody ratio (DAR) in an ADC comprising an anti-ROR1/anti-B7-H3 bispecific antibody using size-exclusion liquid chromatography-mass spectrometry (LC/MS) according to an embodiment.

[0330] FIG. 12 is a graph demonstrating the binding capability of an ADC comprising an anti-ROR1/anti-B7-H3 bispecific antibody to the antigen (ROR1) according to an embodiment.

[0331] FIG. 13 is a graph illustrating the cytotoxicity of an ADC comprising an anti-ROR1/anti-B7-H3 bispecific antibody against cancer cells expressing ROR1 and B7-H3 according to an embodiment.

MODE FOR INVENTION

[0332] Hereinafter, the following examples will be described in detail to aid in the understanding of the present invention. However, these examples are provided to illustrate the present invention and are not to be construed as limiting the scope of the present invention. The Examples of the present application are provided to more fully describe the present application to the person with ordinary skill in the art.

Example 1. Anti-ROR1 Antibody

1.1. Manufacture of Anti-ROR1 Antibody

[0333] From the anti-ROR1 antibody clone sequence disclosed in International Publication No. WO2019-225992 A1, peptide mapping (Accurate Determination of Succinimide Degradation Products Using High Fidelity Trypsin Digestion Peptide Map Analysis, Anal. Chem. 2011, 83 (15): 5912-5919) was employed to identify posttranslational modification (PTM) sites and introduce mutations to remove these sites, thereby manufacturing antibodies with improved physicochemical properties and developability. Among the anti-ROR1 antibodies disclosed in WO2019-225992 A1, the BA6 antibody (WT) (based on IgG1) was chosen as the target for PTM site removal, and the amino acid sequences of the complementarity-determining regions (CDRs) of the heavy chain CDR1 (HCDR1), CDR2 (HCDR2), and CDR3 (HCDR3), as well as the light chain CDR1 (LCDR1), CDR2 (LCDR2), and CDR3 (LCDR3) of the BA6 antibody, are summarized in Table 6 below.

TABLE-US-00007 TABLE6 SEQID Aminoacidsequence(N.fwdarw.C) NO:# HCDR1 NYDMS 1 HCDR2 AIYHSGSSKYYADSVKG 2 HCDR3 GGNGAWDTGFDY 9 LCDR1 SGSSSNIGSNDVS 4 LCDR2 YDNNRPS 10 LCDR3 GAWDDSLSGYV 6 VH EVQLLESGGGLVQPGGSLRLSCAASGFTF 11 SNYDMSWVRQAPGKGLEWVSAIYHSGSSK YYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARGGNGAWDTGFDYWGQGTL VTVSS VL QSVLTQPPSASGTPGQRVTISCSGSSSNI 12 GSNDVSWYQQLPGTAPKLLIYYDNNRPSG VPDRFSGSKSGTSASLAISGLRSEDE ADYYCGAWDDSLSGYVFGGGTKLTVL

[0334] Peptide mapping identified PTMs in LCDR2 and HCDR3. To pinpoint the actual hot spots of PTM within the BA6 antibody sequence, peptide mapping was conducted using the BA6 antibody. After storing the antibody at 4 C. and 37 C. for two weeks, the samples were digested and fragmented with Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS; Genovis) and dithiothreitol (DTT), followed by LC/MS analysis, which confirmed the occurrence of PTMs in CDRL2 and CDRH3. Since there were two PTM hot spots, the approach involved dividing them into two groups, inducing single mutations, and then, if the binding activity was found to be similar to the wild-type antibody, combining mutations to create double, triple mutants, etc.

[0335] A total of 10 PTM mutants were constructed to verify ligand binding activity. Specifically, to obtain IgGs of each anti-ROR1 antibody clone with PTM removal mutations, mutagenesis primer sets (Macrogen, South Korea) targeting CDRL2 and CDRL3, the PTM hot spots of the above clone, to introduce mutation sequences, were synthesized, and nucleic acid sequences coding for the light and heavy chains were obtained through site-directed mutagenesis using PCR techniques.

[0336] For transfection, ExpiCHO-S cells (Thermo Fisher) were prepared at a concentration of 6.010.sup.6 cells/mL in a 500 mL flask. Vectors expressing the heavy and light chains (each cloned into separate pcDNA3.4 vectors (Thermo Fisher) for plasmid preparation before use in transfection) and ExpiFectamine (Thermo Fisher) were diluted in OptiPRO medium (Thermo Fisher) to form concentrations of 1 g/mL and 3.2 L/mL, respectively, then the diluted vector and ExpiFectamine mixture were mixed and allowed to stand at room temperature for 5 minutes, then added to the flask containing the prepared cells, then the mixture was agitated well and cultured under conditions of 8% CO.sub.2, 37 C., and 120 rpm. The day after performing the transfection, enhancer (Thermo Fisher) at 600 L and feed (Thermo Fisher) at 24 mL were each added, and the culture was continued under conditions of 8% CO.sub.2, 37 C., and 120 rpm for 7 days. For detailed experimental protocols, reference was made to the manufacturer's guide of the ExpiCHO expression system provided by Thermo Fisher.

[0337] Through the method, a total of 10 mutants (M1-M10) were yielded and used as candidate clones for the anti-ROR1 antibody. These clones each comprised mutations confirmed in PTM sites CDRL2 or CDRH3. Clones M1 to M4 had mutations introduced at L51 in CDRL2 and clones M5-M10 had mutations introduced in CDRH3, and Clone M11 incorporated mutations from M3 (mutation at L51 in CDRL2) and M9 (mutation at H97 in CDRH3), which were selected as leading clones among M1 to M10. The specific information for these candidate clones is summarized in Table 7 below.

TABLE-US-00008 TABLE 7 PTM deN & IsoD at L51/52 deD at H97 Hot spot Yes Yes CDR or FR region CDRL2 CDRH3 Kabat numbering L51 L52 H97 H98 Location n n + 1 n n + 1 Residue D N N G Pass M1 A O M2 T X M3 E O M4 S X M5 Y X M6 V X M7 R X M8 G X M9 S 0 M10 Y X M11 E S (deD: Asp Deamidation, deN: Asn Deamidation, isoD: Aspartate Isomerization; Pass: evaluated based on ligand(antigen) binding activity)

1.2. Assay for Binding Specificity of Candidate Clones to Extracellular Domain of ROR1 (ELISA)

[0338] Given that the PTM hotspots in the BA6 antibody were identified within the CDR regions, removing PTMs could potentially decrease the binding activity to the antigen compared to WT (wild-type, BA6 antibody) due to sequence changes in the CDR regions, which in turn could reduce the antibody's potency. Thus, the ultimate goal of PTM engineering was to identify clones with equal or superior binding activity to WT. Therefore, the specific binding ability of candidate clones M1 to M11 produced in Example 1.1 to the ROR1 antigen was analyzed as follows.

[0339] The binding affinity of the anti-ROR1 antibody-antigen was evaluated using an ELISA-based solution binding assay. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with ROR1 protein (ROR1-His; sino biological, Catalog #13968-H08H) at a concentration of 1 g/ml in PBS solution at 4 C. for 16 hours, and non-specific binding sites were blocked with 1% (v/v) BSA (bovine serum albumin) for 2 hours.

[0340] Subsequently, on the 96-well microtiter plates, anti-ROR1 antibodies at concentrations stated in FIGS. 1a to 1c were added to the microtiter plates, and their binding capabilities were analyzed by ELISA as follows. Specifically, after incubating at 37 C. for 2 hours, the plates were washed five times with PBS containing 0.05% (v/v) Tween 20, then the HRP-conjugated Fab polyclonal antibody reagent (Pierce, Catalog #31414) was diluted at a 1:30,000 ratio and added to the washed microtiter plates, then reacted for 1 hour at 37 C., then detected the bound anti-ROR1 antibodies. After reaction, the color was developed using TMB (tetramethylbenzidine, Sigma, T0440). The enzymatic reaction was stopped with 0.5 mol/L sulfuric acid, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0341] The results obtained were presented in FIGS. 1a to 1c. As understood from data of FIGS. 1a to 1c, based on ELISA analysis, M1 and M3 showed similar ligand (antigen) binding activity compared to WT, however, M1 was excluded from the final clone selection due to a lower saturation point than WT. Additionally, none of M7, M8, and M10 showed comparable binding activity to WT. M3 and M9 exhibited comparable binding activity to WT. Consequently, a double mutant M11 clone (D51E (LC)+N97S (HC)) combining M3 and M9 was produced using the same method as in the Example. The M11 clone thus produced was confirmed to exhibit similar binding activity compared to WT and M3, leading to its selection as the final candidate clone.

1.3. Analysis for Developability of Candidate Clone

[0342] The clone BA6M11 (also denoted as BA6M11) clone (derived from BA6 through PTM engineering to remove PTM liability) the efficacy of which was confirmed in Example 1.2 was assayed for stability under stressed conditions, compared to BA6. More specifically, after storing both the BA6 antibody and the BA6M11 antibody at 4 C. (normal conditions) and 37 C. (stressed conditions) for two weeks, SE-HPLC, icIEF, and LC/MS analyses were conducted on each sample to check for purity and impurity content. Furthermore, to determine whether changes in the content of the substance also affected efficacy, an antigen-antibody binding ability analysis (ELISA; see Example 1.2) was conducted.

[0343] The results thus obtained are summarized in Table 8, below.

TABLE-US-00009 TABLE 8 BA6 IgG (WT) BA6 IgG (M11) Content Method Acceptance Criteria 4 C. 37 C. 4 C. 37 C. Purity (%) SEC purity % 1.5% 99.6 98.4 99.1 98.2 1.2 0.9 Charge icIEF Basic Information only 27.7 24.1 10.2 17.0 heterogeneity Major 35.3 27.1 74.5 69.0 Acidic 37.0 48.9 15.3 14.0 Major % 8.2 5.5 Protein ELISA SACE 70% Ratio 130%, 0.217 0.261 0.166 0.129 Binding (EC50 to similar curve fit 83.1% 128.6% Human ROR1), nM) PTM LC/MS Report result 30.1 47.1 **N/D **N/D (succinimide D %) 17.0 *N/A (*N/A: Not applicable; **N/D: Not detected)

[0344] As shown in Table 8, it was observed that the purity of the BA6M11 clone remained high and comparable to BA6 when stored at both 4 C. and 37 C. Moreover, compared to BA6, during the production process, the formation of charge variants in the BA6M11 clone significantly decreased in both basic and acidic conditions, and the formation of charge variants under stressed conditions was also significantly reduced (Major %). In the BA6M11 clone, peptide mapping analysis confirmed the disappearance of succinimide formation at problematic residues, leading to a reduction in charge variants. The decrease in charge variants indicates a reduced risk of antibody quality variation during production and storage/distribution processes and enhances the reliability of purification processes using ion exchange resins. It also reduces the risk of increased immunogenicity when administered to humans by preventing the hydrolysis of succinimide intermediates, which could lead to antibody chain cleavage, lower potency, and increased immunogenicity. Furthermore, the antigen-antibody binding capacity, verified using human recombinant ROR1 protein (ROR1-His; Sino biological, Catalog #13968-H08H), showed no significant difference in binding ability between samples stored for two weeks at 4 C. and 37 C. These results confirmed that BA6M11 maintained potency comparable to BA6 while improving physicochemical properties in terms of developability.

[0345] Ultimately, the BA6M11 clone not only showed comparable binding to WT but also clearly improved developability through PTM engineering.

[0346] The amino acid sequences of the novel anti-ROR1 antibody, BA6M11, in the IgG form are listed in Tables 9 and 10.

