MAb11-22.1 conjugate, an anti-TfR1 antibody for cancer treatment.

Abstract

The present invention provides a therapeutic agent for the treatment, prevention, and diagnosis of cancers associated with cells that overexpress transferrin receptor 1 (TfR1) and its variants on the cell surface, including, but not limited to, AML, ALL, lymphoma, multiple myeloma, breast cancer, gastric cancer, glioblastoma, prostate cancer, urothelial cancer (bladder cancer), pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, and liver cancer. The agent is based on the amino acid sequences of the novel light and heavy chain variable regions of MAb11-22.1, an anti-TfR1 monoclonal antibody (mAb), which is highly specific for tumor cells, in the form of an ADC, and can functionally inhibit the proliferation of several human cancer cell lines and the growth of xenograft tumors derived from AML cell lines in mouse models.

Description

[Technical Field]

[0001] The present invention relates to the use of antibodies to bind to transferrin receptor 1 (TfR1) and its isoforms in human cancer cells and to modulate their internalization and activity. The present invention also relates to antibody-drug conjugates (ADCs) with small molecules used for in vitro, in-situ, and / or in vivo diagnosis and / or treatment of mammalian cells or pathological conditions associated with TfR1 and its isoforms. [Background technology]

[0002] Transferrin receptor 1 (TfR1, CD71) is a type II transmembrane glycoprotein consisting of 760 amino acids. It is linked by two disulfide bonds to form a 180-kDa homodimer, which plays a crucial role in regulating iron uptake and cell growth (BOMFORD and MUNRO. Hepatology. 5:870-875, 1985). When diiron transferrin (Tf) binds to TfR1 on the cell surface, the holo-Tf-TfR1 complex is internalized by clathrin-coated pits and delivered to acidic endosomes. There, the iron-Tf-TfR1 complex undergoes a conformational change induced by low pH, followed by the release of iron and its transport into the cytoplasm. Next, the apo-Tf / TfR1 complex (apo-Tf / TfR1) returns to the cell surface through recirculation, where apo-Tf dissociates from its receptor (WARD. Invest Radiol. 22:74-83, 1987; DANIELS et al. Clin Immunol. 121:144-158, 2006). TfR1 expression increases in rapidly proliferating cells that strongly require iron for heme synthesis, such as blood cells, hepatocytes, and keratinocyte precursors, but its expression decreases or is absent in non-dividing cells. TfR1 is overexpressed in primary and metastatic cancer cells originating from lymphocytes, pancreas, stomach, colon, lung, breast, bladder, and skin (GATTER et al. J Clin Pathol.36:539-545,1983; FAULK et al. Lancet.2:390-392,1980; SUTHERLAND et al. Proc Natl Acad Sci USA 78:4515-4519,1981; DANIELS et al. Clin Immunol.121:144-158,2006; JEONG et al. Biochem Biophys Res Commun.471:373-379,2016; PEER et al. Nat Nanotechnol.2:751-760,2007; QIAN et al. Pharmacol Rev.54:561-587,2002; RICHARDSON et al. Biochim Biophys Acta Gen Subj.1790:702-717,2009).Since cancer cells are thought to be more sensitive to iron deprivation, targeting Tf or TfR1 by blocking iron binding or interfering with the internalization of the holo-Tf / TfR1 complex will induce iron deprivation and kill malignant cells.

[0003] The literature over the past 30 years has described numerous attempts to treat malignancies by developing anti-human TfR1 antibodies or TfR1-binding peptides to compete with TfR1 in interfering with receptor binding or internalization (for an overview, see TORTORELLA and KARAGIANNIS.J Membr Biol.247:291-307,2014; CANDELARIA et al. Front.Immunol.17 March 2021.doi.org / 10.3389 / fimmu.2021.607692). TROWBRIDGE and LOPEZ (Proc. Natl Acad Sci USA, 79, 1175-1179, 1982; U.S. Patent Nos. 4,434,156) reported a mouse anti-TfR1 antibody named 42 / 6 that can inhibit the growth of human T-cell leukemia cell lines in vitro by blocking the binding of Tf and TfR1 through a non-competitive mechanism. While 42 / 6 was well-tolerated by patients in a Phase Ia clinical trial, its lack of efficacy was observed because, being a mouse IgA isotype, it induced human anti-mouse antibodies (HSMS) and was rapidly eliminated by the kidney (BROOKS et al. Clin Cancer Res. 1:1259-1265, 1995). MOURA et al. (J Exp Med, 194, 417-425, 2001) reported a high affinity (K) for TfR1. DA more potent neutralizing mouse anti-TfR1 IgG2b antibody (A24) has been reported that directly competes with Tf by binding at a strength of 2.7 nM, reducing TfR expression and inhibiting TfR recirculation, thereby inhibiting T cell proliferation. In contrast to 42 / 6, which exerts its antiproliferative effect by inhibiting cells in the S phase of the cell cycle, A24 acts by inducing apoptosis in target cells, inhibiting the extracorporeal proliferation of adult T-cell leukemia / lymphoma (ATLL), acute myeloid leukemia (AML), and mantle cell lymphoma (MCL) cells (Moura et al. Blood. 103:1838-1845, 2004; CALLENS et al. Leukemia. 22:42-48, 2008; LEPELLETIER. Cancer Res. 67:1145-1154, 2007).

[0004] In recent years, several chimeric antibodies, humanized antibodies, or fully human antibodies have been developed to overcome the lack of efficacy in humans and the HAMA response. For example, the mouse-human IgG3 chimeric antibody ch128.1 has shown in vivo anticancer activity in cell line-derived xenograft (CLDX) models of human multiple myeloma (MM) and AIDS-associated non-Hodgkin lymphoma (AIDS-NHL) (DANIELS.J Immunother.34:500-508,2011;DANIELS-WELLS.J Immunother.38:307-310,2015.), and its humanized version hu128.1(IgG1) is also potent in the AIDS-NHL CLDX model (DANIELS-WELLS.Cancer Res.80(16 Suppl):5655,2020.). The anticancer activity of ch128.1 and hu128.1 is thought to depend on Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). Three fully human neutralizing antibodies against IgG1 isotypes have been described. PPMX-T003 and H7-IgG1 inhibited tumor growth and extended mouse survival in various leukemia or lymphoma models (SHIMOSAKI et al. Biochem Biophys Res Commun. 485:144-151, 2017; ZHANG et al. Cancer Res. 77(13 Suppl):5586, 2017). Even more surprisingly, the anti-TFRC developed by NAGAI et al. (Cancer Med.3:108-1099,2014) inhibited the proliferation of oral squamous cell carcinoma (OSCC) cells both in vitro and in vivo (NEIVEYANS et al. MAbs.11:593-605,2019), suggesting that TfR1 has potential for use in the treatment of solid tumors in addition to hematopoietic malignancies.

