Anti-ROR1 antibody and method of use

Abstract

This invention relates to an antibody that binds to ROR1.

Description

[Technical Field]

[0001] [Referencing sequence listings submitted as standard-compliant XML 1.0 format files (.xml)] In accordance with the EFS-Web legal framework and 37 CFR §§1.821-825 (see MPEP §2442.03(a)), Rule 30 EPC, and §11 PatV, an electronic sequence listing in XML 1.0 format file, compliant with WIPO standard ST.26, is filed concurrently with this application, and the entire contents of the sequence listing are incorporated herein by reference. To avoid misunderstanding, in the event of any inconsistency between sequences mentioned herein and in the electronic sequence listing, the sequences mentioned herein shall be considered the correct sequences.

[0002] [Field of Invention] The present invention relates to antibodies that bind to ROR1, fragments or derivatives thereof, and methods for using them.

[0003] [Integration by reference] All publications, patents, patent applications, and other documents cited herein are incorporated herein by reference in their entirety for all purposes to the same extent that each publication, patent, patent application, or other document is individually indicated as being incorporated by reference for all purposes. In the event of any inconsistency between one or more of the references incorporated herein and the teachings of this disclosure, the teachings of this disclosure shall prevail. [Background technology]

[0004] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the ROR family, which also includes ROR1 and ROR2. The ROR1 protein contains an extracellular immunoglobulin-like (Ig) domain at the amino terminus, followed by a cysteine-rich domain known as the frizzled domain (FZD), and then a kringle domain (KRD). The intracellular portion of ROR1 consists of a tyrosine kinase domain for cell signaling, two serine / threonine-rich domains, and a proline-rich domain. ROR was discovered and extensively studied in the context of embryonic and neurogenesis, but more recently, increasing literature has established ROR1 as a marker for cancer, such as CLL and other hematological malignancies. More importantly, ROR1 has been shown to be critically involved in the progression of multiple hematological and solid tumors. For example, ROR1 has been shown to inhibit apoptotic signaling, enhance EGFR signaling, and induce epithelial-mesenchymal transition (EMT).

[0005] ROR1 expression is present during normal embryonic development, but is absent in most mature tissues. Low levels of ROR1 expression have been observed in adipose tissue, and even lower levels have been observed in subsets of pancreatic, lung, thyroid, stomach, and intermediate B cells (Baskar et al., 2008; Hudecek et al., 2010; Bicocca et al., 2012+ et al.).

[0006] However, ROR1 expression has been reported to be upregulated in numerous hematological and solid malignancies. Positive ROR1 expression was initially identified in B-cell chronic lymphocytic leukemia (CLL). Primary CLL cells express high levels of ROR1 but not ROR2, and ROR1 expression increases throughout CLL progression. According to the CLL Research Consortium, flow cytometry analysis shows that ROR1 is expressed on the surface of CLL cells from 94% of patients with CLL. While ROR1 is not the only biomarker for CLL, it may serve as a potential prognostic indicator (Daneshmanesh et al., 2013). Constitutive phosphorylation of STAT3 is characteristic of CLL and has been shown to bind to multiple sites on the ROR1 promoter. Furthermore, ROR1 expression can be induced by IL-6 in a STAT3-dependent and dose-dependent manner (Frank et al., 1997; Li et al., 2010). Since the discovery of elevated ROR1 expression in CLL, increased levels of ROR1 have been reported in a variety of hematological malignancies, including acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), and myeloid malignancies (Daneshmanesh et al., 2008; Barna et al., 2011; Daneshmanesh et al., 2013). Furthermore, ROR1 expression has been shown in ovarian cancer, breast cancer, particularly triple-negative breast cancer, and lung cancer.

[0007] The importance of ROR1 in cancer cell survival and proliferation has been confirmed in clinical trials, and girobertamab (also known as sirumutuzumab, UC-961) has shown promising results in patients with CLL or MCL. Girobertamab is a ROR1 antagonist that inhibits the proliferation and migration of tumor cells. However, girobertamab does not activate immune cells and does not induce ADCC (antibody-dependent cell-mediated cytotoxicity).

[0008] While ROR1 antibodies that induce ADCC have been reported previously, their efficacy is relatively low. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a novel therapeutic method that increases the effectiveness of anti-ROR1 therapy.

[0010] The objective of this invention is to provide novel and improved therapeutic methods for treating cancer.

[0011] These and other problems are resolved by embodiments shown in the independent claims of this specification. Dependent claims disclose preferred embodiments. [Brief explanation of the drawing]

[0012] [Table 2] This table shows the kinetic parameters of the interaction with recombinant extracellular hROR1, as analyzed by BLI / SPR. The apparent K is derived from the 1:2 divalent analyte model. D The following were evaluated: EC50 binding to hROR1-transfected CHO cells, analyzed by flow cytometry and domain-binding analysis by BLI; cross-reactivity analysis to recombinant mouse ROR1, analyzed by BLI / SPR; and binding to human recombinant ROR-2, analyzed by BLI / SPR and flow cytometry analysis of CHO-ROR2 cells.

[0013] [Figure 1]Figures 1A to 1D are graphs showing binding curves for the evaluation of the EC50 of the tested antibodies against CHO cell lines overexpressing ROR1, ROR2, and the control cell line CHO-EV. (A) The PB004.22.0357.aF defucosylated antibody binds only to the cell line overexpressing ROR1 with an EC50 of 0.933x10-9 M. (B) The PB004.22.0372.aF defucosylated antibody binds only to the cell line overexpressing ROR1 with an EC50 of 0.617x10-9 M. (C) The PB004.22.0405.aF defucosylated antibody binds only to the cell line overexpressing ROR1 with an EC50 of 0.557x10-9 M. (D) The PB004.22.0408.aF defucosylated antibody binds only to the cell line overexpressing ROR1 with an EC50 of 0.233x10-9 M. The bars represent the standard deviation calculated based on two technical replicates.

[0014] [Table 3] This is a table showing the comparison of the affinity between standard IgG1 or defucosylated IgG1 and the FcyRIIIA receptor.

[0015] [Figure 2A]Figure 2 is a graph showing FcγRIII-dependent ADCC. (A) ADCC against JeKo-1 cells using the Promega ADCC reporter kit to measure FcγRIII interaction. Target cells were incubated for 6 hours in the presence of each antibody and reporter cells and analyzed by a luminescence reader. Flow cytometry-based analysis of NK cell-dependent induction (ADCC) of B-F cancer cell death. NK cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Samples were incubated for 4 h with various concentrations of zilovertamab, R12hIgG1 (a humanized variant of rabbit monoclonal R12 described in Yang et al., 2011), anti-CD20 antibody rituximab, defucosylated isotype control and selected antibodies (PB004.22.0357.aF, PB004.22.0372.aF, PB004.22.0405, PB004.22.0405.aF, PB004.22.0408.aF), and target cells (Jeko-1, NK:Jeko-1 ratio; effector:target 10:1). [Figure 2B] (B) Representative ADCC induction for PB004.22.0405 and PB004.22.0405.aF. [Figure 2C] (C) ADCC comparison of PB004.22.0405.aF, rituximab, zilovertamab and R12hIgG1. [Figure 2D] (D) Representative ADCC results for PB004.22.0357.aF. [Figure 2E] (E) Representative ADCC results for PB004.22.0372.aF. [Figure 2F] (F) Representative ADCC for PB004.22.0408.aF. Bars in Figure 2 represent standard deviations calculated based on two technical replicates.