TABLE-US-00010 TABLE9 SEQ ID Aminoacidsequence(N.fwdarw.C) NO:# BA6_HCDR1 NYDMS 1 BA6_HCDR2 AIYHSGSSKYYADSVKG 2 BA6_HCDR3 GGNGAWDTGFDY 9 BA6_VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSW 11 VRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCARGGNGAWDTGF DYWGQGTLVTVSS BA6M11_HCD NYDMS 1 R1 BA6M11_HCD AIYHSGSSKYYADSVKG 2 R2 BA6M11_HCD GGSGAWDTGFDY 3 R3 BA6M11_VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSW 7 VRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGF DYWGQGTLVTVSS HeavyChain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT 104 constant VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS region LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC (WT) PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK HeavyChain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT 105 constant VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS region LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC (NA) PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID Nucleicacidsequence(5.fwdarw.3) NO:# BA6M11_VH GAGGTGCAGCTGCTGGAGTCCGGCGGCGGCCTGGT 106 GCAGCCCGGCGGCTCCCTGCGGCTGTCCTGCGCCG CCTCCGGCTTCACCTTCTCCAACTACGACATGTCCT GGGTGCGGCAGGCCCCCGGCAAGGGCCTGGAGTG GGTGTCCGCCATCTACCACTCCGGCTCCTCCAAGTA CTACGCCGACTCCGTGAAGGGCCGGTTCACCATCT CCCGGGACAACTCCAAGAACACCCTGTACCTGCAG ATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTA CTACTGCGCCCGGGGCGGCTCCGGCGCCTGGGACA CCGGCTTCGACTACTGGGGCCAGGGCACCCTGGTG ACCGTGTCCTCC HeavyChain GCCTCCACCAAGGGCCCCTCCGTGTTCCCCCTGGCC 107 constant CCCTCCTCCAAGTCCACCTCCGGCGGCACCGCCGC region CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGC (WT) CCGTGACCGTGTCCTGGAACTCCGGCGCCCTGACC TCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGTCC TCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTG CCCTCCTCCTCCCTGGGCACCCAGACCTACATCTGC AACGTGAACCACAAGCCCTCCAACACCAAGGTGGA CAAGAAGGTGGAGCCCAAGTCCTGCGACAAGACC CACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTG GGCGGCCCCTCCGTGTTCCTGTTCCCTCCTAAGCCC AAGGACACCCTGATGATCTCCCGGACCCCCGAGGT GACTTGCGTGGTGGTGGACGTGTCCCACGAGGACC CCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCACAACGCCAAGACCAAGCCCCGGGAGG AGCAGTACAACTCCACCTACCGGGTGGTGTCCGTG CTGACCGTGCTGCACCAGGACTGGCTGAACGGCAA GGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGC CCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAG GGCCAGCCCCGGGAGCCCCAGGTGTACACCCTGCC CCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGT CCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCG ACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCC GAGAACAACTACAAGACCACCCCCCCCGTGCTGGA CTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGAC CGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGT TCTCCTGCTCCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGC AAG

TABLE-US-00011 TABLE10 SEQ ID Aminoacidsequence(N.fwdarw.C) NO:# BA6_LCDR1 SGSSSNIGSNDVS 4 BA6_LCDR2 YDNNRPS 10 BA6_LCDR3 GAWDDSLSGYV 6 BA6_VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQ 12 QLPGTAPKLLIYYDNNRPSGVPDRFSGSKSGTSASLAI SGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVL BA6M11_LCD SGSSSNIGSNDVS 4 R1 BA6M11_LCD YENNRPS 5 R2 BA6M11_LCD GAWDDSLSGYV 6 R3 BA6M11_VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQ 8 QLPGTAPKLLIYYENNRPSGVPDRFSGSKSGTSASLAI SGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVL LightChain GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV 108 constantregion TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS SEQ ID Nucleicacidsequence(5.fwdarw.3) NO:# BA6M11_VL CAGTCCGTGCTGACCCAGCCCCCCTCCGCCTCCGGC 109 ACCCCCGGCCAGCGGGTGACCATCTCCTGCTCCGG CTCCTCCTCCAACATCGGCTCCAACGACGTGTCCTG GTACCAGCAGCTGCCCGGCACCGCCCCCAAGCTGC TGATCTACTACGAAAACAACCGGCCCTCCGGCGTG CCCGACCGGTTCTCCGGCTCCAAGTCCGGCACCTCC GCCTCCCTGGCCATCTCCGGCCTGCGGTCCGAGGA CGAGGCCGACTACTACTGCGGCGCCTGGGACGACT CCCTGTCCGGCTACGTGTTCGGCGGCGGCACCAAG CTGACCGTGCTG LightChain GGCCAGCCCAAGGCCGCCCCCTCCGTGACCCTGTT 110 constantregion CCCCCCCTCCTCCGAGGAGCTGCAGGCCAACAAGG CCACCCTGGTGTGCCTGATCTCCGACTTCTACCCCG GCGCCGTGACCGTGGCCTGGAAGGCCGACTCCTCC CCCGTGAAGGCCGGCGTGGAGACCACCACCCCCTC CAAGCAGTCCAACAACAAGTACGCCGCCTCCTCCT ACCTGTCCCTGACCCCCGAGCAGTGGAAGTCCCAC CGGTCCTACTCCTGCCAGGTGACCCACGAGGGCTC CACCGTGGAGAAGACCGTGGCCCCCGCCGAGTGCT CC

Example 2. Anti-ROR1/Anti-4-1BB Bispecific Antibody

2.1. Manufacture of Improved Anti-ROR1/Anti-4-1BB Bispecific Antibody

[0347] An anti-ROR1/anti-4-1BB bispecific antibody was manufactured based on the improved anti-ROR1 antibody clone BA6M11, produced and selected in Example 1, and one of the anti-4-1BB antibody clones disclosed in International Publication Number WO2020/111913 A1, namely 41B01.03 (also referred to as 1A10M12).

[0348] In this Example, a bispecific antibody comprising an anti-ROR1 IgG (full-length) antibody and a 4-1BB scFv connected to the C-terminus of the heavy chain of the anti-ROR1 IgG (full-length) antibody was constructed.

[0349] To construct the anti-ROR1/anti-4-1BB bispecific antibody, DNA segment 1 having a nucleotide sequence coding for the heavy chain of the anti-ROR1 IgG antibody (BA6M11) was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 1) while DNA segment 2 having a nucleotide sequence coding for the light chain of the IgG antibody in the anti-ROR1/anti-4-1BB bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 2).

[0350] Subsequently, DNA segment 3 encoding the scFv was fused at the portion corresponding to the C-terminal of the Fc region of the IgG antibody inserted in plasmid 1, using DNA segment 4 encoding a 20 amino acid-length peptide linker composed of (GGGGS) 4 (SEQ ID NO: 114), or DNA segment 5 encoding an 18 amino acid-length linker peptide composed of (GS) 9 (SEQ ID NO: 113), to construct a vector for the expression of the bispecific antibody.

[0351] Additionally, the anti-4-1BB scFv (named 1A10M12) took a form in which VL103-VH44 (VL103: VL with a G (Gly).fwdarw.C (Cys) mutation introduced at position 103; VH 44: VH with a G.fwdarw.C mutation introduced at position 44) at the C-termini of the light chain variable region (VL) and heavy chain variable region (VH) respectively, were fused through the linker ((GGGGS) 4, SEQ ID NO: 114). These mutations stabilized the scFv and enable the formation of disulfide bridges.

[0352] The amino acid sequences of the bispecific antibody thus prepared are listed in Table 11 below.

TABLE-US-00012 TABLE11 SEQID Aminoacidsequence(N.fwdarw.C) NO:# Lightchain QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWY 111 BA6M11 QQLPGTAPKLLIYYENNRPSGVPDRFSGSKSGTSASL AISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA VTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYL SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS HeavychainBA6M11(WT)x1A10M12 Heavychain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSW 112 BA6M11(WT) VRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDT GFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK Linker1 GSGSGSGSGSGSGSGSGS 113 1A10M12 QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWY 46 (VL) QQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASL AISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL Linker2 GGGGSGGGGSGGGGSGGGGS 114 1A10M12 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSW 37 (VH) VRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMR EFDYWGQGTLVTVSS Full-length EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSW 115 VRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDT GFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGS QSVLTOPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQL PGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRS EDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGG GSGGGGSGGGGSEVOLLESGGGLVQPGGSLRLSCAA SGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYY ADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYC ARDAQRNSMREFDYWGQGTLVTVSS HeavychainBA6M11(NA)x1A10M12 Heavychain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSW 116 BA6M11(NA) VRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDT GFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK Linker1 GSGSGSGSGSGSGSGSGS 113 1A10M12 QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWY 46 (VL) QQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASL AISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL Linker2 GGGGSGGGGSGGGGSGGGGS 114 1A10M12 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSW 37 (VH) VRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMR EFDYWGQGTLVTVSS Full-length EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSW 117 VRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDT GFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGS QSVLTQPPSASGTPGORVTISCSGSSSNIGNNYVTWYQ QLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAI SGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGG SGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSI YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARDAQRNSMREFDYWGQGTLVTVSS (Linker1: BA6M11(IgG) linked to 1A10M12(scFv) (C-terminus of heavy chain of BA6M11 linked to N-terminus of VL of 1A10M12); Linker2: VL linked to VH in 1A10M12 (scFv) (C-terminus of VL linked to N-terminus of VH)

[0353] The coding nucleic acid sequence (5.fwdarw.3) of the bispecific antibody is as follows:

TABLE-US-00013 [Heavychain_BA6M11(WT)x1A10M12] (SEQIDNO:118) gaggtgcagctgctggagtccggcggcggcctggtgcagccccgcggctccctgcggctgtcctgcgccgcctcc ggcttcaccttctccaactacgacatgtcctgggtgcggcagccccccggcaagggcctggagtggctctccccc atctaccactccggctcctccaagtactacgccgactccgtgaagggccggttcaccatctcccgggacaactcc aagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgcccggggcggc tccggcgcctgggacaccggcttcgactactggggccagggcaccctggtgaccgtgtcctctgcttctaccaag ggaccctctgtgttccctctggctccttccagcaagtctacctctggtggaacccctgctctgggctgcctggtc aaggattactttcctgagcctgtgacagtgtcctggaactctggtgctctgacctctggcgtgcacacctttcca gctctcctgcagtcctctggcctgtactctctgtcctctgtcgtgaccgtgccatcttctagcctgggcacccag acctacatctgcaacgtgaaccacaagccttccaacaccaaggtggacaagaaggtggaacccaagtcctgcgac aagacccacacctgtcctccatgtcctgctccagaactgctcggcggtccctccgttttcctgtttccacctaag cctaaggacaccctgatgatctctcggacccctgaagtgacctgcgtggtggtggatgtgtctcacgaggatccc gaagtgaagttcaattggtacgtggacggcgtggaagtccacaacgccaagaccaagcctagagaggaacagtac aactccacctacagagtggtgtctgtcctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgc aaggtgtccaacaaggccctgcctgctcctatcgaaaagaccatctccaaggctaagggccagcctcgggaacct caagtgtacaccctgcctcctagcagagaagagatgaccaagaaccaggtgtccctgacctgcctggtcaagggc ttctacccttccgatatcgccgtggaatgggagtccaatggccagcctgagaacaactacaagaccacacctcct gtgctggactccgacggctcattcttcctgtactccaagctgaccgtggacaagtccagatggcagcagggcaac gtgttctcctgctccgtgatccacgaggccctgcacaatcactacacccagaagtccctgtctctctcccctggc aaaggctccggatctggttctggatccggaagcggttctggcagcggatctggatctcagtccgtgctgactcag cctccttctgcttctggcacccctggccaaagagtgacaatctcttgctccggctcctcctccaacatcggcaac aactacgtgacctggtatcagcagctgcccggcacagctcccaaactgctgatctacgccgactctcacagacct tccggcgtgcccgatagattctccggctctaagtctggcacctctgccagcctggctatctccggcctgagatct gaggacgaggccgactactactgcgccacctgggattattccctgtccggctacgtgttcggctgcggcacaaaa ctgacagtgcttggcggcggaggatctggcggaggtggaagcggaggcggaggaagcggtggcggcggatctgaa gttcagctgttggaaagtggcggaggcctggttcaacctggcggatctctgagactgtcttgtgccgcctccggc tttaccttctcctcctacgacatgtcctgggtccgacagcctcctggcaagtgtctggaatgggtttcctggatc tcctactccggcggctccatctactacgccgattccgtgaagggcagattcaccatcagccgggacaactccaag aacaccctgtacctccagatgaactccctgagagccgaggacaccgccgtgtactactgtgctagagatgcccag cggaacagcatgagagagttccactattcggcccagggcaccctggtcacagtctcttct [Lightchain_BA6M11] (SEQIDNO:119) cagtccgtcctgacccagcccccctccgcctccggcacccccggccagcgggtgaccatctcctgctccgcctcc tcctccaacatcggctccaacgacgtgtcctggtaccagcagctgcccggcaccccccccaagctgctgatctac tacgaaaacaaccggccctccggcgtgcccgaccggttctccggctccaagtccggcacctccgcctccctggcc atctccggcctgcggtccgaggacgaggccgactactactgcggcgcctgggacgactccctgtccggctacgtg ttcggcggcggcaccaagctgaccgtgctgggccagcccaaggccgccccctccgtgaccctcttccccccctcc tccgaggagctgcaggccaacaaggccaccctggtgtgcctgatctccgacttctaccccggcgccgtgaccctg gcctggaaggccgactcctcccccgtgaaggccggcgtggagaccaccaccccctccaagcagtccaacaacaag tacgccgcctcctcctacctgtccctgacccccgagcagtggaagtcccaccggtcctactcctgccaggtgacc cacgagggctccaccgtggagaagaccgtggcccccccccagtcctcc