[0005] Due to the ubiquitous expression profile of TfR1 in dividing cells, anti-TfR1 antibodies may possess universal antiproliferative activity that is untolerable in cancer patients. Mild to moderate toxicity of anti-TfR1 antibodies against erythrocyte (RBC) progenitor cells and myeloid progenitor cells has been reported in animal models (CANDELARIA et al. Front.Immunol. 17 March 2021). In addition to 42 / 6, many anti-human TfR1 antibodies have shown promising preclinical results, but only the anti-TfR1 antibody-drug conjugate (ADC) CX-2029 has progressed to the clinical stage, demonstrating dose-dependent hematological toxicity (anemia, neutropenia, and leukopenia) manageable with red blood cell transfusions in a Phase I clinical trial (JOHNSON et al. Clin Cancer Res. 27:4521-4530, 2021). Therefore, for cancer treatment, an anti-TfR1 antibody is needed that specifically targets cancer cells and minimizes cross-reactivity with normal cells. [Overview of the project]

[0006] MAb11-22.1 mAb is a monoclonal antibody (IgG1, κ) produced by one of the hybridomas obtained from mice immunized with multiple acute myeloid leukemia (AML) live cell lines, and was identified as a specific mAb against human TfR1 expressed in AML and other cancer cells. The affinity between MAb11-22.1 mAb and TfR1 is very high (K D <1×10 -12 M). In vitro assays showed that MAb11-22.1 mAb has a slight inhibitory effect on cancer cell proliferation. In one in vivo study, a chimeric antibody of MAb11-22.1 (MAb11-22.1-S239C-DM1) in the form of an antibody-drug conjugate (ADC) bound to meltansine (DM1, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) significantly inhibited the growth of OCI / AML2 xenograft tumors.

[0007] The coding sequence of the variable region of the light chain of MAb11-22.1 mAb is as shown in sequence number 1 below. 1 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga 50 51 gaaggtcact atgagctgca agtccagtca gagtctgtta aatagtggaa 100 101 atcaaaagaa ctacttgacc tggtaccagc agaaaccagg acagcctcct 150 151 aaactgttga tctactgggc atccacttgg gaatctaggg tccctgatca 200 201 cttcacaggc agtggatctg gaacagattt cactctcacc atcagcagtg 250 251 tgcaggctga agacctggca gtctattact gtcagaatga ttatagttat 300 301 cctctcacgt tcggtgctgg gaccaagctg gagctgaaac gggctgatgc 350 351 tgca (Sequence ID 1)

[0008] The translated amino acid sequence in the variable region of the light chain is predicted as Sequence ID No. 2 below, with the three antigen-determining regions (CDR1-CDR3, from left to right) highlighted in bold and underlined. IVMTQSPSSL TVTAGEKVTM SCKSS QSLLN SGNQKNYLT W YQQKPGQPPK LLIY WASTWE SR VPDHFTGS GSGTDFTLTI SSVQAEDLAV YYC QNDYSYP LT FGAGTKLE LKRADAA(Sequence ID 2)

[0009] The coding sequence of the variable region of the heavy chain of MAb11-22.1 mAb is as shown in sequence number 3 below. 1 gaggtccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc 50 51 agtgaggatt tcctgcaaga cttctggcta caccttcaca aactactata 100 101 tacactggat gaagcagagg cctggacagg gacttgagtg gattggatgg 150 151 atttatcctg gagatggtaa ttctcattac aatgagaagt tcaagggcaa 200 201 gaccacactg actgcagaca aatcctccag cacaggctac atattgctca 250 251 gcagcctgac ctctgaagac tctgcagtct atttctgtac aagagattat 300 301 gataactacg ggggatttgc ttactggggc caagggactc tggtcactgt 350 351 ctct(SEQ ID NO: 3)

[0010] The translated amino acid sequence in the variable region of the heavy chain is predicted as SEQ ID NO: 4 below, and three antigenic determinant regions (CDR1 - CDR3, from left to right) are highlighted in bold and underlined. EVQLQQSGPE LVKPGASVRI SC KTSGYTFT NYYIH WMKQR PGQGLEWIG WI YPGDGNSHYN EKFKG KTTLT ADKSSSTGYI LLSSLTSEDS AVYFC TRDYD NYGGFAY WGQG TLVTVSA(SEQ ID NO: 4)

[0011] The present invention relates to a binder (defined below) comprising an antibody (defined below), such as a monoclonal antibody, its fragments, and derivatives, comprising one or more light chain CDR regions that are at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical to the light chain CDR region of an anti-TfR1 specific mAb named MAb11-22.1, and a light chain variable region that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical to SEQ ID NO: 2. Optionally, the heavy chain variable region of the binder is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical to SEQ ID NO: 4. The binder may include a region such as the light chain CDR1 of MAb11-22.1 that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical to the sequence QSLLNSGNQKNYLT (SEQ ID NO: 5). The binder may also include a region such as the light chain CDR2 of MAb11-22.1 that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical to the sequence WASTWESR (SEQ ID NO: 6). The binder may also include a region such as the light chain CDR3 of MAb11-22.1 that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical to the sequence QNDYSYPLT (SEQ ID NO: 7). The binder may include a region such as heavy chain CDR1 of MAb11-22.1 that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical to the sequence KTSGYTFTNYYIH (SEQ ID NO: 8). The binder may also include a region such as heavy chain CDR2 of MAb11-22.1 that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical to the sequence WIYPGDGNSHYNEKFKG (SEQ ID NO: 9).The binding agent may include a region such as the heavy chain CDR3 of MAb11-22.1 that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%) identical to the sequence TRDYDNYGGFAY (SEQ ID NO: 10).

[0012] This application relates to the treatment of multiple types of cancers overexpressing TfR1 using MAb11-22.1 and binding agents related to MAb11-22.1 (defined below), wherein the binding agent is derived from one or more of the sequences of the CDRs of the light and heavy chains of MAb11-22.1 or includes a portion that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%) identical to one or more of the sequences of the CDRs of the light and heavy chains of MAb11-22.1. MAb11-22.1 specifically binds to the human TfR1 protein with high affinity and induces internalization of this protein.

[0013] The binding agent is preferentially used for the treatment of tumor-related diseases including primary and metastatic cancers such as, for example, leukemia, lymphoma, multiple myeloma, breast cancer, glioblastoma, prostate cancer, urothelial cancer (bladder cancer), esophageal cancer, colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, gastric cancer (including gastric squamous cell carcinoma, gastric adenocarcinoma, gastric small cell carcinoma, gastric squamous epithelial carcinoma, gastric carcinoid tumor, and gastric-duodenal cancer).