[0016] [Figure 3A]Figure 3 is a graph showing the potent but safe induction of ADCC by the tested antibodies. Either whole-cell PBMCs or isolated NK cells isolated from peripheral blood mononuclear cells (PBMCs) of a healthy donor were used as effector cells. Samples were incubated for 4 hours with various concentrations of PB004.22.0405.aF, girobertamab, rituximab, defucosylated isotype controls, and target cells (Jeko-1, primary CLL material, or naive B cells) and NK cells. Combination experiments were performed by incubation with either venetoclax or ibrutinib. (A) Representative ADCC induction of JeKo-1 cells using whole-cell PBMCs. [Figure 3B] (B) Pooled data on ADCC induction in primary CLL patient substances. [Figure 3C] (C)PB004.22.0405.aF, Biomarker analysis in the form of NK cell activation by girobertamab or rituximab. [Figure 3D] (D) Additive / synergistic ADCC effect of PB0004.22.405 with venetoclax. [Figure 3E] (E) Additive / synergistic ADCC effect of PB0004.22.405 with ibrutinib. [Figure 3F] (F)PB0004.22.405 does not kill naive B cells compared to rituximab.

[0017] [Figure 4A]Figure 4 is a graph showing further modes of action. (A) Flow cytometry-based analysis of antibody-dependent cell phagocytosis (ADCP). Samples were incubated for 4 hours with 50 nM isotype control, R12hIgG1 antibody, girobertamab, rituximab, and selected leads (PB004.22.0357.aF, PB004.22.0372.aF, PB004.22.0405.aF, PB004.22.0408.aF) and Jeko-1 cells (macrophage:Jeko-1, effector:target ratio 2:1). Percentage cell phagocytosis was calculated using the formula: number of double-stained positive target cells (cells phagocytosed by macrophages; violet+ / CFSE+) ÷ total number of target cells (CFSE+). [Figure 4B] (B) CDC-inducing ability was assessed using C1q-binding ELISA. The tested antibodies were coated onto ELISA plates ON along with the control, followed by washing and blocking, and recombinant C1q was added to serial dilutions. C1q binding was detected using anti-C1q-HRP antibody, and the plates were analyzed with a plate reader after TMB substrate.

[0018] [Figure 5A] Figure 5 is a graph showing the antitumor efficacy of the tested antibodies against subcutaneous JeKo-1 tumors. (A) CD34+ humanized NCG-hIL15 mice were subcutaneously inoculated with JeKo-1, and from the time the tumor volume reached an average of approximately 85 mm3, they were treated twice weekly at 10 mg / kg with six doses of media: PB004.22.0357, PB004.22.0372, PB004.22.0405, PB004.22.0408, girobertamab, or rituximab. Graphs represent mean ± SEM, n=8, and statistical significance was examined using a two-way ANOVA with Bonferroni multiple comparisons *p<0.01, ****p<0.0001. [Figure 5B](B) Specific activation of tumor NK cells. At the end of the experiment, the tumor and spleen were removed and processed into a single cell along with the final bleeding for flow cytometry analysis. Data represent mean ± SD, n=8, and statistical significance was examined using Kruskal-Wallis, nonparametric ANOVA, **p<0.001.

[0019] [Table 4] Shows antitumor efficacy against JeKo-1 tumors. CD34 + Humanized NCG-hIL15 mice were subcutaneously inoculated with JeKo-1 tumor cells, and the average tumor volume was approximately 85 mm². 3 From that point onward, the subjects were treated twice a week at 10 mg / kg with six different antibody doses. At the end of the treatment, tumor volume was compared to that of the media. The table shows the tumor volume inhibition compared to the media-treated group.

[0020] [Figure 6A] This figure shows the antitumor efficacy in a leukemia model. SCID mice were intravenously inoculated with MEC1-ROR1 cells, a CLL cell line overexpressing human ROR1. One week later, the mice were intravenously treated with either the medium, PB004.22.0405.aF, or girobertamab at a dose of 10 mg / kg twice weekly for a total of six doses. Four weeks after inoculation, the mice were sacrificed, and the bone marrow and spleen were analyzed for CD19 and ROR1-positive CLL cells. Graphs represent mean ± SD, n=4-5, and statistical significance was examined using one-way analysis of variance (ANOVA) with Kruskal-Wallis multiple comparisons *p<0.01. [Figure 6B] This figure shows the antitumor efficacy in a leukemia model. SCID mice were intravenously inoculated with MEC1-ROR1 cells, a CLL cell line overexpressing human ROR1. One week later, the mice were intravenously treated with either the medium, PB004.22.0405.aF, or girobertamab at a dose of 10 mg / kg twice weekly for a total of six doses. Four weeks after inoculation, the mice were sacrificed, and the bone marrow and spleen were analyzed for CD19 and ROR1-positive CLL cells. Graphs represent mean ± SD, n=4-5, and statistical significance was examined using one-way analysis of variance (ANOVA) with Kruskal-Wallis multiple comparisons *p<0.01.

[0021] [Figure 7A] This graph shows the in vivo safety evaluation. HuHSC-NCG-IL15 mice were subcutaneously inoculated with JeKo-1 cells, and established tumor-bearing mice were treated intravenously every other week with six injections of 10 mg / kg. The safety of the antibody was evaluated by (A) body weight measurements, (B) GvHD score (measuring body weight, body position, fur texture, and skin integrity), and (C) serum cytokines in plasma 6 or 24 hours after treatment. The graph represents the three pooled representative cytokines measured. The graph shows the mean ± SEM for A and B, and ± SD for C. No statistical significance was observed for any of the measured parameters (Kruskal-Wallis). [Figure 7B] This graph shows the in vivo safety evaluation. HuHSC-NCG-IL15 mice were subcutaneously inoculated with JeKo-1 cells, and established tumor-bearing mice were treated intravenously every other week with six injections of 10 mg / kg. The safety of the antibody was evaluated by (A) body weight measurements, (B) GvHD score (measuring body weight, body position, fur texture, and skin integrity), and (C) serum cytokines in plasma 6 or 24 hours after treatment. The graph represents the three pooled representative cytokines measured. The graph shows the mean ± SEM for A and B, and ± SD for C. No statistical significance was observed for any of the measured parameters (Kruskal-Wallis). [Figure 7C] This graph shows the in vivo safety evaluation. HuHSC-NCG-IL15 mice were subcutaneously inoculated with JeKo-1 cells, and established tumor-bearing mice were treated intravenously every other week with six injections of 10 mg / kg. The safety of the antibody was evaluated by (A) body weight measurements, (B) GvHD score (measuring body weight, body position, fur texture, and skin integrity), and (C) serum cytokines in plasma 6 or 24 hours after treatment. The graph represents the three pooled representative cytokines measured. The graph shows the mean ± SEM for A and B, and ± SD for C. No statistical significance was observed for any of the measured parameters (Kruskal-Wallis).

[0022] [Figure 8] This graph shows the pharmacokinetic profile of PB004.22.0405 as measured in mouse serum by ELISA. BALB / cJRj mice were injected with a single IV bolus of 10 mg / kg of PB004.22.0405, blood was drawn at various time points, and plasma was analyzed by ELISA using ROR1-coated plates. Detection was performed using anti-FC-HRP antibody. Data are expressed as mean ± SD. [Figure 9] This diagram shows the domain structure of ROR1. [Modes for carrying out the invention]

[0023] According to a first aspect of the present invention, these are the heavy / light chain variable domain (HCVD / LCVD) pairs shown below: Sequence ID 1 and Sequence ID 2, Sequence ID 3 and Sequence ID 4, Sequence IDs 5 and 6, and / or Sequence IDs 7 and 8 The present invention provides an antibody that binds to ROR1, or a target-binding fragment or derivative thereof that maintains its target-binding ability.