[0354] The coding DNA of the bispecific antibody cloned as described in the foregoing were transiently expressed using the ExpiCHO system (Thermo Fisher) according to the manufacturer's protocol. The culture with expressed antibodies was cleared of suspensions using centrifugation and filtration, and the antibody was purified using affinity chromatography with Mabselect Sure resin and size exclusion chromatography with Superdex200 resin. The purity of the purified antibody was analyzed using HPLC with TSKgel SuperSW3000, and its purity was found to be excellent, exceeding 95%.

2.2. Evaluation of Anti-ROR1/Anti-4-1BB Bispecific Antibody

2.2.1. Dual-Antigen Capture ELISA (DACE)

[0355] The binding affinities of the anti-ROR1/anti-4-1BB bispecific antibodies comprising either the BA6M11 clone (PTMs removal clone, test group) or the BA6 clone (parental clone, control group) to ROR1 and 4-1BB were compared and analyzed using the ELISA method. Two versions of the test group BA6M11 clone were used: BA6M11 (NA), which includes the N297A (NA) mutation that reduces ADCC, and BA6M11(WT), which does not include such mutation.

[0356] Sequences of the anti-ROR1/anti-4-1BB bispecific antibody comprising BA6 clone used as control are as follows:

TABLE-US-00014 LightchainBA6 (SEQIDNO:124) QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAP KLLIYYDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCG AWDDSLSGYVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKAT LVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS HeavychainBA6x1A10M12 (SEQIDNO:125) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGL EWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARGGNGAWDTGFDYWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSGSS SNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTS ASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSG GGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSS YDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS

[0357] Briefly, 96-well microtiter plates (NUNC, 446612) were coated overnight at 4 C. with human ROR1-Fc protein (Sinobio, 13968-H02H1) in PBS at 10 ng/well, then the plates were incubated with 1% BSA buffer (1PBS, 1% (v/v) BSA) at 200 l/well for 2 hours at 37 C. to prevent nonspecific binding. After blocking nonspecific binding, the wells were washed, and serial dilutions of the bispecific antibody test materials (starting at 100 nM) were added to each well. After incubation for 2 hours at 37 C., the plates were washed with PBS-T (1PBS/0.05% (v/v) Tween-20). Then, human 4-1BB-His protein (Sinobio, 10041-H08H) dissolved in 1% (v/v) BSA was added to each well at 100 ng/well and incubated for 1 hour at 37 C. After washing the plates with PBS-T, anti-His HRP (Roche, Cat: 11965085001) was added and incubated for 1 hour at 37 C. After washing, color development was conducted TMB (tetramethylbenzidine, Sigma, T0440) and the reaction was stopped with 0.5M sulfuric acid (Samchun Chemicals, S1410), followed by reading absorbance (OD) at 450-650 nm in a spectrophotometer. Four-parameter logistic curve analysis was performed using GraphPad software.

[0358] The results obtained through the experiments are presented in Table 12 and FIGS. 2a and 2b.

TABLE-US-00015 TABLE 12 ROR1x4-1BB BA6x1A10M12 BA6M11(NA)x1A10M12 EC50(nM) 0.386 0.268

[0359] As shown in FIG. 2a and Table 12, the bispecific antibody comprising BA6M11 did not show any decrease in binding affinity compared to the parental clone BA6, and instead, an improvement in binding affinity was observed as indicated by the lower EC50.

[0360] Additionally, when measuring the difference in binding affinity due to the presence or absence of the NA mutation, BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 showed no difference in binding activity to human ROR1 and 4-1BB.

[0361] Considering the fact that it is often challenging to maintain the binding affinity of a parental clone upon changes to its CDR sequences, the finding that the antigen-binding affinity of BA6M11, which had its sequence altered by PTM removal, was equivalent to or better than that of the parental clone BA6, demonstrates desirable characteristics of BA6M11 and its potential and advantages for further development as a therapeutic antibody.

2.2.2. Binding Assay to Cell Surface Antigen (Flow Cytometry)

[0362] Using various cells expressing human ROR1 or human 4-1BB on their surface, the binding affinity of anti-ROR1/anti-4-1BB bispecific antibodies comprising either the BA6M11(WT) or BA6M11 (NA) clone was compared by measuring with a flow cytometry device (BD).

[0363] In particular, Jurkat cells overexpressing human 4-1BB (Promega) and NCI-N87 cells, a human ROR1 positive cell line (ATCC), were seeded into V-bottom 96-well plates (96 Well Plate-RV, Bioneer, 910D09) and washed with 200 l/well of 1% BSA buffer. Fourfold dilutions of the test materials (bispecific antibodies) starting from 100 nM were added to each well and incubated for 1 hour at 4 C. The plates were then washed with 1% BSA buffer. To each well, FITC-conjugated anti-human IgG (Fc specific) (Sigma, F9512) dissolved in 1% BSA buffer was added and incubated for 1 hour at 4 C. The plates were washed again with 1% BSA buffer. The mean fluorescence intensity (MFI) of FITC was measured by flow cytometry. Four-parameter logistic curve analysis was performed on the obtained MFI values using GraphPad software.

[0364] As shown in FIGS. 2c and 2d, both BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 bound similarly to Jurkat cells overexpressing human 4-1BB and ROR1-positive NCI-N87 cells, demonstrating that there is no difference in binding activity to human 4-1BB and human ROR1.

2.2.3. Binding Affinity Assay Using Surface Plasmon Resonance (SPR)

[0365] The binding affinity of both BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 clones, which are anti-ROR1/anti-4-1BB bispecific antibodies comprising BA6M11 clone, was assessed using surface plasmon resonance (SPR). The antigens used for this assessment were human ROR1 protein (13968-H02H1, Fc tag) from Sino Biological and human 4-1BB protein (9220-4B-100, his tag) from R&D Systems. The measurements were conducted on a Biacore T200 instrument (Cytiva).

[0366] Candidate materials were measured for binding affinity for human ROR1 as follows.

[0367] The CM5 chip (Cytiva, BR100530) was coated with a 5 g/mL human ROR1 protein dilution in Acetate 4.0 (Cytiva, BR100349) using the amine coupling method (Cytiva, BR100050) to achieve a surface density of about 80 RU. The BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 samples were two-fold serially diluted in HBS-EP buffer (1) (Cytiva, BR100669) to form concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 0 nM. These diluted antibody samples were then flowed over the human ROR1 protein-immobilized CM5 chip for approximately 60 seconds to conjugate the sample to the antigen, followed by flowing HBS-EP buffer (1) over the sample-antigen complex for 180 seconds to dissociate the sample from the antigen. The chip surface was regenerated by flowing Glycine-HCl pH1.5 (Cytiva, BR100354) over the chip for 30 seconds. The assay was performed with HBS-EP (1) as the running buffer, at a flow rate of 30 L/min and at a temperature of 25 C. Binding affinity was analyzed using the bivalent analyte model to derive the primary association rate constant (Ka1) and primary dissociation rate constant (Kd1). The equilibrium dissociation constant (KD) was calculated by dividing the primary dissociation rate constant by the primary association rate constant, and this value was used to represent the final binding affinity.

[0368] Candidate materials were measured for binding affinity for human 4-1BB as follows: 10 g/mL anti-Fc antibody (Invitrogen, 31125) diluted in 5.0 (Cytiva, BR100351) was fixed at about 15000 RU onto a CM5 chip (Cytiva, BR100530) using the amine coupling method. BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 diluted in HBS-EP (1) buffer were then bound at 300 RU to the anti-Fc antibody-immobilized CM5 chip. Human 4-1BB antigen was serially two-fold diluted in HBS-EP (1) buffer to form concentrations of125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, and 0 nM. These diluted human 4-1BB samples were then flowed over the CM5 chip for approximately 60 seconds to conjugate the sample to the antigen, followed by flowing HBS-EP buffer (1) over the sample-antigen complex for 180 seconds to dissociate the sample from the antigen. The chip surface was regenerated by flowing Glycine-HCl pH1.5 (Cytiva, BR100354) over the chip for 30 seconds. The assay was performed with HBS-EP (1) as the running buffer, at a flow rate of 30 L/min and at a temperature of 25 C. The binding affinity was determined using a 1:1 binding model to derive the association rate constant (Ka) and dissociation rate constant (Kd). The equilibrium dissociation constant (KD) was calculated by dividing the dissociation rate constant by the association rate constant, and this value was used as the final measure of binding affinity.

[0369] The results thus obtained are summarized in Table 13, below:

TABLE-US-00016 TABLE 13 Antibody Ka Kd KD Rmax Antigen sample (1/Ms, 10.sup.5) (1/s, 10.sup.3) (M, 10.sup.9) (RU) Human BA6M11(WT)x1A10M12 2.408 1.456 6.049 40.56 ROR1 0.001 0.066 0.277 0.74 BA6M11(NA)x1A10M12 2.289 1.522 6.654 38.87 0.047 0.129 0.701 0.14 Antibody Ka Kd KD Rmax Antigen sample (1/Ms, 10.sup.5) (1/s, 10.sup.4) (M, 10.sup.9) (RU) Human BA6M11(WT)x1A10M12 2.236 10.12 4.527 43.52 4-1BB 0.022 0.31 0.098 0.48 BA6M11(NA)x1A10M12 2.207 9.924 4.497 39.77 0.027 0.292 0.078 0.43

[0370] As indicated in Table 13, both BA6M11(WT)1A10M12 and BA6M11 (NA)x1A10M12 exhibited similar levels of binding to human ROR1 and human 4-1BB, confirming that there was no difference in antigen-binding activity between the WT and NA cell lines.