[0014] The binder complex preferably comprises an antitumor agent, for example, a cytotoxin (meytansine or its derivatives), auristatin or its derivatives, epothilone or its derivatives, paclitaxel or its derivatives, or a vinca alkaloid compound, or further comprising combretastatin A-4 phosphate, combretastatin A-4 or its derivatives, an indole-sulfa compound, or a vinca alkaloid compound (vinblastine). e) Vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrous vinblastine, dorastatin 10 and its analogues, halichondrin B and eribulin, indole-3-oxalylamide, substituted indole-3-oxalylamide, podophyllotoxin, 7-di Ethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discormolide, laulimalide, etc.); DNA topoisomerase inhibitors (camptothecin and its derivatives, mitoxantrone, etc.); mitoguazone; nitrogen mustard analogs (chlorambucil, chlormaphazine, cyclophosphatidyl Examples include cyclophosphamide, estramustine, ifosfamide, mustine, nitromin, melphalan, novembichin, fenamet, phenesterine, prednimustine, trophosfamide, and uramustine.Nitrosourea (carmustine, streptozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.); antibiotics (engine antibiotics, dynemicin, esperamicin, neocartinostatin, aclacinomycin, actinomycin) Actinomycin, Anthroamycin, Azaserine, Bleomycin, Actinomycin C, Carabicin, Idarubicin, Carzinophilin, Carminomycin, Actinomycin D, Daunorubicin, Doxorubicin, 6-Diazo-5-oxo-L-norleucine, Adli Adriamycin, Epirubicin, Esorubicin, Idarubicin, Marcellomycin, Mitomycin, Mycophenolic acid, Nogalamycin, Olivomycin, Peplomycin, Bofeimeisu, Puromycin, Adriamycin-Fe, Rodorubicin icin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, etc.; folic acid analogs (denopterin, methotrexate, pteropterin, trimethrexate, edatrexate, etc.);Purine analogs (fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.); pyrimidine analogs (ancitabine, gemcitabine, enoxaparin, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, deoxy-fluorouridine, fluorouridine, etc.); androgens (calsterone, dromostanolone propionate) (propionate), epithiostanol, mepitiostane, testolactone, etc.); adrenocorticotropic hormone compounds (aminoglutethimide, mitotane, trilostane, etc.); trichothecene (T-2 toxin, verracurin A, roridin A and anguidine, etc.); aziridine (benzodopa, carboco This includes (Carboquone, Meturedopa, and Uredopa, etc.); platinum analogs (Cisplatin, Carboplatin, Oxaliplatin, Miriplatin, Etoposide, etc.); antiandrogens (Flutamide, Nilutamide, Bicalutamide, Leuprolide, and Goserelin, etc.); protein kinases and proteasome inhibitors.

[0015] Preferably, the binder is bound to the antitumor agent by a linker. The binding may be carried out by site-directed binding, as described below for mertansine (DM1), i.e., by binding the mercapto group of the binder to the antitumor agent. Many other binding methods are available.

[0016] In some embodiments, the linker may consist of flexible residues such as glycine and serine (one example being four repeating glycine and one serine residue, or simply repeating glycine residues) and may be used to bind the complex, thereby allowing adjacent protein domains to move freely relative to each other. If it is desired to maintain distance between domains and prevent them from interacting, a rigid linker is preferred, one example being (repeated): glutamic acid, three alanine residues, lysine. Another rigid linker is (repeated): any amino acid residue and proline. The linker may be non-cleavable linkers (e.g., thioether, SMCC, PEG linkers) or cleavable linkers (valine-citrulline (Val-Cit, VC) dipeptides, glutamic acid-valine-citrulline (Glu-Val-Cit, GVC) tripeptides, and disulfide linkers). U.S. Patent No. 9,310,373, "Molecular Conjugate," discloses multiple hydrazide thiol linkers and the preparation of complexes using them. U.S. Patent No. 8,518,891 discloses linkers having aminoarylmethyl or aminoheteroaryl moieties. Bifunctional linkers are also described. See U.S. Patent No. 11,040,084.

[0017] Appropriate dosage ranges and administration plans can be estimated from the results of in vivo and in vitro studies described herein. While conjugates are preferentially delivered as therapeutic pharmaceutical formulations, they may also be used for diagnostic, purification, or screening purposes to select other conjugates. [Brief explanation of the drawing]

[0018] The patent or application documents include at least one color drawing. A copy of this patent or patent application publication containing the color drawing will be provided by the Patent Office upon request and payment of the required fees.