[0024] Antibodies having the VH / VL sequences of SEQ ID NOs. 1 and 2 are referred to herein as PB004.22.0357.

[0025] Antibodies having the VH / VL sequences of SEQ ID NOs. 3 and 4 are referred to herein as PB004.22.0372.

[0026] Antibodies having the VH / VL sequences of SEQ ID NOs. 5 and 6 are referred to herein as PBA0405, AF0405, PB0405, or PB004.22.0405.

[0027] Antibodies having the VH / VL sequences of SEQ ID NOs. 7 and 8 are referred to herein as PB004.22.0408.

[0028] The defucosylated variants of these antibodies have the tag "af".

[0029] The tyrosine-protein kinase transmembrane receptor ROR1 (UniProt:Q01973), also known as neurotrophic tyrosine kinase or receptor-associated 1 (NTRKR1), is an enzyme encoded by the ROR1 gene in humans. ROR1 is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family. The protein encoded by the ROR1 gene is a receptor tyrosine kinase that regulates neurite outgrowth in the central nervous system. It is a type I membrane protein and belongs to the ROR subfamily of cell surface receptors. ROR1 is currently being studied for its role in cancer cell metastasis.

[0030] Recently, ROR1 has been shown to be expressed in ovarian cancer stem cells, and it is thought that ROR1 plays a functional role in promoting migration / invasion or spheroid formation in vitro, as well as tumor engraftment in immunodeficient mice.

[0031] Human ROR1 consists of an immunoglobulin-like domain (IG), two cysteine-rich domains, a frizzled domain (FZD), and a kringle domain (KRD). Intracellularly, ROR1 has a tyrosine kinase domain (TKD), two serine / threonine-rich domains (Ser / Thr), and a proline-rich domain (PRD).

[0032] In one embodiment, the antibody is a monoclonal antibody.

[0033] As used herein, the term “monoclonal antibody (mAb)” refers to an antibody composition having a homogeneous population of antibodies, i.e., a homogeneous population of all immunoglobulins, or fragments or derivatives thereof that maintain target-binding ability. Particularly preferred, such antibodies are selected from the group consisting of IgG, IgD, IgE, IgA and / or IgM, or fragments or derivatives thereof that maintain target-binding ability.

[0034] As used herein, the term “fragment” means a fragment of such an antibody that maintains its target-binding ability, for example CDR (Complementarity Determination Area) Hypervariable region, Variable domain (Fv) IgG or IgM heavy chain (consisting of VH, CH1, hinge, CH2, and CH3 regions) IgG or IgM light chain (consisting of VL and CL regions), and / or Fab and / or F(ab)2 This refers to...

[0035] As used herein, the term “derivative” refers to a protein construct that is structurally different from the usual antibody concept, e.g., scFv, Fab and / or F(ab)2, and double, triple, or higher-order specific antibody constructs, but still maintains some structural relationship to the usual antibody concept and further preserves target-binding ability. All of these items are described below.

[0036] Other antibody derivatives known to those skilled in the art include bispecific antibodies, camel antibodies, nanobodies, domain antibodies, bivalent homodimers having two chains consisting of scFv, IgA (two IgG structures linked by a J chain and secretory components), shark antibodies, antibodies consisting of New World primate frameworks and non-New World primate CDRs, dimerized constructs containing CH3+VL+VH, and antibody conjugates (e.g., antibodies, fragments, or derivatives conjugated to toxins, cytokines, radioisotopes, or labels). These types are well described in the literature and are available to those skilled in the art pursuant to this disclosure without any further inventive activity.

[0037] A method for generating hybridoma cells was disclosed by Kohler & Milstein (1975).

[0038] Methods for generating and / or selecting chimeric or humanized mAbs are known in the art. For example, U.S. Patent No. 6,331,415 by Genentech describes the generation of chimeric antibodies, U.S. Patent No. 6,548,640 by the Medical Research Council describes a CDR implantation technique, and U.S. Patent No. 5,859,205 by Celltech describes the generation of humanized antibodies.

[0039] Methods for generating and / or selecting fully human mAbs are known in the art. These may involve the use of transgenic animals immunized with the respective protein or peptide, or the use of a suitable display technique, such as yeast display, phage display, B cell display, or ribosome display, in which the library-derived antibody is screened against human iRhom2 in the stationary phase.

[0040] Among the many available in vitro antibody libraries, those disclosed are U.S. Patent No. 6,300064 by MorphoSys and U.S. Patent No. 6,248516 by MRC / Scripps / Stratagene. Phage display techniques are disclosed, for example, U.S. Patent No. 5,223409 by Dyax. Transgenic mammalian platforms are described, for example, in European Patent Application Publication No. 1480515A2 by TaconicArtemis.

[0041] IgG, IgM, scFv, Fab, and / or F(ab)2 are antibody types well known to those skilled in the art. Relevant implementation techniques are available in their respective textbooks.

[0042] As used herein, the term "Fab" refers to an IgG / IgM fragment containing an antigen-binding region, wherein the fragment consists of one constant and one variable domain derived from each heavy and light chain of the antibody.

[0043] As used herein, the term "F(ab)2" refers to an IgG / IgM fragment consisting of two Fab fragments linked to each other by a disulfide bond.

[0044] As used herein, the term "scFv" refers to a single-chain variable fragment, which is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked by a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide.

[0045] According to one embodiment, the antibody or its fragment or derivative has an improved ability to induce ADCC compared to naturally occurring antibodies or fragments or derivatives.

[0046] The primary strategies for improving the ADCC-inducing ability of IgG are site-directed mutagenesis to alter the Fc moiety of the antibody to increase binding affinity to activated FcγRIIIA, alter Fc domain glycosylation, and / or prevent Fc domain fucosylation. Mutagenesis to create IgG variants with improved binding to activated FcγR has been an effective strategy for increasing the ADCC efficiency of IgG antibodies (Shields et al. 2000, Tang et al. 2007, Zahavi et al. 2018, all of which are incorporated herein by reference for authorization). In addition to Fc residue modification, asymmetric manipulation of the Fc moiety to create heterodimers of different heavy chains has resulted in more stable antibodies with improved ADCC functionality (Liu et al. 2013, which is incorporated herein by reference for authorization).

[0047] According to one embodiment, the antibody or a fragment or derivative thereof contains a defucosylated Fc domain.

[0048] Defucosylated antibodies are monoclonal antibodies that have been modified so that the oligosaccharides in the Fc region of the antibody do not contain any fucose sugar units. When an antibody is of the defucosylated IgG type, its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) is increased.

[0049] Technically, defucosylation is, For example, by overexpressing the enzyme GnTIII (see, e.g., Davies et al. 2001) or by knocking out FUT8Y (fucosyltransferase 8, Yamane-Ohnuki et al. 2004), the addition of fucose to existing glycans can be inhibited. Chemical or enzymatic removal of fucose already attached to existing sugar chains. Redirecting fucose synthesis by using heterologous enzymes that deplete the intracellular fucose pool (see also ProBioGen-based GlyMaxx technology, Chung et al., 2012), and / or Use an expression system that does not add fucose residues to the sugar chain, such as a ciliate-based expression system (see, for example, European Patent Application Publication No. 2542575A1). This can be achieved by one of the following means.

[0050] In one embodiment, the antibody according to the present invention was produced by expression in a FUT8-KO CHO (Chinese hamster ovary) cell line containing a knockout of the FUT8 gene. The antibody thus produced lacks fucose and is therefore defucosylated. In the examples shown herein, the defucosylated antibody according to the present invention was produced in that manner. However, it should be noted that similar effects can be expected with the antibody according to the present invention that has been defucosylated by other methods, such as those described earlier.