2.2.4. Assay for Developability of Anti-ROR1/4-1BB Bispecific Antibody

[0371] Anti-ROR1/4-1BB bispecific antibodies comprising BA6 clone or BA6M11 clone were assayed for developability.

[0372] For the analysis of developability, capillary isoelectric focusing (cIEF) or imaged capillary isoelectric focusing (icIEF) were conducted. Both methods analyze changes in charge variants using the isoelectric point (pI) value.

[0373] Samples for the developability analysis were prepared by exposing them to temperatures of 4 C. and 37 C. (or 40 C.) for two weeks. The storage temperatures were 4 C. or 37 C. for the BA6x1A10M12 clone and 4 C. or 40 C. for both BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 clones.

[0374] The cIEF analysis was carried out using a PA 800 plus instrument from Beckman Coulter, equipped with a UV detector. A neutral capillary cartridge (50 m I.D.) was crafted to a total length of 30.2 cm and mounted in the instrument.

[0375] The bispecific antibody samples used for analysis were diluted with a formulation buffer (20 mM histidine, 7% (w/v) trehalose, pH 6.0) to for a final concentration of 5 mg/mL. The master mix solution was prepared using a 3M urea solution including gel (Urea: GE Healthcare, 17-1319-01; cIEF Gel: Beckman Coulter, 477497), ampholyte 3-10 (Pharmalyte 3-10; GE Healthcare, 17-0456-01), an anodic stabilizer solution (200 mM iminodiacetic acid; Sigma, 220000), a cathodic stabilizer solution (500 mM L-arginine; Sigma, A5006), and synthetic peptide isoelectric point markers (10, 9.5, 7.0, 5.5, 4.1; cIEF Peptide Marker Kit; Beckman Coulter, A58481). 10 L of analysis sample (bispecific antibody) was mixed with 239 L of the master mix solution and centrifuged. 200 L of the mixture was transferred to a PCR tube, placed in a plastic vial, and prepared on the sample tray. Distilled water, anolyte solution (200 mM phosphoric acid; Sigma, 345-245), catholyte solution (300 mM sodium hydroxide; Sigma, 79724), capillary cleaning solution (4.3 M urea; GE Healthcare, 17-1319-01), cIEF gel solution (Beckman Coulter, 477497), and chemical mobilizer solution (350 mM acetic acid; Sigma, 695092) were prepared in individual plastic vials on the buffer tray for analysis.

[0376] For the icIEF analysis, the Maurice instrument and capillary cartridge from ProteinSimple were utilized. The Maurice cIEF Cartridge (ProteinSimple, PS-MC02-C) was filled with 2 mL of catholyte solution (100 mM sodium hydroxide in 0.1% methyl cellulose; ProteinSimple) and 2 mL of anolyte solution (0.08 M phosphoric acid in 0.1% methyl cellulose; ProteinSimple) and then mounted in the instrument.

[0377] The bispecific antibody samples for analysis were diluted in distilled water to form a final concentration of 1 mg/mL. The master mix solution was prepared using an 8 M Urea solution (Sigma, U6504), 1% methyl cellulose (Methyl Cellulose; ProteinSimple), ampholytes (pH 3-10) (Pharmalyte pH 3-10; ProteinSimple, 042-684 or GE Healthcare, 17-0456-01), distilled water (UltraPure DNase/RNase-Free Distilled Water; Invitrogen, 10977015), and peptide isoelectric point markers (5.85, 9.50; ProteinSimple). 20 L of the analysis sample (bispecific antibody) was mixed with 80 L of the master mix solution, centrifuged, and then transferred to a 96-well plate for analysis.

[0378] Distilled water, 0.5% methyl cellulose (ProteinSimple), fluorescence calibration standard (ProteinSimple), and air were prepared in individual vials and placed in their respective positions for analysis.

[0379] The results of this analysis are presented in Table 14, below:

TABLE-US-00017 TABLE 14 BA6 BA6M11(WT) BA6M11(NA) 1A10M12 1A10M12 1A10M12 Content Method 4 C. 37 C. 4 C. 40 C. 4 C. 40 C. Charge cIEF Acidic 24.7 27.3 22.5 36.7 21.0 35.8 heterogeneity or Main 51.6 34.6 75.4 59.3 77.5 60.2 icIEF Basic 23.7 38.1 2.1 3.9 1.5 4.0

[0380] The developability assessment results presented in Table 14 indicate that the main peak content of BA6x1A10M12 was about 52% under storage conditions at 4 C. This clone showed a decrease of about 17% in the content of the main peak under storage conditions at 37 C. compared to 4 C., while the basic peak content increased by about 14%.

[0381] Assay results for developability of the two BA6M11 clone variants with modified BA6 clone are as follows: The main peak content at 4 C. was approximately 75% for BA6M11(WT)x1A10M12 and about 78% for BA6M11 (NA)x1A10M12. Both clones showed a decrease of about 16-17% in the content of the main peak under storage conditions at 40 C. compared to 4 C., with an increase of about 2-3% in the content of the basic peaks.

[0382] The BA6M11 clones tested showed improved characteristics in the content of basic charge variants compared to BA6. A decrease in the content of basic charge variants was confirmed through the analysis of samples stored at 4 C., indicating a reduction during production as well, and the rate of change under high-temperature storage conditions (40 C.) was also reduced, and the two mutant clones, based on samples stored at 4 C., showed that the WT and NA forms have similar main peak contents. From these results, it was confirmed that the BA6M11 clones possess superior developability compared to the parent BA6 clone.

2.2.5. Analysis for Serum Stability of Anti-ROR1/Anti-4-1BB Bispecific Antibody

[0383] To evaluate the serum stability of the anti-ROR1/anti-4-1BB bispecific antibodies comprising either the BA6 clone (parental clone) or the BA6M11 clone (PTMs removed), they were stored in human and monkey serum and then analyzed and compared using the ELISA method.

[0384] In this analysis, the anti-ROR1/anti-4-1BB bispecific antibodies were stored in human and monkey serum at 37 C. for 2 days, 7 days, and 14 days.

[0385] A 96-well microtiter plate (NUNC, 446612) was coated overnight at 4 C. with human ROR1-Fc protein (Sinobio, 13968-H02H1) at 100 ng/well in PBS. The plate was then incubated with 1% BSA buffer (1PBS, 1% (v/v) BSA) at 200 l/well at 37 C. for 2 hours to block non-specific binding. Thereafter, the blocking solution was discarded and fourfold dilutions of each bispecific antibody test material (starting from 100 nM) were added to the wells. The plate was incubated at 37 C. for 2 hours and then washed with PBS-T (1PBS/0.05% (v/v) Tween-20). Subsequently, human 4-1BB-His protein (Sinobio, 10041-H08H) dissolved in 1% BSA buffer was added at 100 ng/well and incubated at 37 C. for 1 hour. After washing with PBS-T, the plate was incubated with anti-His HRP (Roche, Cat: 11965085001) at 37 C. for 1 hour. After washing, color development was performed with TMB (tetramethylbenzidine, Sigma, T0440) and the color development was stopped with 0.5M sulfuric acid (Samchun Chemicals, S1410), and the absorbance (OD) at 450-650 nm was read using a spectrophotometer. Four-parameter logistic curve analysis was performed using GraphPad software.

[0386] The results obtained are presented in Table 15 and FIGS. 3a to 3d.

TABLE-US-00018 TABLE 15 BA6 1A10M12 BA6M11(NA) 1A10M12 EC50 EC50 Incubation EC50 Ratio Incubation EC50 Ratio Serum days (nM) (%) Serum days (nM) (%) Human 0 day 0.228 100 Human 0 day 0.211 100 serum 2 days 0.365 62 serum 3 days 0.220 96 7 days 0.420 54 7 days 0.236 89 14 days 0.457 50 14 days 0.233 91 Monkey 0 day 0.240 100 Monkey 0 day 0.208 100 serum 2 days 0.360 67 serum 3 days 0.238 87 7 days 0.427 56 7 days 0.257 81 14 days 0.419 57 14 days 0.273 76

[0387] As indicated in Table 15 and FIGS. 3a to 3d, the anti-ROR1/anti-4-1BB bispecific antibodies comprising BA6M11 maintained stability over a longer period in both human and monkey serum compared to the bispecific antibodies comprising the parental BA6 clone, demonstrating improved serum stability. Serum stability is a crucial property that significantly affects the efficacy of antibody drugs when administered in vivo, particularly in terms of binding affinity and specificity, and should be considered in antibody development and production. Thus, these results confirm that BA6M11 significantly contributes to enhancing the serum stability of the antibodies compared to BA6.

2.2.6. 4-1BB Signal Activation of Anti-ROR1/Anti-4-1BB Bispecific Antibody (4-1BB Reporter Bioassay)

2.2.6.1. 4-1BB Signal Activation Depending on ROR1 Expression

[0388] To measure, compare, and analyze the 4-1BB signal activation of anti-ROR1/4-1BB bispecific antibodies, comprising the BA6 clone or BA6M11 clone, a 4-1BB NFB luciferase reporter bioassay was conducted. The 4-1BB reporter bioassay used a human ROR1-positive cell line, the gastric cancer cell line AGS, and a hamster ovarian epithelial cell CHOK1 stably overexpressing human ROR1 (CHOK1-hROR1).

[0389] In this analysis, the GloResponse NFB-luc2/4-1BB Jurkat cell line (Promega, cat #CS196004), genetically engineered to stably express human 4-1BB and luciferase downstream of the response element, was used as the effector cell.

[0390] In brief, AGS (ROR1 positive gastric cancer cell line, ATCC, CRL-1739, 2.510.sup.4) or CHOK1-hROR1 (ROR1 overexpressing hamster ovarian epithelial cells, BPS Bioscience, 79609-H, 2.510.sup.4) were each dispensed into white 96-well assay plates containing 100 L of culture medium (10% (v/v) FBS, RPMI 1640) and incubated overnight at 37 C. in a 5% CO.sub.2 humidified incubator. After overnight incubation, 100 L of culture medium was removed, and 25 L of assay medium (1% (v/v) FBS, RPMI 1640) was dispensed onto the pre-attached target cells (AGS or CHOK1-hROR1) as previously described. 25 L of bispecific antibody (starting from 40 nM and serially diluted 5-fold) was added to the plate. 25 L of GLORESPONSE NFB-luc2/4-1BB Jurkat cell suspension (Promega, CS196004) was dispensed into the plate to achieve 2.510.sup.4 cells per well and incubated for 6 hours at 37 C. in a 5% CO.sub.2 humidified incubator. During the incubation period, the BIO-GLO Luciferase Assay System (Promega, G7940) was reconstituted according to the manufacturer's instructions. After 6 hours of incubation, 75 L of BIO-GLO Reagent was added to each well of the assay plate. Five 5 minutes later, luminescence was measured using a microplate reader. The four-parameter logistic curve was evaluated using GraphPad software. The results of the experiments using AGS and CHOK1-hROR1 cells are presented in Table 16 and FIG. 4a, respectively.

TABLE-US-00019 TABLE 16 EC50 (nM) Clone Name AGS CHOK1-ROR1 BA6M11(WT)x1A10M12 0.162 0.015 BA6M11(NA)x1A10M12 0.149 0.014 BA6x1A10M12 0.200 0.018

[0391] As shown in Table 16 and FIG. 4a, the anti-ROR1/4-1BB bispecific antibody comprising BA6M11 exhibited lower EC50 (nM) values in ROR1-expressing cells compared to the bispecific antibody comprising the parental clone, BA6, indicating that the anti-ROR1/4-1BB bispecific antibody comprising BA6M11 induces a stronger 4-1BB signal activation in the presence of the ROR1 antigen. Specifically, the anti-ROR1/4-1BB bispecific antibody comprising BA6M11 showed superior 4-1BB signal activation effects in both ROR1 positive tumor cells (AGS) and human ROR1 overexpressing cells (CHOK1-hROR1), and these results were observed regardless of the presence or absence of the NA mutation.