[0019] [Figure 1A-1C] MAb11-22.1 mAbs directly bind to AML cell lines but do not bind to healthy human peripheral blood mononuclear cells (PBMCs). [Figure 1A] Fluorescence-activated cell sorting (FACS) assays showed that MAb11-22.1 mAb interacted with nine human AML cell lines on the cell surface but not with human PBMCs (left panel). In this assay, isotype control antibodies were used as negative controls (right panel). Detection was performed using 1:800 dilution of fluorescein-labeled AffiniPure goat anti-mouse IgG, Fcγ fragment-specific antibody (anti-Mo IgG Fc-FITC, Jackson ImmunoResearch Laboratories). The assay was performed at 4°C. MFI is the mean fluorescence intensity. [Figure 1B] Images of immunocytochemistry (ICC) assays of MAb 11-22.1 mAb conjugated to the surface of NB4 or THP-1 cells are shown (upper panel), and isotype control antibodies did not stain these cells (lower panel). AML cells were fixed to glass slides by cytospin in 4% paraformaldehyde. After antigen retrieval, cells were blocked in PBST containing 1% bovine serum albumin (BSA), then incubated with primary antibodies for 1 hour, followed by incubation with 1:1000 dilution peroxidase-labeled AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3), Fcγ fragment-specific antibody (Jackson ImmunoResearch Laboratories), for 1 hour. Cells were stained with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. [Figure 1C]This Western blot shows that MAb11-22.1 mAbs hybridized with protein bands present in whole cell lysates of ALL cells (Raji) and AML cells (NB4, OCI / AML2, and THP-1). The target protein was approximately 250 kDa under non-reducing conditions (left panel) or 110 kDa under reducing conditions (right panel). Chinese hamster ovary (CHO) cell lysates were used as a negative control. β-actin was simultaneously detected by specific mouse mAbs as a sample loading control. Peroxidase-labeled AffiniPure goat anti-mouse IgG Fcγ at a 1:10,000 dilution was used for detection. [Figure 2A-2D] This paper demonstrates the identification and confirmation of transferrin receptor 1 (TfR1) as a target for MAb11-22.1 mAbs. [Figure 2A] This is a representative silver-stained SDS-PAGE image of an immunoprecipitation (IP) sample obtained by reaction of MAb11-22.1 mAb with AML whole cell lysates. The IP sample was reduced and loaded evenly into lanes 1 and 2, and the target band of approximately 110 kDa shown was excised for LC-MS / MS analysis. Reduced MAb11-22.1 mAb was loaded at 8 ng in lane 4 as a control. Protein bands were detected using the ProteoSilver® silver staining kit (MilliporeSigma) according to the manufacturer's instructions. MW is the molecular weight marker, HC is the heavy chain, and LC is the light chain. [Figure 2B] Western blots of unreduced ALL and AML whole cell lysate samples simultaneously probed with MAb11-22.1 mAbs and anti-β-actin mAbs are shown. The left lane is loaded with 1.2 μg of recombinantly expressed TfR1 extracellular domain (rTfR1-ECD, ACRO Biosciences) as a positive control, and the two lanes on the right are loaded with CHO cell lysates at two concentrations (1× and 2×) as negative controls. Peroxidase-labeled AffiniPure goat anti-mouse IgG Fcγ at a dilution of 1:20,000 was used for detection. [Figure 2C]This study demonstrates the direct interaction between MAb11-22.1 mAb and the extracellular domain of TfR1, confirmed by ELISA. Plates were coated with 0.1 μg / mL purified rTfR1-ECD containing a C-terminal 6×His tag. Mouse anti-6×His mAb and isotype control antibodies were used as positive and negative controls, respectively. All primary antibodies were serially diluted 3-fold at a starting concentration of 3 μg / mL. Peroxidase-labeled AffiniPure goat anti-mouse IgG Fcγ at a dilution of 1:6,000 was used for detection. [Figure 2D] The results of FACS assays of CHO cells with and without recombinant expression of human TfR1-GFP fusion protein (rTfR1-GFP) on the cell membrane are shown. CHO cells did not react with MAb11-22.1 mAb #1 or #2 (two different purified batches) or a commercially available anti-human TfR1 control mAb (R&D systems). However, after reconstitution of TfR1 on the cell membrane, MAb11-22.1 mAb #1, #2 and the anti-TfR1 control mAb were able to bind to the cell surface. MAb11-22.1 mAb had a higher affinity than the anti-TfR1 control mAb. The secondary antibody used was R-phycoerythrin (R-PE) labeled AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3), Fcγ fragment-specific antibody (Jackson ImmunoResearch Laboratories), at a dilution of 1:800. [Figure 3A-3C] This is data output from a ForteBio Octet® QK system, showing the affinity and dynamics between MAb11-22.1 mAb and rTfR1-ECD. [Figure 3A]Aligned sensorgram traces showing binding and dissociation curves for MAb11-22.1 mAb binding to 20 nM rTfR1-ECD (left panel) or 300 nM goat anti-mouse (H+L)Fab (positive control, right panel), extracted from raw data by fortebio software. The ForteBio Octet® QK system was equipped with an anti-mouse Fc capture (AMC) biosensor for immobilizing 10 nM or 50 nM MAb11-22.1 mAb. The duration of the main steps was only 60 seconds baseline in dynamic buffer, 450 seconds MAb11-22.1 mAb immobilization, 120 seconds baseline / wash in dynamic buffer, 450 seconds antigen binding, followed by 3000 seconds dissociation in dynamic buffer. The upper curve is for 10 nM mAb, and the lower curve is for 50 nM mAb. [Figure 3B] The dynamical data of the binding of MAb11-22.1 mAb to rTfR1-ECD, serially diluted 2.5 times from 20 nM, are shown. Within the observation period (3000 seconds), binding occurred immediately, and no dissociation was detected. [Figure 3C] To verify the reliability of the instrument, we present the kinetic data of binding of MAb 11-22.1 mAb in serial dilutions to the positive control, goat anti-mouse IgG(H+L)Fab. [Figure 4] These are the results of a competitive ELISA demonstrating that MAb11-22.1 mAb and transferrin (Tf) bind to different sites on rTfR1-ECD. ELISA plates were coated with 0.1 μg / mL rTfR1-ECD and incubated with arrays of five concentrations of purified Tf protein (X axis) individually mixed with seven concentrations of HRP-conjugated MAb11-22.1 mAb, serially diluted four-fold from 2 μg / mL, and with different concentrations of Tf alone. MAb11-22.1 mAb-HRP bound to rTfR1-ECD in a dose-dependent manner, independent of the presence of Tf. Data represent the mean ± standard deviation (SD) of overlapping samples. [Figures 5A-5C]These are FACS results of the binding of MAb11-22.1 mAb to PBMCs and bone marrow (BM) cells from healthy adults, and BM cells from AML patients. [Figure 5A] MAb11-22.1 mAbs did not bind to normal human PBMCs (Stanford Blood Center) or normal human BM cells (Human Cells (Fremont, California)), regardless of mAb concentration. Isotype control antibodies served as negative controls. mAbs were detected in 1:800 dilution anti-mouse IgG Fc-FITC, serially diluted 8-fold from 20 μg / mL. [Figure 5B] MAb11-22.1 mAb demonstrated dose-dependent binding to BM cells from AML patients. [Figure 5C] This is a scatter plot of PBMC and BM cells stained with different mAbs. The cutoff values ​​for determining whether cell surface markers are positive or negative are shown by solid lines, dividing the graph into four quadrants. The presence of stem cells in the BM sample was confirmed by FITC-labeled mouse anti-human CD34 mAb (Miltenyi, Cambridge, Massachusetts), which is specific to hematopoietic stem cells (lower left panel). MAb11-22.1 mAbs and isotype control antibodies stained a small number of BM cells due to their adhesion. [Figure 5D] The erythrocytes present in PBMC and BM cell populations stained with anti-human CD233-PE mAb (Miltenyi) did not react with MAb11-22.1 mAb or isotype control antibody. [Figure 5E] This indicates that erythrocytes and erythroblasts present in PBMC or BM cell populations stained with anti-human CD235a-PE (Miltenyi) did not react with MAb11-22.1 mAb or isotype control antibody. [Figure 6A-6C] These are the results of an internalization assay of MAb11-22.1 mAb using cancer cell lines. [Figure 6A]The fluorescence intensity on the cell surface of three leukemic cell lines (OCI / AML2, NB4, and Raji) individually stained with MAb11-22.1 mAb, mouse anti-human CD20 mAb, or isotype control antibody was compared under different conditions in a FACS assay. Cells were incubated with each mAb at 4°C for 1 hour, followed by washing with cold PBS and incubation in medium at 4°C or 37°C. Cell staining was performed with anti-Mo IgG Fc-FITC for 30 minutes, 1 hour, and 2 hours, respectively. At 4°C, MAb11-22.1 mAb dose-dependently bound to the surface of two AML cell lines but not to Raji cells, and was immediately internalized upon transfer to 37°C (left panel). Anti-CD20 mAb, which interacted with Raji cells but not with AML cells, was partially internalized by Raji cells at 37°C (middle panel). Isotype control antibodies that do not bind to either cell line were used as negative controls (right panel). [Figure 6B] These are representative fluorescence images of MAb11-22.1 mAb bound to and internalized by the OCI / AML2 cell line. Cells from duplicate 96-well culture plates were incubated at 4°C for 1 hour with different concentrations of CF488-labeled MAb11-22.1 mAb or CF488-labeled isotype control antibody in DMEM medium, followed by washing with ice-cold PBS to remove the mAb. Cells from one plate were resuspended in ice-cold PBS in preparation for imaging at 400× magnification using a Keyence fluorescence microscope, while cells from the duplicate plate were resuspended in warm SFM and incubated at 37°C for 1 hour to allow for mAb internalization. MAb11-22.1 mAb-CF488 bound to the surface of OCI / AML2 cells at 4°C, but was primarily internalized after incubation at 37°C for 1 hour. As a control, the CF488-labeled isotype control mAb did not stain the cells and was not internalized by the cells (right panel). All images were taken using the same settings. The scale bar represents 100 μm. [Figure 6C]This is a representative image showing a dose-dependent increase in the binding and internalization of MAb11-22.1 mAb-CF488 by MDA-MB-231 cells. The assay procedure was the same as in Figure 6B, except that a higher concentration (5 μg / mL) of mAb was used. At 4°C, MAb11-22.1 mAb-CF488 was present not only on the surface of MDA-MB-231 cells but also intracellularly, indicating a high tendency for internalization of MAb11-22.1 mAb-CF488 by the MDA-MB-231 cell line. After incubation at 37°C for 1 hour, the majority of MAb11-22.1 mAb-CF488 was internalized. CF488-labeled isotype control mAb-CF488 did not stain the cells or was not taken up by the cells (right panel). Images were taken at 400× magnification with the same exposure time. Scale bar represents 100 μm. [Figure 7] This study demonstrates the partial inhibitory effect of MAb11-22.1 mAb on the proliferation of OCI / AML2 cells. Cells (2 × 10⁴ cells / mL) were cultured at 37°C in a humidified 5% CO₂ atmosphere in the presence of 100 μg / mL of MAb11-22.1 mAb or isotype control antibody. Cell viability, reflected by absorbance at 450 nm (OD450), was monitored every 24 hours by treatment with 10 μL of CCK-8 solution for 4 hours. Data show the mean ± SD of three overlapping samples. *P<0.05. [Figures 8A-8B] This data output from the ForteBio Octet® QK system shows the affinity and dynamics between the MAb11-22.1 chimeric antibody (cAb) and rTfR1-ECD. [Figure 8A]Fitting views and data tables of binding and dissociation curves for the binding kinetics between MAb11-22.1 cAb and rTfR1-ECD at 2.5-fold serial dilutions (50, 20, 8, 3.2, and 1.28 nM) are shown. The Octet QK system was equipped with an anti-human Fc capture (AHC) biosensor for immobilizing 50 nM or 10 nM MAb11-22.1 cAb. The duration of the main steps was 60 seconds for baseline in kinetic buffer, 450 seconds for MAb11-22.1 cAb immobilization, 120 seconds for baseline / wash in kinetic buffer, 450 seconds for binding of antigen to immobilized MAb11-22.1 cAb, followed by 3600 seconds for dissociation in kinetic buffer. Since no decay of the binding signal was detected during the period of permissible dissociation, the KD of MAb11-22.1 cAb and rTfR1-ECD was less than 1 pM. [Figure 8B] This is a fitted view of the kinetic analysis of the positive control goat anti-human (H+L)Fab for three-fold serial dilutions (300, 100, 33.3, 11.1, and 3.7 nM) in binding to 50 nM MAb11-22.1 cAb. The calculated KD was 2.18 nM. [Figure 9] This bar graph shows the effect of MAb11-22.1 cAb and isotype control antibodies with or without the S239C mutation on the proliferation of various cancer cell lines in vitro. OCI / AML2, Raji, HCC38, and MDA-MB-231 cell lines were cultured for 5 days with individual antibodies at optimal doses and then analyzed by the CCK-8 assay. Two doses (100 and 300 μg / mL) of MAb11-22.1 cAb (shaded bars) and MAb11-22.1-S239C cAb (gray bars) were treated with DM1 and indicated with a "+" sign below the X-axis, while antibodies not treated with DM1 are indicated with a "-" sign. Unbound isotype control antibodies (white bars) reflect the baseline proliferation of each cell line. Data show the mean ± SD of three overlapping samples. [Figure 10]This study demonstrates the in vivo efficacy of MAb11-22.1-S239C-DM1 against tumor growth in a xenograft (CLDX) mouse model derived from OCI / AML2 cell lines. MAb11-22.1-S239C-DM1 was administered intraperitoneally (ip) at a dose of 10 mg / kg (low dose, n=5) or 20 mg / kg (high dose, n=6) on a Q4×7 schedule (days 0 to 24). The negative control group received isotype control antibody (without S239C mutation) treated with DM1 at 20 mg / kg / Q4×7 / ip (n=5). Tumor volume was measured every 4 days. The left panel plots the tumor volume of individual mice in each group, and the right panel compares the mean tumor volume ± SD between different groups. From day 16 after four antibody treatments, the mean tumor volume in the low-dose group was significantly different from that of the isotype control DM1 group (*P<0.05). [Modes for carrying out the invention]