[0051] According to one embodiment, the antibody or its target-binding fragment or derivative is of the IgG type, preferably of the IgG type having the heavy chain / light chain variable domain (HCVD / LCVD) pair of SEQ ID NOs. 1 and 2, SEQ ID NOs. 3 and 4, SEQ ID NOs. 5 and 6 and / or SEQ ID NOs. 7 and 8.

[0052] According to one embodiment, the antibody or its target-binding fragment or derivative induces increased ADCC compared to girobertamab. Girobertamab is an anti-ROR1 antibody supplied by Oncternal Therapeutics.

[0053] Similar findings apply to the antibody R12hIgG1 (Yang et al. (2021), which is currently used as scFv in CAR-T cells but not as a naked antibody).

[0054] [Table 1]

[0055] In one embodiment, an antibody or its target-binding fragment or derivative is administered to the same or a different subject under equivalent conditions to increase ADCC. As used herein, the term "administered to a subject under equivalent conditions" means equivalent subject parameters (size, weight, sex, age, medical history) and equivalent administration conditions (dosage, timing, interval).

[0056] In one embodiment, an antibody or its target-binding fragment or derivative is tested in vitro against girobertamab to show an increase in ADCC. Suitable cell-based ADCC assays are commercially available from private laboratory suppliers, and the principles of such assays are described in Parekh et al. (2012) or Alpert et al. (2012), and elsewhere in this specification.

[0057] According to another aspect of the present invention, (i) Whether the antibody or its target-binding fragment or derivative as described above competes for binding to ROR1, (ii) Or bind to the same ROR1 epitope as the antibody or its target-binding fragment or derivative described above, A target-binding molecule is provided.

[0058] In one embodiment, the target-binding molecule is an antibody or a target-binding fragment or derivative thereof, as defined elsewhere in this specification.

[0059] As used herein, the term “competing for binding” is used with respect to a target-binding molecule that has the activity to bind to the same substrate to which an antibody or its target-binding fragment or derivative binds. The efficiency of the binding molecule (e.g., kinetic or thermodynamic) may be the same as, greater than, or less than the efficiency of substrate binding by the antibody or its target-binding fragment or derivative. For example, their equilibrium binding constants (Kj) for binding to the substrate may differ. mWhen used herein, " is the Michaelis-Menten constant for an enzyme, and is defined as the concentration of a specific substrate at which a given enzyme produces half of its maximum rate in an enzyme-catalyzed reaction.

[0060] As used herein, the term “binding to the same epitope” for two or more binding molecules means that, as determined by a given method, the molecules bind to the same portion of an amino acid residue. Techniques for determining whether one antibody binds to the same epitope as another antibody include, for example, epitope mapping, e.g., X-ray analysis of the antigen:antibody complex crystals, and hydrogen / deuterium exchange mass spectrometry (HDX-MS), which provide atomic resolution of the epitope. Other methods involve monitoring binding to antigen fragments or variant forms of antigens, where loss of binding due to alteration of amino acid residues within the antigen sequence is often considered to indicate an epitope component. Furthermore, computer combinatorial methods for epitope mapping are also available. These methods depend on the ability of the antibody in question to affinity isolate specific short peptides from combinatorial phage display peptide libraries.

[0061] According to another aspect of the present invention, a pharmaceutical composition is provided comprising an antibody or a target-binding fragment or derivative thereof, or a target-binding molecule, as described above, and one or more pharmaceutically acceptable excipients of any choice.

[0062] According to another aspect of the present invention, a combination is provided comprising (i) an antibody or a target-binding fragment or derivative thereof or a target-binding molecule as described above, and (ii) one or more further therapeutically active compounds.

[0063] According to the embodiment, such a combination is Bcl-2 inhibitors, and / or Bruton's tyrosine kinase (BTK) inhibitors It includes at least one of the following.

[0064] Bcl-2 (B-cell lymphoma 2) is encoded in humans by the BCL2 gene and is a member of the Bcl-2 family of regulatory proteins that control cell death (apoptosis) by either inhibiting (anti-apoptotic) or inducing (pro-apoptotic) apoptosis.

[0065] BCL-2 is localized to the outer membrane of mitochondria, where it plays a crucial role in promoting cell survival and inhibiting the action of pro-apoptotic proteins. Furthermore, BCL-2 is known to regulate mitochondrial dynamics and is involved in the control of mitochondrial fusion and fission.

[0066] Damage to the Bcl-2 gene has been identified as a cause of several cancers, including melanoma, breast cancer, prostate cancer, chronic lymphocytic leukemia, and lung cancer. Damage to the Bcl-2 gene is also a cause of resistance to cancer treatments.

[0067] Bcl-2 inhibitors, in particular, Oblimersen (antisense oligonucleotide) (G3139) ABT-737 Navitocrax (ABT-263) Venetoclaus (ABT-199) Sonrotokraks (BGB-11417) That is the case.

[0068] Bruton's tyrosine kinase (abbreviated as Btk or BTK), also known as tyrosine-protein kinase BTK, is a tyrosine kinase encoded by the BTK gene in humans. BTK plays a crucial role in B cell development. It is necessary for signaling from pre-B cell receptors formed after successful immunoglobulin heavy chain rearrangement, thus playing a vital role in B cell development. BTK also plays a role in mast cell activation via high-affinity IgE receptors. Mutations in the BTK gene are sometimes associated with X-linked agammaglobulinemia (Bruton's agammaglobulinemia), a primary immunodeficiency disorder sometimes abbreviated as XLA and selective IgM deficiency.

[0069] Patients with XLA have a normal pre-B cell population in the bone marrow, but these cells cannot mature and enter circulation. The Btk gene is located on the X chromosome (Xq21.3~q22). At least 400 mutations in the BTK gene have been identified. Of these, at least 212 are thought to be disease-causing mutations.

[0070] BTK inhibitors, in particular, Ibrutinib (Imbruvica) Acalabrutinib (Calcens) Zanubrutinib (Burkinza) Tirabrutinib (berexible) Piltobrutinib (Jaiparka) Olerabrutinib Evobrutinib Trebultinib Remibrutinib Fenebrutinib (RG7845) ABBV-105 Fenebrutinib (GDC-0853) Chirabltinib (GS-4059) Speblutinib (AVL-292, CC-292) HM71224 and / or Luxeptinib That is the case.

[0071] According to such a combination embodiment, Bcl-2 inhibitors include venetoclax and / or Ibrutinib is an inhibitor of Bruton's tyrosine kinase.

[0072] Venetoclax (CAS number 1257044-40-8) is a drug that binds to a protein called Bcl-2. The Bcl-2 protein is abundant in CLL cancer cells, where it helps cells survive longer in the body and makes them resistant to cancer drugs. By binding to Bcl-2 and blocking its action, venetoclax causes cancer cell death, thereby slowing disease progression. Venetoclax is used, in particular, to treat adults with chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), or acute myeloid leukemia. The inventors of this invention have invented the co-administration of venetoclax and the anti-ROR1 antibody according to the present invention and have shown that a synergistic effect exists from this combination.