[0392] The data demonstrates that the anti-ROR1/4-1BB bispecific antibody comprising BA6M11 specifically targets ROR1-expressing cancer cells.

[0393] To further validate that the activation of 4-1BB by BA6M11 (NA)x1A10M12 and BA6M11(WT)x1A10M12 is dependent on the expression of ROR1, a mixture of parental CHOK1 cells and CHOK1 cells overexpressing human ROR1 (CHOK1-hROR1) was used to measure 4-1BB signal activation as described previously. For comparison, Urelumab (BMS-663513), an anti-4-1BB antibody, was used.

[0394] The results are presented in Table 17 and FIG. 4b

TABLE-US-00020 TABLE 17 Clone EC50 (nM) Name CHO-hROR1 100% BA6M11(WT)x1A10M12 0.016 BA6M11(NA)x1A10M12 0.015 Urelumab 0.235

[0395] As illustrated in FIG. 4b, both BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 demonstrated a higher Max fold induction as the proportion of CHOK1-ROR1 cells increased. This indicates that the more cells express ROR1, the more 4-1BB activation is induced. As shown in FIG. 4b and Table 17, BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 induced 4-1BB activation at similar levels.

2.2.6.2. 4-1BB Signal Activation of Anti-ROR1/Anti-4-1BB Bispecific Antibody Depending on ROR1 Expression Level

[0396] The cell surface expression levels of ROR1 in various cancer cell lines were quantified using the QIFIKIT quantification kit (Dako, K0078) according to the manufacturer's recommendations. Briefly, the cancer cells used in the test were stained at saturation with an unlabeled anti-ROR1 mouse monoclonal antibody (BD, 564464) or a purified mouse IgG2b isotype control antibody (BD, 555740). After washing, the stained cells and calibration beads were simultaneously labeled with the same FITC-conjugated goat anti-mouse IgG secondary antibody. Both the calibration beads and the secondary antibody are included in the kit. The labeled cells and calibration beads were analyzed by flow cytometry. Linear regression analysis was performed using the MFI values of the calibration beads. Using the regression line derived from the linear regression analysis, the Antibody Binding Capacity (ABC) for both the anti-ROR1 antibody and the isotype control antibody was calculated, and the specific ABC (sABC) value was obtained by subtracting the value of the isotype control antibody from the ABC value of the anti-ROR1 antibody.

[0397] The results obtained are presented in Table 18.

TABLE-US-00021 TABLE 18 Cell lines ROR1 sABC MFI Fold CHO-k1/ROR1 BPS Bioscience, 79609-H 765,378 836 HCC1187 KCLB, 9S1187 22,824 10.3 NCI-N87 ATCC, CRL-5822 21,512 31 Kasumi-2 DSMZ, ACC 526 10,156 26.8 MDA-MB-231 ATCC, HTB-26 8,902 10 AGS ATCC, CRL-1739 7,209 11.5 697 DSMZ, ACC 42 6,081 22 DLD1 ATCC, CCL-221 5,327 9.6 HCC1954 ATCC, CRL-2338 1,874 1.9 MCF-7 ATCC, HTB-22 57 1 CHO-k1 ATCC, CRL-1469 4 1 Jurkat ATCC, TIB-152 1 1 BT474 ATCC, HTB-20 1 1 MC38 Kerafast, ENH204 0 1

[0398] The correlation between the obtained ROR1 expression levels (ROR1 sABC) and the 4-1BB-induced NF-kB signal by the antibody was tested.

[0399] The ROR1 levels measured in Table 18 were standardized to the ROR1 expression levels in NCI-N87, and the levels of 4-1BB activation induced by the bispecific antibodies BA6M11(WT)x1A10M12, BA6M11 (NA)x1A10M12, and the control antibody Urelumab were determined as the maximum fold change compared to the control group in the 4-1BB NF-kB luciferase reporter assay. The method used was the same as described in Example 2.2.6.1. The shaded area represents the confidence interval for the linear fit.

[0400] The results obtained are presented in FIG. 4c. As shown in FIG. 4c, compared to the control antibody, the 4-1BB activation induced by the anti-ROR1/4-1BB bispecific antibodies BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 showed a high correlation with ROR1 cell surface expression.

2.6.3. 4-1BB Signal Activation According to FcRI Engagement

[0401] 4-1BB activation signals were evaluated using CHO-K1 cells (Promega) expressing FcRI, FcRIIb, or FcRIIIa. The method used was the same as described in Example 2.2.6.1. Four-parameter logistic curve analysis was performed using GraphPad Prism software.

[0402] As shown in FIG. 4d, only CHO-K1 cells overexpressing FcRI induced the 4-1BB signal of BA6M11(WT)x1A10M12. From this data, it was determined that for BA6M11 (NA)x1A10M12, which has an NA mutation in the Fc, the binding to FcRI is weakened could not induce 4-1BB signal activation, however, BA6M11(WT)x1A10M12, without the mutation introduced in the Fc, was found to induce strong 4-1BB signal activation in the presence of FcRI. CHO-K1 cells overexpressing FcRIIb or FcRIIIa, which have weaker binding to the Fc region compared to FcRI, did not induce 4-1BB signal activation.

2.2.7. Induction of T Cell Immune Response and Cytokine Secretion Activity by Anti-ROR1/Anti-4-1BB Bispecific Antibody in ROR1-Expressing Cells (In Vitro Efficacy Test)

2.2.7.1. PBMC Activation Test Using CHOK1 Cells Artificially Overexpressing ROR1

[0403] The 4-1BB signal activation ability of anti-ROR1/4-1BB bispecific antibodies comprising either the BA6 clone or BA6M11 clone to activate human peripheral blood mononuclear cells (PBMCs) was evaluated by co-culturing these bispecific antibodies with hamster ovarian epithelial cells (CHOK1-hROR1) stably expressing human ROR1. Interferon gamma (IFN-gamma), known as a cytokine, is primarily produced by natural killer (NK) cells, CD4+ helper T cells (Th1), and CD8+ cytotoxic T cells. Interferon gamma was measured to assess PBMC activation in this Example.

[0404] Briefly, a 96-well plate was coated with 5 g/mL of anti-human CD3 (Biolegend, 300438) (in PBS) at 37 C. for 2 hours. After removing the anti-human CD3 solution, human PBMCs (Peripheral Blood Mononuclear Cell; CTL-UP-1) were added at a density of 3x10.sup.4 cells per well, and CHOK1-hROR1 cells were added at a density of 3x10.sup.4 cells per well. The test antibody (starting at 50 nM, diluted 5-fold) was added to the wells of the plate. The plate was then incubated for 72 hours at 37 C. in a 5% CO.sub.2 humidification incubator. After incubation, the concentration of interferon gamma (IFN-gamma) released into the supernatant was measured using the Human IFN-gamma Duoset ELISA Kit (R&D Systems, DY285) according to the manufacturer's protocol. Four-parameter logistic curve was evaluated using GraphPad software.

[0405] As shown in FIG. 5a, the anti-ROR1/4-1BB bispecific antibody BA6M11 (NA)x1A10M12 induced the release of IFN-gamma at lower concentrations compared to BA6x1A10M12, and at the same concentration, it induced a higher release of IFN-gamma, thus, the bispecific antibody comprising BA6M11 demonstrated superior PBMC activation efficacy compared to the parental clone, BA6. Furthermore, bispecific antibodies comprising BA6M11, such as BA6M11 (NA)x1A10M12 and BA6M11 (NA)x1A10M12, induced the release of IFN-gamma at similar levels.

2.2.7.2. PBMC Activation Test Using Gastric Cancer Cell Line Expressing ROR1

[0406] NCI-N87, a gastric cancer cell line expressing human ROR1, was used to evaluate whether the 4-1BB signal activation activity of the bispecific antibodies of the present disclosure can activate human peripheral blood mononuclear cells (PBMCs) when co-cultured with these human ROR1 expressing gastric cancer cells. In brief, a 96-well plate was coated with 5 g/mL of anti-human CD3 (Biolegend, 300438) dissolved in PBS at 37 C. for 2 hours. Subsequently, human PBMCs (CTL) and NCI-N87 (ATCC) were added to the plate. Four-fold diluted solutions of each test antibody (starting at 20 nM) were added to each well and incubated for 72 hours. After 72 hours of incubation, the concentration of IFN-gamma in the supernatant was measured using the Human IFN-gamma Duoset ELISA Kit (R&D System, DY285B). The viability of NCI-N87 was measured with the cell counting kit-8 (Dojindo, CK04-20).

[0407] The results obtained are shown in FIGS. 5b and 5c. As indicated in FIGS. 5b and 5c, the anti-ROR1/4-1BB bispecific antibody comprising BA6M11, compared to the control antibody (urelumab), had a lower EC50 (nM) value, induced IFN-gamma secretion at lower concentrations, and induced cell death in NCI-N87 at lower concentrations. This data indicates a superior PBMC activation capability in the presence of the human ROR1 antigen compared to the control antibody. Furthermore, the anti-ROR1/4-1BB bispecific antibody comprising BA6M11, in both WT and NA forms, demonstrated superior PBMC activation capability compared to the combination of BA6M11 alone and 1A10M12 alone (BA6M11+1A10M12). From this observation, it was confirmed that the anti-ROR1/4-1BB bispecific antibody of the present disclosure exhibits a superior synergistic effect compared to the simple combination of monospecific antibodies.

2.3. In Vivo Efficacy of Anti-ROR1/Anti-4-1BB Bispecific Antibody

2.3.1. Anti-Tumor Efficacy

[0408] In vivo anti-tumor efficacy of the anti-ROR1/anti-4-1BB bispecific antibodies BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 was evaluated using human 4-1BB knock-in mice (Biocytogen). Briefly, MC38 colon cancer cells, stably expressing human ROR1 (hROR1-expressing MC38 cells, 510.sup.5, Biocytogen), were subcutaneously injected into the right flank of the mice. The tumor-bearing humanized mice were divided into three groups based on tumor size (average tumor size: about 89 mm.sup.3, n=5 mice/group). After grouping, on day 0, a single intravenous dose of human IgG1 antibody (hIgG1 antibody, 3 mg/kg), BA6M11(WT)x1A10M12 (4 mg/kg), or BA6M11 (NA)x1A10M12 (4 mg/kg) was administered. Tumor size was measured twice a week using a digital caliper, and tumor volume was calculated using the formula: V (mm.sup.3)=a*b2/2 (where a and b represent the long and short dimensions of the tumor, respectively). The tumor growth inhibition (TGI) percentage was calculated based on tumor volume using the formula: TGI % (Day i)=[1(T.sub.Day(i)T.sub.Day0)/(V.sub.Day(i)V.sub.Day0)]100% (T: tumor volume of the treated group; V: tumor volume of the control group).

[0409] The results of the anti-tumor efficacy are presented in FIG. 6a and Table 19.

TABLE-US-00022 TABLE 19 Survival Dosing TGI (%) rate (%) Test articles Dosage Time (Day 23) (Day 44) hIgG1 3 mg/kg 1 0 0 BA6M11(WT)x1A10M12 4 mg/kg 1 98.6 100.0 BA6M11(NA)x1A10M12 4 mg/kg 1 69.9 80.0

[0410] As shown in FIG. 6a and Table 19, BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 demonstrated significant tumor suppression effects. On the 23.sup.rd day after grouping, the TGI for the groups treated with BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 were 98.6% and 69.9%, respectively, and on the 44th day, the survival rates for the BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12 treated groups were 100% and 80%, respectively, while the hIgG1 control group had no survivors. This confirms that both bispecific antibodies, BA6M11(WT)x1A10M12 and BA6M11 (NA)x1A10M12, exhibited superior anti-tumor efficacy and high survival rates even with a single administration.