[0020] Unless otherwise specified in the context, please understand that the singular forms "a / an" and "the" can also be interpreted as plural. Monoclonal antibodies are sometimes referred to as "mAb." The singular and plural forms of the terms "conjugate" and "antibody" are interchangeable.

[0021] The binders of the present invention include antibodies, antibody fragments, fusion proteins, or antibody-derived or modified chimeric antibody receptors (CARs) that are similar to or derived from MAb11-22.1, and contain an intracellular signaling domain, such as a single-chain variable fragment (scFv) fused with the ζ chain of CD3 (CD3ζ). The CARs are preferably expressed in CAR immune effector cells, including but not limited to T cells, NK cells, and macrophages.

[0022] The term “conjugate” includes antibodies, which are immunoglobulin molecules capable of binding to a target antigen, e.g., an antigen on gastric cancer cells, by at least one antigen-recognition site located in the variable region of the immunoglobulin molecule (both terms are used interchangeably in the plural). As used herein, the term “antibody” includes not only complete (i.e., full-length) polyclonal or monoclonal antibodies, but also their antigen-binding fragments (Fab, Fab', F(ab')2, Fv, Fd, rIgG, single-chain (scFv) or sc(Fv)2), their variants, fusion proteins containing the antibody portion, humanized antibodies, chimeric antibodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antigen-binding (SDAB) molecules, VH or VL domains or VHH domains, and any other modified configurations of immunoglobulin molecules containing antigen-recognition sites of the required specificity, wherein the modified configurations include glycosylated variants of antibodies, amino acid sequence variants of antibodies, and antibodies modified by covalent bonding. Antibody-drug conjugates are also included.

[0023] Antibodies include all classes of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and an antibody does not need to belong to any particular class. Immunoglobulins may be assigned to different classes based on the antibody amino acid sequence of the constant domain of their heavy chain. There are five major classes of immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0024] The antibodies described herein may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies, such as those described in U.S. Patent No. 7317091B2, and such antibodies produced by affinity maturation). In some examples, the antibodies include a modified constant region, such as an immunologically inactive constant region that does not induce complement-mediated lysis or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity may be evaluated by the method disclosed in U.S. Patent No. 5,500,362.