[0073] Ibrutinib (CAS number 936563-96-1) is a small molecule drug that inhibits B cell proliferation and survival by irreversibly binding to the protein Bruton's tyrosine kinase (BTK). By blocking BTK, it inhibits the B cell receptor pathway, which is often abnormally active in B cell cancers. Therefore, ibrutinib is used to treat such cancers, including mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström macroglobulinemia. [6][7] Ibrutinib also binds to C-terminal Src kinase, which is an off-target receptor for BTK inhibitors. Ibrutinib binds to these receptors and inhibits the kinase from promoting cell differentiation and proliferation. This leads to a variety of side effects, such as left atrial enlargement and atrial fibrillation, during treatment for chronic lymphocytic leukemia. Ibrutinib is indicated for the treatment of mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), Waldenström macroglobulinemia (WM), marginal zone lymphoma (MZL), and chronic graft-versus-host disease (cGVHD). The inventors of this invention have invented the co-administration of ibrutinib and the anti-ROR1 antibody according to the present invention and have demonstrated the existence of a synergistic effect resulting from this combination.

[0074] According to another aspect of the present invention, neoplastic diseases Have you been diagnosed with, • To suffer from or • There is a risk of developing the disease. The use of antibodies or their target-binding fragments or derivatives, target-binding molecules, pharmaceutical compositions, or combinations (for the manufacture of pharmaceuticals) as described above, for the treatment of human or animal subjects, or for the prevention of such conditions, is provided.

[0075] This phrasing is considered to encompass both the Swiss-type claim phrasing, which is acceptable in some countries (in which case parentheses are not considered to exist) and the EPC2000 phrasing (in which case parentheses and their contents are not considered to exist).

[0076] According to another aspect of the present invention, a method for treating or preventing a neoplastic disease is provided, comprising administering to a human or animal subject a therapeutically sufficient dose of an antibody or its target-binding fragment or derivative, target-binding molecule, pharmaceutical composition, or combination thereof as described above. [Examples]

[0077] Although the present invention has been illustrated and described in detail in the drawings and the foregoing description, such illustrations and descriptions should be considered illustrative or representative, but not restrictive. The present invention is not limited to the embodiments disclosed. Other variations of the disclosed embodiments can be understood and implemented by those skilled in the art in carrying out the claimed invention from a consideration of the drawings, this disclosure, and the appended claims. In the claims, the phrase "including" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude plurals. The mere fact that certain means are enumerated in different dependent claims does not indicate that combinations of these means cannot be used advantageously. Reference numerals in the claims should not be construed as limiting the scope.

[0078] All amino acid sequences disclosed herein are shown from the N-terminus to the C-terminus.

[0079] Materials and methods CHO-cell line binding (barcoding method) - major candidates Materials and methods CellTrace® CFSE preservation solution (Invitrogen, #C34554) at a concentration of 5 mM was thawed by RT before analysis. 1 μL of CellTrace® CFSE was added to 10 mL of DPBS to make a 0.5 μM preservation solution. Tenfold serial dilutions of CFSE were prepared by transferring 10 mL from the 0.5 μM solution to 90 mL of DPBS, thereby obtaining usable concentrations of 0.05 and 0.005 μM.

[0080] 80 million CHO cells (overexpressing ROR1, overexpressing ROR2, or transfected with an empty vector) were washed twice with 20 mL of DPBS and resuspended in the respective solutions: CHO-EV (empty vector) in 90 mL of DPBS; CHO-ROR1 in 90 mL of 0.05 μM CFSE solution; and CHO-ROR2 in 90 mL of 0.005 μM CFSE solution. The cells were incubated at RT for 30 minutes with agitation, protected from light.

[0081] After incubation, 10 mL of cell culture medium containing 10% FBS was added, and the cells were incubated at RT for 10 minutes with agitation. The cells were centrifuged (450 rcf, 5 minutes), washed twice with 20 mL of DPBS, and resuspended in 22.5 mL of staining medium (DPBS + 5% FBS) at a density of 4 mln cells / mL. CHO-ROR1, CHO-ROR2, and CHO-EV cell lines were mixed in a 1:1:1 ratio (22 mL each). The mixed cells were divided equally into 96-well plates (150 μL - 600,000 cells per well).

[0082] For staining with the tested antibody, serial dilutions were prepared in DPBS + 5% FBS to obtain final concentrations of 50, 10, 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001 μg / mL. The equally divided cells were centrifuged (450 rcf, 5 minutes), the supernatant was discarded, and the cells were resuspended in 100 μL of the preferred tested antibody dilution and incubated on ice for 30 minutes.

[0083] For secondary antibody staining, cells were washed twice with DPBS + 0.5% FBS 200 μL, resuspended in DPBS + 0.5% FBS 100 μL containing allophycocyanin (APC) AffiniPure goat anti-human IgG, Fcγ fragment-specific antibody (Jackson ImmunoResearch, #109-135-098) (0.4 μL per sample), and incubated on ice for 30 minutes. After incubation, cells were washed twice with DPBS + 0.5% FBS 200 μL, resuspended in DPBS 200 μL containing 1 μL of Sytox blue (Invitrogen, #S34857) per 1 mL of DPBS, and stored on ice until analysis.

[0084] Samples were measured using an Attune® NxT focusing cytometer (Thermo Fisher Scientific) equipped with a CytKick autosampler, and analysis was performed using Attune® software. Performance testing was conducted prior to analysis. After removing dead cells and doublets, 20,000 events were captured for the tested samples in the “target population” gate.

[0085] Results and Conclusions The defucosylated antibodies tested bound to target ROR1 in a dose-dependent manner in CHO cells. All of the antibodies exhibited very low EC50 values ​​in the sub-nanomolar range.

[0086] KD Evaluation - BLI (hROR1-Fc, mROR1-Fc, hROR2-Fc) The binding activity of the antibody was determined using an Octet RED384 instrument (Sartorius). In a FAB2G (anti-hIgG CH1 capture) biosensor (Sartorius), the antibody was captured at a concentration of 5 μg / mL. Serial dilutions (200 - 3.13 nM) of recombinant hROR1 / Fc, hROR2 / Fc, mROR1 / Fc (R&D Systems) and mROR2 / Fc (Acro Biosystems) were prepared in 10× kinetic buffer (Sartorius). Binding of the analyte was measured for 200 seconds, followed by dissociation for 500 seconds in 10× kinetic buffer. The biosensor was regenerated with 10 mM glycine, pH 1.7 (Cytiva) after each binding cycle. Sensorgrams were referenced by subtracting the blank (buffer), and the binding (k on ) and dissociation (k off ) rate constants were evaluated from global fitting based on a 1:2 (bivalent analyte) binding model using Octet Analysis Studio 12.2 software (Sartorius). The apparent dissociation constant was calculated from the equation: K D = k off1 / k on1 .

[0087] KD Evaluation - SPR (FcyR) (CAP or SA) The affinity of the antibody was determined using surface plasmon resonance in a BIACORE 8K instrument (Cytiva). Recombinant FcγR protein (R&D Systems) was biotinylated according to the supplier's instructions and immobilized on a CAP chip. Molecules were injected onto the chip surface to a capture level of approximately 70 RU. Serial dilutions of the antibody as the analyte were prepared in PBS-P + buffer (Cytiva) and injected at a flow rate of 30 μL / min. Data generated by double-reference subtraction (reference cell subtraction and blank subtraction) were analyzed using BIAevaluation software (Cytiva). Binding (k on ) and dissociation (k off ) rate constants were evaluated based on a 1:1 Langmuir binding model, and the equilibrium dissociation constant was calculated from the equation: K D = k off / k on .

[0088] Epitope Binning - BLI Epitope binning was performed using an Octet RED384 instrument (Sartorius). Antibody (5 μg / mL in 10 mM acetate buffer, pH 6.0) was immobilized on an AR2G sensor. The resulting antibody sensor was incubated in 1000 nM ROR1-His (for 100 seconds), followed by 1000 nM ROR1-His (for 100 seconds) pre-mixed with 500 nM competing antibody, and dissociation was measured for 200 seconds. Sensorgrams were evaluated using Octet Analysis Studio 12.2 software (Sartorius).