2.3.2. Dose-Dependent Anti-Tumor Efficacy

[0411] To confirm the dose-dependent effects of the bispecific antibody BA6M11(WT)x1A10M12, in vivo anti-tumor efficacy was evaluated in human ROR1-expressing MC38-bearing mice. Specifically, tumor cells (hROR1-expressing MC38 cells, 510.sup.5 cells) were subcutaneously injected into the right flank of human 4-1BB knock-in mice to prepare the human ROR1-expressing MC38-bearing mice. These prepared mice were randomly grouped based on a tumor volume (about 80 mm.sup.3). On day 0 of grouping, a single intravenous injection of hIgG1 antibody (10 mg/kg) or BA6M11(WT)x1A10M12 (0.4 mg/kg, 1 mg/kg, or 4 mg/kg) was administered. The anti-tumor efficacy was measured referring to Example 2.3.1.

[0412] As shown in FIG. 6b, BA6M11(WT)x1A10M12 exhibited dose-dependent anti-tumor efficacy. Additionally, treatment with BA6M11(WT)x1A10M12 increased the number of mice with complete tumor regression (0% in the hIgG1 treated group vs. 50% in the BA6M11(WT)x1A10M12 4 mg/kg treated group), and the mice with complete regression survived for more than 100 days without signs of tumor recurrence after the initial tumor implantation.

2.3.3. Memory Response Upon Serial Tumor Re-Challenge

[0413] To evaluate the long-lasting memory response, tumor re-challenge was performed in mice that had been cured of their tumors by BA6M11(WT)x1A10M12. The tumor re-challenge was performed 100 days after the last dose of the antibody in cured mice. Specifically, hROR1-expressing MC38 cells (2.510.sup.6 cells, 5 times the initial inoculation) were subcutaneously injected into both the cured mice and control nave mice (mice that had not undergone tumor inoculation and antibody treatment) for a secondary inoculation. 59 days after the secondary inoculation, surviving mice were re-challenged with the tumor (2.510.sup.6 cells, tertiary inoculation). The anti-tumor efficacy was measured referring to Example 2.3.1.

[0414] As shown in FIG. 6c, mice previously treated with the bispecific antibody demonstrated resistance to repeated secondary and tertiary tumor re-challenges, while control nave mice did not exhibit tumor reduction. This confirmed that treatment with BA6M11(WT)x1A10M12 induces a sustained anticancer memory response.

2.3.4. In Vivo Immune Modulation

[0415] To understand the effects of the bispecific antibody on immune cells, tumor and peripheral tissues (blood and liver) were analyzed using flow cytometry.

[0416] In particular, human ROR1-expressing MC38 tumor-bearing humanized mice used in the previous example were randomly grouped based on a tumor volume of about 100 mm.sup.3. On days 0 and 4 after grouping, mice were intraperitoneally administered with hIgG1 antibody (7.5 mg/kg), urelumab (7.5 mg/kg), or the bispecific antibody BA6M11(WT)x1A10M12 (10 mg/kg). On day 7 after grouping, immune cells were analyzed using flow cytometry. In the tumor and peripheral blood, the proportions of leukocytes (CD45.sup.+) and Treg cells (CD45.sup.+CD3.sup.+CD4.sup.+FOXP3.sup.+) and the expression of 4-1BB on T cells (CD45.sup.+CD3.sup.+) were analyzed. In the liver, the proportions of T cells, CD8.sup.+ T cells (CD45.sup.+CD3.sup.+CD8.sup.+), and macrophages (CD45.sup.+CD11b.sup.+F4/80.sup.+) were analyzed. The results are shown in FIG. 6d (A: Tumor; B: Peripheral Blood; C: Liver).

[0417] As illustrated in FIG. 6d, a significant increase in tumor-infiltrating leukocytes (CD45+/Live) was observed in the BA6M11(WT)x1A10M12 treated group. Additionally, unlike urelumab, BA6M11(WT)x1A10M12 specifically induced the depletion of immunosuppressive Treg cells (Treg/CD45+) in the tumor, not in the systemic circulation. The expression of 4-1BB on T cells (4-1BB+/CD3+) was measured at higher levels in the tumor compared to the blood, and its levels were increased by treatment with BA6M11(WT)x1A10M12. These results confirm the tumor-specific T cell activation effects of the bispecific antibody of this application. Furthermore, while urelumab's systemic activation was accompanied by a massive infiltration of immune cells into the liver, the bispecific antibody of this application did not show an increase in T cells and macrophages in the liver. These results confirmed that the bispecific antibody of the present application exerts a strong anti-tumor effect through tumor-specific immune modulation while minimizing the risk of peripheral toxicity.

2.3.5. Additional Demonstration of Tumor-Specific T Cell Activation by Anti-ROR1/Anti-4-1BB Bispecific Antibody

[0418] To prove that the bispecific antibody of the present application specifically activates T cells only in cells expressing ROR1 (e.g., tumor cells) and does not act on normal cells and tissues that do not express ROR1, the cytokine secretion activity induced by the anti-ROR1/anti-4-1BB bispecific antibody was verified using human PBMCs in the absence of ROR1-expressing cells.

[0419] In particular, five types of human PBMCs (CTL, CTL-UP1) were thawed and then seeded in a 96-well plate at a concentration of 210.sup.5/well in 10% RPMI medium. As a positive control, anti-human CD3 antibody (Biolegend, 317326) was diluted in RPMI with 10% FBS to concentrations of 0.01 g, 0.1 g, 1 g, 25 g, and 50 g and added to each well. For the test group, 1 g, 25 g, and 50 g of the bispecific antibody (BA6M11(WT)x1A10M12) were added to each well.

[0420] The plate was incubated in a 37 C., 5% CO2 incubator for 48 hours. After 48 hours, the plate was centrifuged to separate the supernatant. ELISA analysis was performed according to the manufacturer's protocol to measure the concentrations of IFN-gamma (R&D System, DY285B), IL-2 (R&D System, DY202), IL-6 (R&D System, DY206), and TNF-alpha (R&D System, DY210) in the culture supernatant. The results are presented in FIG. 6e.

[0421] As shown in FIG. 6e, compared to the positive control, BA6M11(WT)x1A10M12 did not induce cytokine release in the absence of ROR1. This means that the bispecific antibody of the present application activates T cells only in tumor tissues expressing ROR1 and not in normal cells and tissues, suggesting a reduced risk of unexpected toxicity upon administration in the body.

Example 3. ROR1xB7-H3 Bispecific Antibody and Antibody-Drug Conjugate (ADC) Comprising Same

3.1. Design and Construction of Improved Anti-ROR1/Anti-B7-H3 Bispecific Antibody

[0422] Based on the anti-ROR1 antibody clone BA6M11 selected in Example 1 and the anti-B7-H3 antibody clone B5 disclosed in the international publication WO2019-226017 A1, an anti-ROR1/anti-B7-H3 bispecific antibody was manufactured that comprises both an anti-ROR1 antibody and an anti-B7-H3 antibody.

[0423] This bispecific antibody was designed in an IgG-scFv fusion format, where the scFv antibody fragment against one of the antigens (either ROR1 or B7-H3) is fused to the C-terminus (heavy chain C-terminus) of the full-length IgG antibody against the other antigen. In this Example, a bispecific antibody comprising the anti-ROR1 IgG antibody and the B5 scFv linked to the C terminus of each of the heavy chains of the anti-ROR1 IgG antibody was constructed.

[0424] To construct the anti-ROR1/anti-B7-H3 bispecific antibody, a DNA segment 1 comprising the nucleotide sequence coding for the heavy chain of the anti-ROR1 IgG antibody (BA6M11) was inserted into pcDNA 3.4 (Invitrogen, A14697; Plasmid 1), and a DNA segment 2 comprising the nucleotide sequence coding for the light chain of the IgG antibody in the anti-ROR1/anti-B7-H3 bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; Plasmid 2). Subsequently, a DNA segment 3 coding for the scFv was fused to a part of DNA segment 1 corresponding to the C-terminus of the Fc region of the IgG antibody in Plasmid 1 using DNA segment 4 coding for a peptide linker composed of 20 amino acids length (GGGGS) 3 (SEQ ID NO: 120), to construct a vector for the expression of the bispecific antibody.

[0425] Additionally, the anti-B7-H3 scFv (named B5) is in a form where the C-terminus of the light chain variable region (VL) and the C-terminus of the heavy chain variable region (VH) are each fused through a linker ((GGGGS) 4) (SEQ ID NO: 114) with VL103-VH44 (VL103: VL with a G(Gly).fwdarw.C(Cys) mutation introduced at position 103; VH44: VH with a G.fwdarw.C mutation introduced at position 44). These mutations stabilize the scFv and enable the formation of disulfide bridges.

[0426] The amino acid sequences of the thus prepared bispecific antibody (BA6M11xB5) are listed in Table 20, below.

TABLE-US-00023 TABLE20 SEQ ID Aminoacidsequence(N.fwdarw.C) NO:# Lightchain QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQ 111 BA6M11 QLPGTAPKLLIYYENNRPSGVPDRFSGSKSGTSASLAIS GLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPAECS HeavychainBA6M11(WT)xB5 Heavychain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWV 112 BA6M11 RQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDN (WT) SKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK Linker1 GGGGSGGGGSGGGGS 120 B5(VL) QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNAVSWYQ 98 QLPGTAPKLLIYYNSHRPSGVPDRFSGSKSGTSASLAIS GLRSEDEADYYCGSWDASLNAYVFGCGTKLTVL Linker2 GGGGSGGGGSGGGGSGGGGS 114 B5(VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWV 86 RQAPGKCLEWVSSISSGSGSIYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCAKNLIPLDYWGQGTL VTVSS Full-length EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWV 121 RQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQ RVTISCSGSSSNIGSNAVSWYQQLPGTAPKLLIYYNSH RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSW DASLNAYVFGCGTKLTVLGGGGSGGGGSGGGGSGGG GSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMS WVRQAPGKCLEWVSSISSGSGSIYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAKNLIPLDYWGQG TLVTVSS (Linker1: linkage between BA6M11(IgG) and B5(scFv) (C-terminus of heavy chain in BA6M11 linked to N-terminus of VL in B5); Linker2: linkage between VL and VH in B5 (scFv) (C-terminus of VL linked to N-terminus of VH)

[0427] Coding nucleic acid sequences of the bispecific antibody (BA6M11xB5) are as follows:

TABLE-US-00024 [Heavychain_BA6M11xB5] (SEQIDNO:122) gaagttcagctgctggaatctggcggcggattggttcaacctggcggctctctgagactgtcttgtgccgcttct ggcttcaccttctccaactacgacatgtcctgggtccgacaggctcccggcaaaggattggaatgggtgtccccc atctaccactccggctcctctaagtactacgccgactccgtgaagggcagattcaccatctctcgggacaactcc aagaacaccctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccagaggcgga tctggcgcctgggacaccggctttgattattggggacagggcaccctggtgaccgtgtcctccgcctccaccaag ggcccctccgtgttccccctggccccctcctccaagtccacctccggcggcaccgccgccctgggctgcctggtg aaggactacttccccgagcccgtgaccgtgtcctggaactccggcgccctgacctccggcgtgcacaccttcccc gccgtgctccagtcctccggcctctactccctctcctccgtcgtgaccgtgccctcctcctccctgggcacccag acctacatctgcaacgtgaaccacaagccctccaacaccaaggtcgacaagaaggtcgagcccaagtcctgcgac aagacccacacctgccctccctgccccgcccccgagctgctgggcggcccctccgtgttcctgttccctcctaag cccaaggacaccctgatgatctcccggacccccgaggtgacttgcgtggtggtggacgtgtcccacgaggacccc gaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaagccccgggaggagcagtac aactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggagtacaagtgc aaggtgtccaacaaggccctgcccgcccccatcgagaagaccatctccaaggccaagggccagccccgggagccc caggtctacaccctgcctcctagcagagaagagatgaccaagaaccaggtgtccctgacctgcctggtcaagggc ttctacccttccgatatcgccgtggaatgggagtccaatggccagcctgagaacaactacaagaccacacctcct gtgctggactccgacggctcattcttcctgtactccaagctgaccgtggacaagtccagatggcagcagggcaac gtcttctcctgctccgtgatgcacgaggccctgcacaatcactacacccagaagtccctctctctgtcccctgga aaaggcggcggaggatctggcggaggtggaagcggagcccgtggatctcagtctgttctgacccagcctccttcc gcttctggcacacctggacagagagtgaccatctcttgctccggctcctcctccaacatcggctctaatgccgtg tcctggtatcagcagctgcctggcacagctcccaaactgctgatctactacaactcccacccgccttccggcgtg cccgatagattttccggctctaagtccggcacctctgccagcctggctatctctggcctgagatctgaggacgag gccgactactactgcggctcttgggatgcctctctgaacgcctacgtgttcggctgtggcacaaagctgacagtg cttggaggtggtggtagtggtggtggcggttcaggtggcggaggaagcggcggaggcggatctgaagttcagctg ttcgaatctcgcggcggactggttcaacctggcggatctctgagactgtcttgtgccgcctctggcttcaccttc tccgactacgctatgtcttgggtccgacagccccctggcaagtgtctggaatgggtttcctccatctcctccggc agcggctctatctactacgccgactctgtgaagggcagattcaccatcagccgggacaactccaagaacaccctg tacctccagatgaactccctgagagccgaggacacccccgtgtactactgtgccaagaatctgatccctctggac tactggggccagggcacactggttacagtgtcctct [Lightchain_BA6M11] (SEQIDNO:123) cagtccgtgctgacccagcccccctccgcctccggcacccccggccagcgggtgaccatctcctgctccggctcc tcctccaacatcggctccaacgacgtgtcctggtaccagcagctgcccggcaccgcccccaagctgctgatctac tacgaaaacaaccggccctccggcgtgcccgaccggttctccggctccaagtccggcacctccgcctccctggcc atctcccgcctgcggtccgaggacgaggccgactactactccggcgcctgggacgactccctgtccggctacgtg ttcggcggcggcaccaagctgaccctcctgggccagcccaaggcccccccctccgtgaccctcttccccccctcc tccgaggagctgcaggccaacaaggccaccctggtgtgcctgatctccgacttctaccccggcgccgtgaccgtg gcctggaaggccgactcctcccccgtgaaggccggcgtggagaccaccaccccctccaagcagtccaacaacaag tacgccgcctcctcctacctgtccctgacccccgagcagtggaagtcccaccggtcctactcctgccaggtgacc cacgagggctccaccgtggagaagaccgtggcccccgccgagtgctcc

[0428] The coding DNAs of the cloned bispecific antibody were transiently expressed using the ExpiCHO system (Thermo Fisher) according to the manufacturer's protocol. The culture medium comprising expressed antibody was clarified using centrifugation and filtration to remove suspensions, and the antibody was purified using affinity chromatography with MabSelect Sure resin and size exclusion chromatography with Superdex200 resin. The purity of the purified antibody was analyzed using HPLC with a TSKgel SuperSW3000 column, and the purity was found to be over 95%.

3.2. Stability Evaluation of Anti-ROR1/B7-H3 Bispecific Antibody Comprising Improved Anti-ROR1 Clone

(1) Binding Specificity Analysis of Anti-ROR1/B7-H3 Bispecific Antibody Under Stressed Conditions (ELISA)

[0429] As a control group, the BA6xB5 bispecific antibody which comprised the parental clone anti-ROR1 antibody BA6 without removed PTM hotspot, and for the experimental group, the BA6M11xB5 bispecific antibody was utilized, and after storing both samples at 4 C. (normal conditions) and 40 C. (stress conditions) for two weeks, the antibody-antigen binding affinity to human ROR1 and human B7H3 recombinant proteins was evaluated using an ELISA-based solution binding assay. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated for 16 hours at 4 C. with 1 g/ml concentration of human ROR1 protein (ROR1-His; Sino Biological, Catalog #13968-H08H) and human B7-H3 protein (B7H3-His; Sino Biological, Catalog #11188-H08H) in PBS solution, and then the plates were then blocked with 1% (v/v) BSA (bovine serum albumin) at 37 C. for 2 hours to prevent non-specific binding.

[0430] Afterwards, the BA6xB5 and BA6M11xB5 bispecific antibodies stored for two weeks were added to the microtiter plates at the concentrations listed in FIGS. 7a-7d, and their binding ability was analyzed by ELISA as follows. Specifically, after incubating at 37 C. for 2 hours, the plates were washed five times with PBS containing 0.05% (v/v) Tween 20, and then HRP-conjugated Fc polyclonal antibody reagent (Pierce, Catalog #31413) was diluted at a 1:30,000 ratio in 1% (v/v) BSA and added to the washed microtiter plates, incubated for 1 hour at 37 C. to allow the reaction, and then used to detect the bound bispecific antibodies. The reaction was developed using TMB (tetramethylbenzidine, Sigma, T0440). The enzymatic reaction was stopped with 0.5 mol/L sulfuric acid, and the absorbance was measured at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0431] The results obtained are presented in FIGS. 7a-7d. As can be seen from FIGS. 7a-7d, upon evaluating the antigen-antibody binding capacity (ELISA; Protein Binding) using human ROR1 protein and human B7-H3 protein, it was confirmed that there was no significant difference in binding ability between the samples stored at 4 C. and 40 C. for two weeks. These results indicate that BA6M11xB5 maintains its potency compared to BA6xB5 while showing improved physicochemical property in terms of its developability. Ultimately, it was confirmed that the BA6M11 clone not only shows binding comparable to WT but also has clearly improved developability through PTM engineering.

(2) Developability Analysis of Anti-ROR1/B7-H3 Bispecific Antibody

[0432] Selection was made of the BA6M11 clone, which were cleared of PTM liability through PTM engineering from BA6, and both BA6M11xB5 bispecific antibody comprising BA6M11 and BA6xB5 bispecific antibody were manufactured to check for increased stability under stressed conditions.

[0433] More specifically, after storing the BA6xB5 bispecific antibody and BA6M11xB5 bispecific antibody at 4 C. (normal conditions) and 40 C. (stressed conditions) for two weeks, analyses such as SE-HPLC, icIEF, and LC/MS were performed on each sample to check for purity and impurity content. Additionally, to determine whether changes in the substance content also affected efficacy, antigen-antibody binding ability analysis (ELISA; refer to Example 3.2 (1)) was conducted.

[0434] The results obtained are presented in Table 21, below.

TABLE-US-00025 TABLE 21 BA6M11 B5 BA6 B5 Content Method Acceptance Criteria 4 C. 40 C. 4 C. 40 C. Purity (%) SEC Purity % 3.0% 99.8 99.2 99.8 99.2 0.6 0.6 Charge icIEF Acidic Information only 49.1 64.9 51.5 45.6 Heterogeneity Main 43.9 29.0 31.8 23.1 Basic 7.0 6.1 16.6 31.2 Main % 14.9 8.7 Protein ELISA SACE 70% Ratio(EC50) 0.152 0.134 0.141 0.180 Binding (EC50 to 130%, similar curve fit 113 78 Human ROR1), nM) SACE 70% Ratio(EC50) 0.232 0.190 0.173 0.186 (EC50 to 130%, similar curve fit 122 93 Human B7-H3), nM) PTM LC/MS Report result *N/A *N/A 9.8 12.6 Succinimide % in Fd N/A 2.8 Report result *N/A *N/A 16.8 45.6 (Succinimide % in LC) *N/A 28.8 (*N/A: Not applicable)

[0435] As indicated in Table 21, the BA6M11xB5 clone maintained a high level of antibody purity when stored at both 4 C. and 40 C., with no significant difference compared to BA6xB5. In terms of charge variants, the BA6M11xB5 clone showed an improvement over the BA6xB5 clone, with a reduction in the basic variant peak related to the formation of succinimide, and analysis using LC-MS also revealed that, compared to BA6xB5, BA6M11xB5 did not show an increase in succinimide in the Fd and LC under 40 C. stressed conditions, and fragments that appeared to be cleaved within the Fd region were not formed. This reduction in charge variants 10 implies an increase in the stability of the antibody quality and the reliability of the purification process, as discussed in Example 1.3. It also suggests that when administered to humans, the bispecific antibody is expected to maintain low immunogenicity and valency.

3.3. Binding Specificity Analysis of Anti-ROR1/B7-H3 Bispecific Antibody to Cell Surface Expressed ROR1 and B7-H3 (FACS)

[0436] To evaluate the cell-binding ability of the anti-ROR1/B7-H3 bispecific antibody, cells expressing both ROR1 and B7-H3 were used to compare the cell-binding ability of the bispecific antibody with that of the monospecific antibodies.

[0437] As a cell line for the comparison of cell-binding ability, CHO-huROR1-huB7-H3 cell line (obtained by transfecting the ROR1-CHO recombinant cell line (BPS Bioscience) with the human B7-H3 gene (Origene) and selecting for the desired gene-inserted single cells using G418), which stably transfected the human ROR1 and human B7-H3 gene to artificially overexpress both human ROR1 and human B7-H3 proteins were used, and analysis was performed using a flow cytometer (LSRFortessa X-20, BD Biosciences). Specifically, after dissociating and washing the overexpressing cell line with PBS, the cell number was counted, and the cells were seeded at a density of 210.sup.5 cells per well into a V-bottom 96-well plate (96 Well Plate-RV, Bioneer, 910D09). Antibodies diluted 4-fold starting from 100 nM in 1% BSA solution were added at 100 L per well to the centrifuged cells and incubated at 4 C. for 1 hour. After 1 hour, the cells were washed twice with the same buffer and then incubated with FITC-labeled Fc-specific antibody (Goat anti-human IgG-FITC antibody produced in goat, Sigma, F9512) diluted 500 times in 1% BSA at 100 L per well at 4 C. for 1 hour. After the reaction, the cells were washed twice with the same buffer, resuspended in 100 L of PBS, and analyzed using a FACS instrument. A total of 10,000 cells were detected per measurement, and the data was analyzed using FlowJo software. The Fold change values on the graphs were represented by dividing the MFI (Mean fluorescence intensity) of the experimental group treated with antibody by the MFI of the control group treated with the secondary antibody only.

[0438] As shown in FIG. 8, the anti-ROR1/B7-H3 bispecific antibody, which has more antigen-binding sites, showed higher binding affinity to the cell line expressing both ROR1 and B7-H3 compared to monospecific antibodies.

[0439] These results confirm that the anti-ROR1/B7-H3 bispecific antibodies provided herein exhibit superior binding affinity by targeting two antigens simultaneously. Such enhanced binding ability demonstrates excellent selectivity for cells expressing two targets (antigens) simultaneously compared to cells expressing a single target (antigen). This feature aligns with the goals of reducing toxicity and increasing efficacy through selectivity, which are important elements in the design of cancer treatment methods.