[0025] A humanized antibody refers to a form of non-human (e.g., mouse) antibody that is its antigen-binding fragment containing a minimal sequence derived from a specific chimeric immunoglobulin, immunoglobulin chain, or non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may also contain residues not found in the recipient antibody or the imported CDR or framework sequence, but included for further improvement and optimization of the antibody's performance. Generally, a humanized antibody contains at least one, typically substantially all, variable domains, provided that all or substantially all CDR regions correspond to those of the non-human immunoglobulin, and all or substantially all FR regions correspond to those of the human immunoglobulin consensus sequence. Humanized antibodies optimally contain at least a portion of the immunoglobulin constant region or domain (Fc), typically at least a portion of the human immunoglobulin constant region. The antibody may have a modified Fc region, as described in International Patent No. WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, and / or six) that are altered relative to the original antibody. Humanized antibodies may be involved in affinity maturation. See Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988).

[0026] In other embodiments, the antibodies described herein are chimeric antibodies that may include heavy chain constant regions and light chain constant regions from human antibodies. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81,6851; Neuberger et al. (1984) Nature 312,604; Takeda et al. (1984) Nature 314:452. A chimeric antibody is an antibody having a variable region or part of a variable region from a first species and a constant region from a second species. Typically, in such chimeric antibodies, both the light chain and heavy chain variable regions are mimics of the variable region of an antibody derived from a certain mammal (e.g., a non-human mammal such as a mouse, rabbit, or rat), and the constant region is homologous to the sequence of an antibody derived from another mammal, such as a human. In some embodiments, amino acid modifications may be made in the variable region and / or constant region. See, U.S. Patent No. 4,816,567.

[0027] The "percentage of identity" between two amino acid sequences may be determined arithmetically by counting and comparing them to shorter sequences described or claimed herein, or by using the algorithm of Karlin and Altschul Proc.Natl.Acad.Set USA 87:2264-68,1990, as modified in Karlin and Altschul Proc.Natl.Acad.Sci.USA 90:5873-77,1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J.Mol.Biol.215:403-10,1990. BLAST protein search may also be performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to the target protein molecule. If a gap exists between two sequences, you may use Gapped BLAST, as described in Altschul et al, Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST program or Gapped BLAST program, you may use the default parameters of the corresponding program (e.g., XBLAST, NBLAST).

[0028] (Preparation of binder) Numerous methods have been developed to produce chimeric antibodies, humanized antibodies, or human antibodies for in vivo therapeutic applications in humans. The most commonly used method involves producing mouse mAbs using the hybridoma method, and then humanizing the mAbs by converting the framework regions of the VH and VL domains and the constant domain of the mAbs to homologous human framework regions of the human VH and VL domains, as well as the constant regions of desired human γ-immunoglobulin isotypes and subclasses. (See U.S. Patent No. 5,225,539.)

[0029] Monoclonal antibodies may be produced by the conventional hybridoma method (Kohler et al., Nature, 256:495 (1975)). In the hybridoma method, mice or other suitable host animals such as hamsters or rabbits are immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro.

[0030] To produce monoclonal antibodies, immune cells are collected from mammals immunized with an antigen, and the increase in the level of the desired antibody in the serum is examined as described above, and the immune cells undergo cell fusion. The immune cells used for cell fusion are preferably obtained from the spleen. Other preferred parent cells to be fused with the above immune cells include, for example, mammalian myeloma cells, and more preferably myeloma cells having acquired properties for drug-induced selection of fusion cells.

[0031] Preferred myeloma cells are those that efficiently fuse, can be used for stable, high-level antibody production by cells that produce selected antibodies, and are sensitive to culture media such as HAT medium. Among these, mouse myeloma cell lines such as MOPC-21 and MPC-11 mouse tumor-derived cell lines (available from Salk Institute Cell Distribution Center (San Diego, California, USA)) and SP-2 cells (available from American Type Culture Collection (Rockville, Maryland, USA)) are preferred myeloma cell lines. Human myeloma cell lines and mouse-human heterozygous myeloma cell lines have also been described as being used for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0032] The above immune cells and myeloma cells may be fused according to known methods, e.g., Milstein et al. (Galfre et al., Methods Enzymol. 73:3-46, 1981). Lymphocytes are fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Hybridomas obtained by cell fusion may be selected by culturing them in a standard selective medium such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Cell culture is typically continued in HAT medium for several days to several weeks, the time being sufficient for all other cells to die except for the desired hybridomas (non-fused cells). Next, standard limiting dilutions are performed to screen and clone hybridoma cells that produce the desired antibody.

[0033] The hybridoma cells thus produced are inoculated into a suitable culture medium and grown. The culture medium preferably contains one or more substances that inhibit the growth or survival of unfused parent myeloma cells. For example, if parent myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibit the growth of HGPRT-deficient cells. The culture medium in which the hybridoma cells grow is then subjected to assays for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays. Enzyme-linked immunosorbent assay (ELISA), enzyme-mediated immunoassay (EIA), radioimmunoassay (RIA), and / or absorbance measurement in immunofluorescence can be used to measure the antigen-binding activity of the antibodies.

[0034] After hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, clones may be subcloned by limiting dilution and grown using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Monoclonal antibodies secreted by the subclones can be appropriately separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods, such as protein A Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Furthermore, hybridoma cells can grow in the body as ascites tumors in the peritoneal cavity of animals, for example, mice.

[0035] The obtained monoclonal antibody may be purified, for example, by ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the protein of the present invention binds.

[0036] The monoclonal antibodies obtained in this manner may also be synthesized by recombinant DNA using genetic engineering techniques (see, for example, Borrebaeck CAK and Larrick JW, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD, 1990). Recombinant antibodies may also be synthesized by cloning the DNA encoding antibodies from immune cells such as antibody-producing hybridomas or immune lymphocytes, inserting it into a suitable vector, and introducing it into host cells. Recombination techniques are described in the following literature. Specifically, Sambrook et al.,MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring Harbor Laboratory Press,1989,and Third edition,2001;Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley &Sons,New York,1987 and periodic updates;the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Kontermann and Dubel, ANTIBODY ENGINEERING, Springer Lab manual, Springer-Verlag Berlin Heidelberg, 2001).

[0037] When the obtained antibodies are administered to humans for therapeutic purposes, human antibodies or humanized antibodies are preferred to reduce their immunogenicity. For example, genetically modified animals having a human antibody gene repertoire may be immunized with antigens selected from whole cells or human cell proteins or their lysates. Next, antibody-producing cells can be collected from the animals and fused with myeloma cells to obtain hybridomas, from which human antibodies against the aforementioned antigens can be produced. Alternatively, immune cells that produce antibodies, such as immune lymphocytes, may be immortalized with oncogenes and used to produce monoclonal antibodies.

[0038] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells are a preferred source of such cDNA. After isolation, the DNA may be placed in an expression vector and then transfused into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, thereby inducing the production of monoclonal antibodies in the recombinant host cells. For a general overview of recombinant expression of antibody-encoding DNA in bacteria, see Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993); Pluckthun, Immunol. Rev., 130:151-188 (1992).