[0089] Results and Conclusions The main compound binds to an epitope (frizzyled domain) distinct from dirobertamab and R12hIgG1 (IgG domain).

[0090] ADCC-Materials and Methods cell culture Raji (CCL-86) was purchased from ATCC, Jeko-1 (ACC553) and MEC-1 (ACC497) were purchased from DSMZ, and MDA-MB-231 (92020424) was purchased from ECACC. Raji and MEC-1 were cultured in RPMI1640 medium (Gibco®, 31870074) containing 10% fetal bovine serum (Sigma Aldrich, F9665-500ML), 1% L-glutamine (Gibco®, 35050061), and 1% penicillin / streptomycin (Gibco®, 15140130). Jeko-1 was cultured in RPMI1640 medium containing 20% ​​fetal bovine serum, 1% L-glutamine, and 1% penicillin / streptomycin. MDA-MB-231 was cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco®, 11965092) containing 10% fetal bovine serum. All cell lines were maintained at 37°C and 5% CO2 and subcultured up to 20 times.

[0091] Isolation of PBMCs and enrichment of human NK cells Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coat samples of healthy donors obtained by leukocyte apheresis and were labeled HIV-, HCV-, and HBV-. Density centrifugation was performed using SepMate®-50 PBMC isolation tubes (Stemcell®, #85460). The isolation tubes were filled with 15 mL of Lymphoprep (Stemcell®, #07811). The buffy coat was transferred to a sterile PETG medium bottle (Fisherbrand®, PBMB125) and diluted with an equal volume of DPBS (Gibco®, 14190144). 17 mL of the diluted buffy coat was gently overlaid on Lymphoprep, and the SepMate® tubes were centrifuged (1200 g, 30 minutes, acc / brake on). After centrifugation, the PBMC layer observed on the plastic membrane of each Sepmate® tube was collected and transferred to a new 50 mL conical tube. The tubes containing the PBMCs were diluted with the same volume of fresh DPBS and centrifuged (800 g, 8 minutes). The supernatant was discarded, and the cell pellet was resuspended in 5 mL of 1X erythrocyte lysis buffer (Biolegend, 420302) and incubated at RT for 10 minutes. The tubes were completely filled with DPBS and centrifuged (350 g, 5 minutes). The supernatant was discarded, and the cell pellet was washed three times with 30 mL of fresh DPBS. The PBMCs were cultured overnight at 37°C and 5% CO2 in Iskov Modified Dulbecco's Medium (IMDM) (Gibco®, 31980030) containing 10% fetal bovine serum, or subjected to NK cell isolation.

[0092] NK cells were isolated from PBMCs by immunomagnetically negative selection using an NK cell isolation kit (Miltenyi Biotec, 130-092-657) according to the manufacturer's protocol. The NK cells were cultured overnight in 10% fetal bovine serum-containing IMDM (Gibco®, 31980030) at 37°C and 5% CO2, and used the following day.

[0093] NK cells used as effector cells in the ADCC assay underwent phenotyping and isolation purity characterization using flow cytometry (Cytek Northern Lights (NL-00020)). The following markers were used: viability (LIVE / DEAD® Fixable Aqua Dead Cell Staining Kit, Invitrogen®, L34966), CD3 (APC-H7 mouse anti-human CD3, BD Bioscience, 560176), CD16 (Alexa Fluor® 647 mouse anti-human CD16, BD Biosciences, 557710), CD56 (Brilliant Violet 605® anti-human CD56 (NCAM), BioLegend®, 362538), CD69 (BV711 mouse anti-human CD69, BD Biosciences, 563836), CD107a (PE mouse anti-human CD107a, BD Farmingen®) Pharmingen (trademark), 555801), NKG2D (BV421 mouse anti-human CD314 (NKG2D), BD Biosciences, 743558).

[0094] ADCC assay Target cells were collected, washed twice with DPBS, and labeled with 0.1 μM CellTrace® CFSE Growth Kit (Invitrogen®, C34554) at 37°C for 20 minutes. After incubation, the cells were washed twice with assay medium (IMDM containing 10% FBS) and 0.2 × 10⁶ cells were incubated in assay medium. 6The cells were resuspended at a concentration of cells / mL. Target cells (10,000 cells per well) in 50 μL volume were seeded into a 96-well cone-bottom plate (Thermo Scientific®, Nunc®, 249935). Subsequently, antibody concentrations of 50 μL in IMDM, adapted for each experiment, were added. 50 μL of assay medium was added to the wells selected for NK background control and target survival control. Target cells were incubated with antibody at 37°C, 5% CO2 for 30 minutes for pre-coating. Meanwhile, NK cells (effector cells) isolated the previous day were centrifuged (350 g, 5 minutes) and 1 × 10⁶ cells were placed in fresh assay medium. 6 The cells were resuspended at a concentration of cells / mL. After the pre-coating step, NK cells were added to each well at a concentration of 100,000 cells / well. 100 μL of assay medium was added to the wells selected for target survival control. The plates were then centrifuged at 100 g for 1 minute and incubated at 37°C at 5% CO2 for 4 hours. The final effector cell to target cell ratio was 10:1.

[0095] After 4 hours, the plate was centrifuged (350g, 5 minutes), and the supernatant was discarded by vigorously inverting the plate. The cell pellet was washed with DPBS, centrifuged (350g, 5 minutes), resuspended in 100 μL of Live / Dead® Fixable Violet Dead Cell Staining Kit solution (1:1000 in DPBS, Invitrogen®, L34964), and subsequently incubated at 4°C for 20 minutes. After that time, 100 μL of FACS buffer (DPBS containing 2% fetal bovine serum) was added, and the plate was centrifuged (350g, 5 minutes). The cell pellet was resuspended at 4°C for 10 minutes in 100 μL of fixation solution of 4% paraformaldehyde (Thermo Scientific®, J19943.K2). After fixation, 100 μL of FACS buffer was added, and the plate was centrifuged (500g, 5 minutes). The cell pellet was resuspended in 80 μL of FACS buffer and subjected to flow cytometry analysis.

[0096] Next, the plates were read using a Cytek Northern Lights (NL-00020) or BD FACSCelesta (trademark) (660344) flow cytometer. The relative percentage of cell lysis was calculated by subtracting the target survival control from the experimental lysis values. 10 The data is presented as relative percentage solubility against antibody concentration (nM) shown on a scale. The dose-response curve was fitted using a 4-parameter nonlinear regression equation. Maximum half-volume effective concentration (EC) 50 ) and other EC values ​​(EC 10 , EC 20 , EC 30 , EC 50 , EC 70 and EC 90 To determine the effective concentration, the antibody dilution range was selected to cover both the upper and lower asymptotes. EC values ​​including at least three biological replicates were calculated. Effective concentration values ​​other than EC50 were calculated as follows: EC F =(F / (100-F)) 1 / H ×EC 50 In the formula, F is the percentage change in response and H is the hill slope value. Data was analyzed using FlowJo (trademark) v10.8.1 software (BD Life Sciences) and GraphPad Prism 7.05 software (GraphPad Software, Inc.).

[0097] Promega ADCC Reporter Assay ADCC was performed according to the manufacturer's instructions.

[0098] Combination ADCC In combined ADCC assays, target cells were incubated with venetoclax or ibrutinib before use in the ADCC assay.