3.4. Comparison of Cellular Internalization Between Monospecific Antibody and Bispecific Antibody

[0440] To compare the cellular internalization rates of the anti-ROR1/B7-H3 bispecific antibody, cells expressing both ROR1 and B7-H3 were used to evaluate the internalization of the bispecific antibody. The experiment was conducted using the Incucyte FabFluor-pH Red antibody labeling reagent (Sartorius, 4722), which fluoresces at low pH values. CHO-huROR1-huB7-H3 cells, the same cell line used in Example 3.3, were plated at a density of 110.sup.4 cells per well in a 96-well plate (96-well Clear Flat Bottom TC-treated Culture Microplate, Falcon, 353072) and cultured for 24 hours. The antibody and Incucyte FabFluor-pH Red antibody labeling reagent were mixed at a 1:1 molar ratio and incubated at 37 C. for 15 minutes before being added to the plate with cells. The plate was then placed in the IncuCyte real-time cell analysis system (IncuCyte S3, Sartorius), and phase-contrast and fluorescence were analyzed at 1-hour intervals through the software. As shown in FIG. 9, an increase in fluorescence signal over time was observed, confirming the internalization process of the antibody entering the cell and reaching the lysosome.

[0441] As shown in FIG. 9, the anti-ROR1/B7-H3 bispecific antibody, which has more antigen-binding sites, increased the fluorescence signal more rapidly in the cell line expressing both ROR1 and B7-H3 compared to monospecific antibodies, conforming that the bispecific antibody is internalized into cells more efficiently than monospecific antibodies due to its ability to target two antigens simultaneously.

[0442] These results indicate that the anti-ROR1/B7-H3 bispecific antibodies provided herein promote efficient cellular internalization by simultaneously targeting two antigens.

[0443] From the described outcomes, it is anticipated that developing the bispecific antibody into an ADC (Antibody-Drug Conjugate) could enhance the efficacy of the ADC selectively in cells that express both targets (antigens). This characteristic was further explored and confirmed in subsequent Examples.

3.5. Manufacturing of Anti-ROR1/Anti-B7-H3 Bispecific Antibody ADC

(1) Manufacturing of Bispecific Antibody ADC

[0444] For the conjugation of the drug to the amino acid position 205 of the antibody light chain, the valine (Valine) at Kabat position 205 in the constant region of the antibody light chain was mutated to cysteine (Cysteine) (V205C), and dithiothreitol (DTT) or a similar reducing agent was reacted to generate a thiol group at antibody light chain V205C, and the antibody was conjugated to the drug through thiosuccinimide formation via Michael addition. Specifically, the antibody was prepared to a concentration of over 5 mg/ml through ultrafiltration/diafiltration (UF/DF), adding 1M tris(hydroxymethyl)aminomethane (Tris-HCl), pH8.8, and 500 mM ethylenediaminetetraacetic acid (EDTA), to adjust the final concentration of the antibody to 5 mg/ml, 75 mM Tris-HCl, and 2 mM EDTA. 100 mM dithiothreitol (DTT) was added to the prepared antibody at an antibody: DTT molar ratio of 1:20 and reacted for 16.5 hours at 25 C. to detach the free cysteine linked through disulfide bonds at cysteine 205 of the antibody light chain. This process, referred to as decapping, was followed by separation of the decapped antibody using cation exchange chromatography (CEX). The reaction product was eluted with SPHP-B buffer [50 mM Tris(hydroxymethyl)aminomethane (Tris-HCl), pH7.5, 0.5M Sodium chloride] after equilibrating on a Hitrap SPHP column (GE Healthcare) with SPHP-A buffer [10 mM succinate, pH5.0]. For the recombination of the decapped antibody, the antibody to be oxidized was prepared in 75 mM Tris(hydroxymethyl)aminomethane (pH7.5) using 1M Tris-HCl, and dehydroascorbic acid (DHAA, oxidized vitamin C) was added at an antibody: DHAA ratio of 1:20, and reoxidized for 2 hours at 25 C. in the dark. Then, the reoxidized antibody was isolated using cation exchange chromatography (CEX), eluting with SPHP-C buffer [10 mM succinate pH 5.0, 0.5 M Sodium chloride]. The purified antibody was concentrated to over 5 mg/ml through ultrafiltration/diafiltration (UF/DF). To create the antibody-drug conjugate, the antibody was prepared in 1M Tris(hydroxymethyl)aminomethane (Tris-HCl, pH7.0) to a final concentration of 5 mg/ml, and the drug was added at an antibody: drug molar ratio of 1:10, reacting for 16.5 hours at 25 C. The antibody-drug conjugate was isolated using cation exchange chromatography (CEX), eluting with SPHP-C buffer. To isolate only DAR2 antibody-drug conjugates, hydrophobic interaction chromatography (HIC) was utilized. For this, the Hitrap Butyl HP column (GE Healthcare) was equilibrated with HIC-A buffer [50 mM Potassium phosphate, pH7.0, 1.0 M Ammonium sulfate] and eluted with HIC-B buffer [50 mM Potassium phosphate, pH7.0, 30% 2-Propanol]. The antibody was exchanged into HS buffer [20 mM Histidine, pH6.0, 240 mM sucrose] through ultrafiltration/diafiltration (UF/DF) to remove 2-Propanol and finalize the antibody-drug conjugate, and the drug used was pyrrolobenzodiazepine (PBD), and the resulting antibody was named antibody (V205C)-T-PBD.

(2) Purity and DAR Measurement of Manufactured Bispecific Antibody ADC

[0445] The purity of the manufactured ADC was measured using size-exclusion high-performance liquid chromatography (SE-HPLC). An Agilent 1200 series HPLC instrument equipped with a Tosoh TSKgel G3000SWxl column (Tosoh Bioscience) was used for the measurement, and the elution positions and the area under the curve (AUC) of each sample were compared to determine the purity of each sample. The main peak's purity of the ADC was over 99.0% (see Table 23 and FIGS. 10a to 10c).

[0446] The drug-to-antibody ratio (DAR) of the ADC was determined using liquid chromatography-mass spectrometry (LC/MS) method. To remove N-glycans, 1 unit of PNGaseF (NEB) per 100 g of ADC (in PBS) at a concentration of 1 mg/ml was added and incubated at 37 C. for 15 hours. A Waters UPLC I-class instrument coupled with a Waters Synapt G2-S LC/MS system was equipped with an Acquity UPLC BEH200 SEC 1.7 m (4.6*150 mm) column and equilibrated with a mobile phase consisting of 30% (v/v) acetonitrile, 0.1% (v/v) formic acid, and 0.05% trifluoroacetic acid (TFA). 5 g of the N-glycan-removed sample was loaded and analyzed by LC/MS. The measurement of the ADC's DAR (drug-to-antibody ratio) by LC/MS confirmed that there were 2 drugs conjugated per antibody molecule (see Tables 22, 23 and FIGS. 11a to 11c).

TABLE-US-00026 TABLE 22 ADC Purity (%) DAR BA6(M11/V205C)xB5-T-PBD 99.63 2.0 BA6(M11/V205C)-T-PBD 99.54 2.0 B5(V205C)-T-PBD 99.57 2.0

TABLE-US-00027 TABLE 23 Theo. Exper. Delta Mass Mass Mass Accuracy ADC (DAR) (Da) (Da) (Da) (ppm) BA6(M11/V205C)xB5-PBD 199162.32 199187.00 24.68 123.92 (DAR2) BA6(M11/V205C)-T-PBD 146561.30 146585.00 23.70 161.71 (DAR2) B5(V205C)-T-PBD (DAR2) 145238.34 145267.00 28.66 197.33

(3) Ligand Binding Activity Test Using Bispecific Antibody ADC

[0447] The ligand binding affinity of the BA6M11 (V205C)-T-PBD monospecific antibody and BA6xB5 (M11) (V205C)-T-PBD bispecific antibody ADCs to human ROR1 recombinant protein was evaluated using an ELISA-based solution binding assay. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated for 16 hours at 4 C. with ROR1 protein (ROR1-His; Sino Biological, Catalog #13968-H08H) at a concentration of 1 g/ml in PBS solution and blocked with 1% (v/v) BSA (bovine serum albumin) at 37 C. for 2 hours to prevent non-specific binding.

[0448] Afterwards, the BA6M11 (V205C)-T-PBD monospecific antibody and BA6xB5 (M11) (V205C)-T-PBD bispecific antibody were added to the microtiter plates at the concentrations listed in FIG. 12, and their binding ability was analyzed by ELISA as follows: After incubating at 37 C. for 2 hours, the plates were washed five times with PBS containing 0.05% (v/v) Tween 20, then HRP-conjugated Fc polyclonal antibody reagent (Pierce, Catalog #31413) diluted at a 1:30,000 ratio in 1% (v/v) BSA was added to the washed microtiter plates, incubated for 1 hour at 37 C. to allow the reaction, and used to detect the bound bispecific antibodies. The reaction was developed using TMB (tetramethylbenzidine, Sigma, T0440). The enzymatic reaction was stopped with 0.5 mol/L sulfuric acid, and the absorbance was measured at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0449] The results displayed in FIG. 12 show that the ligand binding capability of the manufactured BA6xB5 (M11) (V205C)-T-PBD bispecific antibody to human ROR1 protein was on par with that of the manufactured BA6M11 (V205C)-T-PBD monospecific antibody. This confirms that the excellent ROR1 antigen binding ability of BA6M11 is retained even after being manufactured into a bispecific antibody ADC form.

3.6. Comparative Evaluation of Cytotoxicity of Bispecific Antibody ADC

[0450] The anticancer cell death efficacy of ADCs manufactured using the bispecific antibody of the present disclosure was compared with that of monospecific antibody ADCs. The cancer cell lines Calu-3 (KCLB, Catalog No. 30055) and Calu-6 (ATCC, Catalog No. HTB-56), which express both ROR1 and B7-H3, were employed, and the cell death capability was compared by administering ADCs to the cells and measuring the cells' metabolic activity. To determine if the effect of the bispecific antibody was merely a combination of the monospecific antibody targets, a control group treated with both the anti-ROR1 monospecific antibody and the anti-B7-H3 monospecific antibody (combination treatment, indicated with a +) was also evaluated for comparison. The cancer cell lines were cultured using the recommended culture media. 510.sup.3 cells per well were inoculated into a 96-well culture plate with a volume of 50 l and cultured for 4-6 hours, and after this, 50 l of ADCs diluted at various concentrations were dispensed into the 96-well plate with cells. Then, the cells were cultured at 37 C., 5% CO.sub.2 for about 6 days. The number of living cells was quantified using WST-8 (Dojindo, CK04).

[0451] FIG. 13 depicts the cytotoxicity results of the monospecific antibodies or bispecific antibody against cancer cell lines, using B5 as the anti-B7-H3 antibody and BA6M11 as the anti-ROR1 antibody. It was observed that in cancer cell lines (Calu-3, Calu-6) expressing both ROR1 and B7-H3, the cytotoxicity of the bispecific antibody (BA6M11xB5) ADC was greater than the combined cytotoxicity of monospecific antibody (BA6M11, B5) ADCs. Conversely, in the KATOIII cell line (ATCC, Catalog No. HTB-103), which barely expresses ROR1 and B7-H3, ADCs showed minimal cytotoxicity, and there was no significant difference in cytotoxicity between monospecific antibody ADCs and the bispecific antibody ADC. This indicates that the enhanced cytotoxicity of the bispecific antibody ADC compared to monospecific antibody ADCs is antigen-dependent. The results demonstrating higher cytotoxicity with the treatment of the bispecific antibody compared to the combination of monospecific antibodies confirm that the superior cytotoxicity of the bispecific antibody ADC of the present disclosure is more than just the effect of combining monospecific antibodies.

[0452] Although some embodiments have been described herein, it should be understood by those skilled in the art that these embodiments are given by way of illustration only, and that various modifications, variations, and alterations can be made without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be limited only by the accompanying claims and equivalents thereof.