[0039] Genetically modified antibodies may be produced by genetically recombining the DNA encoding antibodies produced by the hybridoma cells described above using conventional techniques. Genetically modified antibodies such as humanized antibodies, chimeric antibodies, single-chain antibodies, diabodies, bispecific antibodies, and multispecific antibodies may be produced using conventional recombination techniques. Next, the DNA may be modified by replacing homologous mouse sequences, for example, by substituting the human heavy chain constant domain and light chain constant domain with coding sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently binding all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Genetically modified antibodies having target antigen binding specificity, such as chimeric antibodies or humanized antibodies, may be produced by the above method.

[0040] Alternatively, certain genetically modified animals (e.g., mice) can produce a complete repertoire of human antibodies through immunization even without the production of endogenous immunoglobulins. For example, the antibody heavy chain binding region (J) in chimeric mice and germ cell mutant mice. HIt has been described that homozygous deletion of the gene completely inhibits the production of endogenous antibodies. When a human germ cell immunoglobulin gene array is transferred into such germ cell mutant mice, human antibodies are produced upon antigen stimulation. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993). See also genetically modified mice (Xenomouse) provided by Amgen, Inc. (Fremont, California), HuMAb-Mouse® and TC Mouse® provided by Medarex, Inc. (Princeton, New Jersey). In another alternative embodiment, antibodies may be produced recombinantly by phage display technology. For example, see U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150; Winter et al., (1994) Annu. Rev. Immunol. 12:433-455; (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991). Alternatively, human antibodies and antibody fragments may be produced in vitro from an immunoglobulin variable (V) domain gene repertoire from an unimmunized donor using phage display technology (McCafferty et al., (1990) Nature 348:552-553).

[0041] The F(ab')2 fragment may be produced from a complete antibody by digestion with pepsin or other proteins (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). The Fab fragment may be produced by reducing the disulfide bond of the F(ab')2 fragment. Currently, such fragments may be produced directly by recombinant host cells. For example, antibody fragments may be isolated from an antibody phage library. Alternatively, the F(ab')2-SH fragment may be directly recovered from E. coli and chemically conjugated to form the F(ab')2 fragment (Carter et al., Bio / Technology 10:163-167 (1992)). By another method, the F(ab')2 fragment may be directly isolated from cultures of recombinant host cells.

[0042] U.S. Patent No. 5,932,448 discloses the preparation of a bispecific antibody having a Fab' portion linked by a leucine zipper, U.S. Patent No. 7,538,196 discloses the preparation of a bispecific antibody in which each portion is linked by a linker, and U.S. Patent No. 8,148,496 discloses a multispecific Fv antibody construct having at least four variable domains linked to each other via a peptide linker.

[0043] U.S. Patent Publication 20170335281 describes the creation of genetically modified T cells expressing a CAR containing an antigen-binding domain that binds to cancer-associated antigens. Similar common techniques may be used to modify T cells or other immune effector cells to express one or more of MAb11-22.1 CDR1, CDR2, and CDR3 as antigen-binding domains for cancer treatment. The antigen-binding domain of the CAR polypeptide molecule may include any antibody, antibody fragment, scFv, Fv, Fab, F(ab')2, single-domain antibody (SDAB), VH or VL domain, or VHH domain.

[0044] Single-domain antibody (sdAb) molecules are disclosed in International Patent No. WO9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. sdAb molecules are naturally occurring single-domain antigen-binding molecules known as light-chain-deficient heavy-chain molecules. In one type of sdAb, the variable domain may be derived from a naturally occurring light-chain-deficient heavy-chain molecule, for example, from camelid species such as camels, llamas, dromedaries, alpacas, and guanacos. Other non-camelid species may also naturally produce light-chain-deficient heavy-chain molecules. sdAb molecules may be recombinant, CDR-grafted, humanized, camelidized, deimmunized, and / or ex vivo-generated (e.g., selected by phage display).

[0045] All of the above binders can be used in antibody-drug conjugates (ADCs) that target cancer cells by binding them to cytotoxins that affect cancer cells. See U.S. Patent No. 9,764,041; U.S. Patent Publication No. 2017,015,1343.

[0046] (High affinity antibody variant) Antibodies having variants of the sequences described herein are within the scope of the present invention. One type of variant is the high-affinity variant described below.

[0047] Antibodies should be humanized while retaining high affinity for antigens and other advantageous biological properties. To achieve this objective, according to preferred methods, humanized antibodies are produced by a process of analyzing the parent sequence and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available. Computer programs are available that display and describe possible three-dimensional conformations of selected candidate immunoglobulin sequences. By examining these displays, it becomes possible to analyze the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, desired antibody properties, such as increased affinity for the target antigen, can be achieved by selecting and combining FR residues from the recipient and import sequences.

[0048] Examples of framework region residues to be modified include those that bind directly to the target noncovalently (Amit et al. Science 233:747-753 (1986)), residues that interact with or affect the conformation of the CDR (Chothia et al. J.Mol.Biol.196:901-917 (1987)), and / or residues involved in the VL-VH interface (European Patent No. EP239400B1). In certain embodiments, modification of one or more such framework region residues leads to improved antibody binding affinity to the target of interest.

[0049] Nucleic acid molecules encoding amino acid sequence variants are prepared by various methods known in this field. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, polymerase chain reaction (PCR) mutagenesis, and cassette mutagenesis of previously prepared variant or non-variant forms of species-dependent antibodies. A preferred method for generating variants is oligonucleotide-mediated synthesis. In certain embodiments, antibody variants have only a single substituted hypervariable region residue, for example, about 2 to about 15 hypervariable region substitutions.

[0050] One method for generating a library of variants is oligonucleotide-mediated synthesis. Three oligonucleotides, each having approximately 100 nucleotides, may be synthesized across the entire light or heavy chain variable region. Each oligonucleotide may contain (1) a 60-amino acid stretch generated by a triplet (NNK).sub.20 (where N is any nucleotide and K is G or T), and (2) an overlap of approximately 15-30 nucleotides with the next oligo or vector sequence at each end. When these three oligonucleotides are annealed in a PCR reaction, polymerase fills the opposite strand to produce a complete double-stranded heavy or light chain variable region sequence. The number of triplets may be adjusted to any length of repeat, and their positions in the oligonucleotide may be selected to substitute only amino acids within a specific CDR or framework region. By using (NNK), all 20 amino acids are possible at each position of the encoded variant. Overlapping sequences of 5-10 amino acids (15-30 nucleotides) are not substituted, but may be selected to fit within the stacking region of the framework, or substituted by a single synthesis round or subsequent synthesis rounds. Methods for synthesizing oligonucleotides are well known in this field and are commercially available. Methods for generating antibody variants from these oligonucleotides are also well known in this field, such as PCR.

[0051] A library of heavy and light chain variants with different random positions within the sequence may be constructed in any expression vector, such as a bacteriophage, each vector containing DNA encoding a specific heavy and light chain variant.