[0099] Cytokine release and degranulation assay CD107a degranulation assay and cytokine release measurement New PBMCs were purified from buffy coat and incubated overnight at 37°C in IMDM supplemented with 10% FBS and 55 μM β-mercaptoethanol. The Jeko-1 cell line was used as the target cell. 500,000 Jeko-1 cells per well were pre-incubated for 30 minutes with 50 nM selected conjugates PB004.22.0357.aF, PB004.22.0372.aF, PB004.22.0405.aF, PB004.22.0408.aF, girobertamab, R12hIgG1, and rituximab, washed, added to the target cells, and co-cultured for 4 hours in a 1:1 (PBMC:target cell) ratio in the presence of APC-conjugate anti-CD107a antibody and protein transport inhibitors (BD GolgiStop and BDGolgiPlug). Subsequently, cells were labeled with Aqua survival dye, BV605 anti-CD56, BV421 anti-CD16, and APC-H7 anti-CD3 (from BD Biosciences or Biolegend) for 25 minutes at 37°C, fixed for 15 minutes at 4°C using the BD Cytofix / Cytoperm® kit, and then intracellularly stained with IFNγ (BB700 anti-IFNγ) and TNFα (BV750 anti-TNFα) for 60 minutes by RT. Background fluorescence was evaluated using isotype-matched antibodies from the same manufacturer. Cells were passed through a FACS Cytek® flow cytometer equipped with standard features. Data were analyzed using FlowJaw v10.

[0100] ADCP Monocyte-derived macrophage cell culture and preparation for phagocytic assays Monocytes were concentrated from frozen peripheral blood mononuclear cell (PBMC) samples using a human whole monocyte isolation kit (Miltenyi Biotech, 130-096-537) according to the manufacturer's instructions. Purity was verified by phenotypic analysis of surface markers: anti-CD14 and anti-CD16. The monocytes concentrated from PBMCs were cultured for 7 days in IMDM 10% FBS in the presence of 50 ng / ml M-CSF + 50 ng / ml IL-4 + 10% human serum. Adherent populations of differentiated macrophages were stained with 0.1 μM violet dye (CellTrace® Violet Cell Growth Kit, for flow cytometry, C34557) and washed twice before ADCP experiments. The following surface markers were used to verify the phenotype of differentiated macrophages: anti-CD163, anti-CD16, anti-CD32, and anti-CD64.

[0101] Target cell culture and preparation Jeko-1 cells were cultured as a suspension in a 75 cm² flask at 37°C and 5% CO2, and maintained at 0.5–1.0 10⁶ cells / mL by adding fresh complete growth medium every 3 days. Before the experiment, Jeko-1 cells were counted, stained with 0.1 μM CFSE dye (CellTrace® CFSE Cell Growth Kit, for flow cytometry, C34554), and washed twice before the ADCP experiment.

[0102] 3-color flow cytometry ADCP assay Stained target cells (Jeko-1, storage solution: 1 M / ml, 50 μl per well) were pre-incubated for 30 minutes with selected conjugates at 50 nM concentrations: isotype control, PB004.22.0357.aF, PB004.22.0372.aF, PB004.22.0405.aF, PB004.22.0408.aF, girobertamab, R12hIgG1, and rituximab. Fluorescently labeled effector monocyte-derived macrophages (violet) differentiated from PBMCs were co-cultured with fluorescently labeled Jeko-1 cells (CFSE) and incubated at 37°C for 4 hours in a 2:1 (effector:target ratio), followed by staining with a survival dye (live / dead fixable near-infrared (780) stain) for 15 minutes. Phagocytosis was measured by analysis using FACS attunement. Percentage cell phagocytosis was calculated using the formula: number of double-stain-positive target cells (cells phagocytosed by macrophages; violet+ / CFSE+) ÷ total number of target cells (CFSE+). Data were analyzed using Flowjaw v10.

[0103] CDC / C1q binding ELISA plates were coated with either the primary compound or control at a concentration of 5 μg / mL, followed by blocking, washing, and serial dilution with recombinant C1q protein. Detection was performed using anti-C1q-HRP antibody, and after adding TMB substrate, the plates were read at 450 nm and 650 nm wavelengths.

[0104] Results and Conclusions PB004.22.0405.aF exhibited strong Cq1 binding, which therefore contributed to the CDC-inducing ability of the ROR1 antibody.

[0105] In vivo antitumor efficacy huHSC-NCG-hIL15 JeKo-1 model Jeko-1 cells resuspended in DPBS were subcutaneously injected into 56 female + surplus huHSC-NCG-hIL15 mice. The huHSC-NCG-hIL15 model was created by reconstitution of CD34+ huHSCs from five independent donors in irradiated female NCG-hIL15 mice. The level of immune reconstitution was confirmed by flow cytometry, and humanization of the mouse model was identified as over 25% hCD45+ / surviving single cells in peripheral blood before tumor cell inoculation. The average tumor size was approximately 82 mm. 3 Upon reaching this point, the mice were randomly divided into groups based on the table above. HuHSC-NCG-hIL15 tumor-bearing mice from various donors were evenly distributed to each experimental group, with each group containing mice from all huHSC donors. Mice were treated every other week with intravenous injection of a medium, 10 mg / kg / of the compound, girobertamab, or rituximab. Blood was collected from the mice 6 and 24 hours after administration and processed as serum for cytokine analysis using CBA detection with the BD CBA Human Th1 / Th2 Kit. At the end of the study, tumors, spleens, and blood were collected, processed for flow cytometry, and analyzed for cellular markers: survival, mCD45, hCD45, hCD3, hCD8, hCD56, hCD69, hCD25, hCD107a, hgranzyme B, and hIFN-γ.

[0106] Results and Conclusions The defucosylated antibodies tested demonstrated antitumor efficacy against JeKo-1 cells in vivo with efficacy similar to or more potent than girobertamab. In contrast to girobertamab, PB004.22.0405.aF was able to activate tumor-infiltrating NK cells to secrete IFN-γ much more potently than rituximab. PB004.22.0405.aF demonstrates safety because activation was observed only in the tumor environment and not systematically, and no activation was observed in blood or splenic NK cells. Furthermore, the compound was as safe as rituximab and girobertamab, as no weight loss, no GvDH score increase, and no increase in serum cytokines were observed.

[0107] MEC-ROR1 leukemia model CB-17 / Icr-Prkdcscid / scid / Rj immunodeficient female mice were intravenously injected with MEC-1-ROR-1 cells (1 × 10⁶). One week after inoculation, intravenous treatment was performed every other week with six injections of 10 mg / kg. Four weeks after tumor cell inoculation, bone marrow derived from the spleen and the femurs and tibias of both hind limbs was isolated from each transplanted mouse. Cell suspensions were prepared from either spleen homogenized with the flat plunger end of a syringe, or from BM-derived cells that had been passed through a cell strainer (70 mM) in complete RPMI. Red blood cells were lysed, and cells were counted. Commercially available anti-human ROR1 (ROR1-PE) and anti-human CD19 (CD19APC) antibodies, along with corresponding isotype controls, were used to analyze spleen and BM-derived cells (1 × 10⁶). 6 Flow cytometry analysis was performed on the cells. Using a 4-laser BD FACSMelody instrument, cells were subjected to data acquisition and multiparametric FACS evaluation. Data were analyzed using FlowJaw software, and after setting the background using each isotype control, both the mean and median readings were used for CD19 + or ROR-1 + I recorded information about the cells.

[0108] Results and Conclusions PB004.22.0405.aF demonstrated significant antitumor efficacy in a leukemia model, showing a reduction in CD19+ROR1+ cells in the spleen and bone marrow. PB004.22.0405.aF showed more significant antitumor efficacy than girobertamab.

[0109] table [Table 2]

[0110] [Table 3]

[0111]

Table 4

[0112] Refer to · Parekh BS, Berger E, Sibley S, Cahya S, Xiao L, LaCerte MA, Vaillancourt P, Wooden S, Gately D. Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay. MAbs. 2012 May-Jun;4(3):310-8. doi: 10.4161 / mabs.19873. Epub 2012 Apr 26. · Alpert MD, Heyer LN, Williams DE, Harvey JD, Greenough T, Allhorn M, Evans DT. A novel assay for antibody-dependent cell-mediated cytotoxicity against HIV-1- or SIV-infected cells reveals incomplete overlap with antibodies measured by neutralization and binding assays. J Virol. 2012 Nov;86(22):12039-52. · Daneshmanesh, Amir Hossein & Hojjat-Farsangi, Mohammad & Khan, A & Jeddi-Tehrani, Mahmood & Akhondi, Mohammad & Bayat, Ali & Ghods, Roya & Mahmoudi, Ahmad-Reza & Hadavi, Reza & Osterborg, A & Shokri, F & Rabbani, Hodjattallah & Mellstedt, Hakan. (2012). Monoclonal antibodies against ROR1 induce apoptosis of chronic lymphocytic leukemia (CLL) cells. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, UK 26. 1348-55. 10.1038 / leu.2011.362. ·          Davies J, Jiang L, Pan LZ, LaBarre MJ, Anderson D, Reff M. Expression of GnTIII in a recombinant anti-CD20 CHO production cell line. Biotechnol Bioeng. 2001 Aug 20;74(4):288-94. PMID: 11410853. · Yamane-Ohnuki, Naoko, et al. “Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.” Biotechnology and bioengineering 87.5 (2004): 614-622. · Chung S, Quarmby V, Gao X, Ying Y, Lin L, Reed C, Fong C, Lau W, Qiu ZJ, Shen A, Vanderlaan M, Song A. Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities. MAbs. 2012 May-Jun;4(3):326-40 · Choi EI, Wang R, Peterson L, Letvin NL, Reimann KA. Use of an anti-CD16 antibody for in vivo depletion of natural killer cells in rhesus macaques. Immunology. 2008 Jun;124(2):215-22. doi: 10.1111 / j.1365-2567.2007.02757.x. Epub 2008 Jan 12. PMID: 18201184; PMCID: PMC2566626. · Shields, R, Namenuk, A, Hong, Ket al. High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR. J Biol Chem 2000; 276: 6591-604. · Tang, Y, Lou, J, Alpaugh, Ret al. Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen. J Immunol 2007; 179: 2815-23. · Zahavi D, AlDeghaither D, O’Connell A, Weiner LM. Enhancing antibody-dependent cell-mediated cytotoxicity: a strategy for improving antibody-based immunotherapy. Antib Ther. 2018 Jun 24;1(1):7-12. · Liu, Z, Gunasekaran, K, Wang, Wet al. Asymmetrical Fc engineering greatly enhances antibody-dependent cellular cytotoxicity (ADCC) effector function and stability of the modified antibodies. J Biol Chem 2013; 289: 3571-90 · Yang J, Baskar S, Kwong KY, Kennedy MG, Wiestner A, Rader C. Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies. PLoS One. 2011;6(6):e21018. · Daneshmanesh AH, Mikaelsson E, Jeddi-Tehrani M, Bayat AA, Ghods R, Ostadkarampour M, Akhondi M, Lagercrantz S, Larsson C, Osterborg A, Shokri F, Mellstedt H, Rabbani H. Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy. Int J Cancer. 2008 Sep 1;123(5):1190-5. · Daneshmanesh AH, Porwit A, Hojjat-Farsangi M, Jeddi-Tehrani M, Tamm KP, Grander D, Lehmann S, Norin S, Shokri F, Rabbani H, Mellstedt H, osterborg A. Orphan receptor tyrosine kinases ROR1 and ROR2 in hematological malignancies. Leuk Lymphoma. 2013 Apr;54(4):843-50.

[0113] Sequence The following sequences form part of the disclosure of this application. A sequence listing conforming to WIPO ST26 is also submitted in conjunction with this application. To avoid misunderstanding, in the event of any discrepancy between the sequences in the following table and the sequence listing, the sequences in this table shall be considered correct.

[0114] In some cases, the signal peptide may be incorporated into the replicated sequence. In such cases, the sequence is to be disclosed with and without the signal peptide. A readily available tool for identifying the signal peptide in a given protein sequence is SignalP-6.0, provided by Dansk Technical University at https: / / services.healthtech.dtu.dk / service.php?SignalP. The same applies to sequences containing the His tag, which are to be disclosed with and without the His tag.

[0115] [Table 5]

Claims

1. The following are the heavy / light chain variable domain (HCVD / LCVD) pairs: Sequence ID 1 and Sequence ID 2, Sequence ID 3 and Sequence ID 4, Sequence IDs 5 and 6, and / or Sequence IDs 7 and 8 An antibody that binds to ROR1, or a target-binding fragment or derivative thereof that maintains target-binding ability.

2. The antibody or fragment thereof according to claim 1, which is a monoclonal antibody or derived from a monoclonal antibody.

3. An antibody or fragment thereof according to claim 1 or 2, having an improved ability to induce ADCC.

4. An antibody, or a fragment or derivative thereof, containing a defucosylated Fc domain.

5. An antibody or a target-binding fragment or derivative thereof according to any one of claims 1 to 4, which is of the IgG type and preferably has heavy chain / light chain variable domain (HCVD / LCVD) pairs of SEQ ID NOs. 1 and 2, SEQ ID NOs. 3 and 4, SEQ ID NOs. 5 and 6 and / or SEQ ID NOs. 7 and 8.

6. An antibody according to any one of claims 1 to 5, or a target-binding fragment or derivative thereof, that induces increased ADCC compared to girobertamab.

7. A target-binding molecule that competes with the antibody or its target-binding fragment or derivative according to any one of claims 1 to 6 for binding to ROR1, or that binds to the same ROR1 epitope as the antibody or its target-binding fragment or derivative according to any one of claims 1 to 6.

8. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 6, or a target-binding fragment or derivative thereof, or a target-binding molecule according to claim 7, and one or more pharmaceutically acceptable excipients of any choice.

9. (i) an antibody or a target-binding fragment or derivative thereof according to any one of claims 1 to 6, or a target-binding molecule according to claim 7, and (ii) a combination comprising one or more additional therapeutically active compounds.

10. Bcl-2 inhibitors, and / or Bruton's tyrosine kinase (BTK) inhibitors The combination according to claim 9, comprising at least one of the following.

11. The Bcl-2 inhibitor is venetoclax, and / or Ibrutinib is an inhibitor of Bruton's tyrosine kinase. The combination described in claim 9.

12. neoplastic diseases Have you been diagnosed with this? - To suffer from or • There is a risk of developing [the condition]. Use of an antibody or a target-binding fragment or derivative thereof according to any one of claims 1 to 6, a target-binding molecule according to claim 7, a pharmaceutical composition according to claim 8, or a combination according to any one of claims 9 to 11 (for the manufacture of pharmaceuticals) in the treatment of a human or animal, or for the prevention of such a condition.

13. A method for treating or preventing a neoplastic disease, comprising administering to a human or animal subject a therapeutically sufficient dose of an antibody or a target-binding fragment or derivative thereof according to any one of claims 1 to 6, a target-binding molecule according to claim 7, a pharmaceutical composition according to claim 8, or a combination according to any one of claims 9 to 11.

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