[0052] After producing antibody variants, the biological activity of the variants against the parent antibody is determined. As mentioned above, this relates to determining the binding affinity of the variants to the target. There are many high-throughput methods for rapidly screening the binding ability of antibody variants to a target of interest.

[0053] Next, in order to obtain a binding affinity that exceeds that of the parental antibody, one or more antibody variants selected by this first screening may be screened. One common method for determining binding affinity is to evaluate the association and dissociation rate constants using a BIAcore surface plasmon resonance system (BIAcore, Inc.). Activate the biosensor chip for covalent attachment of the target according to the manufacturer's (BIAcore) instructions. Next, dilute the target and inject it into the chip to obtain a signal in response units (RU) of the immobilized substance. Since the signal in RU is proportional to the mass of the immobilized substance, this represents the range of the density of the immobilized target on the matrix. Fit the dissociation data to a one-site model to obtain K off ±S.D. (standard deviation of the measurements). By calculating the pseudo-first-order rate constant (Ks) for each binding curve and plotting it as a function of protein concentration, K on ±S.D. (standard error of the fit) is obtained. By SPR measurement, the equilibrium dissociation constant K d is calculated as K off / K on . Since the equilibrium dissociation constant K D is inversely proportional to K off , assuming that the association rate (K on ) is constant for all variants, an improvement in affinity can be estimated.

[0054] Optionally, candidates with the obtained high affinity may be subjected to one or more further biological activity assays, such as being tested in the assays described in the following examples, to confirm that the desired therapeutic properties are still retained in the antibody variants with improved binding affinity. The optimal antibody variants retain the ability to bind to the target with a binding affinity significantly higher than that of the parental antibody.

[0055] The antibody variants thus selected may often undergo further modification depending on the intended use of the antibody. Such modifications may involve further changes in the amino acid sequence, fusion with heterologous polypeptides, and / or covalent modifications as detailed below. For example, any cysteine ​​residue not involved in maintaining the proper conformation of the antibody variant may generally be replaced with serine to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine ​​bonds may be added to the antibody to improve its stability (especially if the antibody is an antibody fragment such as an Fv fragment).

[0056] (formulation) After producing a suitable antibody, it may be prepared as a formulation for administration to a target. A lyophilized formulation is preferred, and for this purpose, the formulation must be prepared as a first step before lyophilization. The amount of antibody in the formulation before lyophilization is determined considering the desired dosage, administration method, etc. Proteins are generally present in solution. For example, proteins may be present in a pH buffer solution with a pH of about 4 to 8, preferably about 5 to 7. Examples of buffers include histidine, phosphate, Tris, citrate, succinate, and other organic acids. The concentration of the buffer may be about 1 mM to about 20 mM, or about 3 mM to about 15 mM, depending on the desired isotonicity of the buffer and the formulation (e.g., the reconstituted formulation). A preferred buffer is histidine, which has cryoprotective properties. Succinate is also a useful buffer.

[0057] A lyophilization protectant is added to the formulation before lyophilization. In preferred embodiments, the lyophilization protectant is a non-reducing sugar such as sucrose or trehalose. The amount of lyophilization protectant in the formulation before lyophilization is generally preferably such that the resulting formulation is isotonic, although hypertonic reconstituted formulations may also be appropriate. Furthermore, the amount of lyophilization protectant should not be so small that an unacceptable amount of protein degradation / aggregation occurs during lyophilization.

[0058] When the lyophilization protective agent is a sugar (such as sucrose or trehalose) and the protein is an antibody, the typical concentration of the lyophilization protective agent in the formulation before lyophilization is about 10 mM to about 400 mM, preferably about 30 mM to about 300 mM, and most preferably about 50 mM to about 100 mM.

[0059] The ratio of protein to lyophilized protective agent is selected for each combination of protein and lyophilized protective agent. When an antibody is selected as the protein and a sugar (e.g., sucrose or trehalose) is used as the lyophilized protective agent to produce a high-protein-concentration isotonic reconstituted preparation, the molar ratio of lyophilized protective agent to antibody may be about 100 to about 1500 moles of lyophilized protective agent per mole of antibody, preferably about 200 to about 1000 moles of lyophilized protective agent per mole of antibody, and may contain about 200 to about 600 moles of lyophilized protective agent per mole of antibody.

[0060] In preferred embodiments, it has been found that it is desirable to add a surfactant to the formulation before freeze-drying. Alternatively, the surfactant may be added to the freeze-dried formulation and / or the reconstituted formulation. Examples of surfactants include polysorbate (e.g., polysorbate 20 or 80), poloxamer (e.g., poloxamer 188), triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine, linoleyl-, myristyl-, or cetyl-betaine, lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl), myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl, or disodium methyl oleyl taurate, and the MONAQUAT® series (Mona Examples of nonionic surfactants include polyethyl glycol, polypropyl glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics®, PF68, etc.), as described by Industries, Inc. (Patterson, New Jersey). The amount of surfactant added is such that it reduces the aggregation of the reconstituted protein and minimizes the formation of particles after reconstitution. For example, the surfactant may be present in the formulation before freeze-drying in an amount of about 0.001 to 0.5%, preferably about 0.005 to 0.05%.

[0061] For the preparation of the formulation before freeze-drying, a mixture of a freeze-drying protective agent (such as sucrose or trehalose) and a bulking agent (such as mannitol or glycine) may be used. The bulking agent allows for the production of a uniform freeze-dried cake without excessive pockets.

[0062] Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), may be included in the formulation before lyophilization (and / or the lyophilized formulation and / or the reconstituted formulation), provided that they do not adversely affect the desired properties of the formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to the recipeen at the doses and concentrations used and include other buffers, preservatives, cosolvents, antioxidants (such as ascorbic acid and methionine), chelating agents (such as EDTA), metal complexes (e.g., Zn-protein complexes), biodegradable polymers (such as polyester), and / or salt-forming counterions (such as sodium).

[0063] The pharmaceutical compositions and formulations described herein are preferably stable to maintain physical and chemical stability and integrity during storage. Various analytical techniques for measuring protein stability are available in the art and are outlined in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991); Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability may be measured over a selected period of time at a selected temperature.

[0064] Preparations used for internal administration must be sterile. This can be easily achieved by filtering through a sterile filtration membrane before or after lyophilization and reconstitution. Alternatively, the entire mixture can be made sterile by autoclaving the components excluding the protein at approximately 120°C for approximately 30 minutes.

[0065] After mixing proteins, freeze-drying protectants, and other optional components, the formulation is freeze-dried. Various freeze-dryers can be used for this purpose, such as the Hull 50® (Hull, USA) or GT20® (Leybold-Heraeus, Germany). Freeze-drying is performed by freezing the formulation and then sublimating the ice from the frozen contents at a temperature suitable for primary drying. Under these conditions, the product temperature is lower than the eutectic or decay temperature of the formulation.

[0066] Typically, the shelf temperature for primary drying is in the range of approximately -30 to 25°C at appropriate pressure (typically about 50–250 mTorr) (assuming the product remains frozen during primary drying). The drying time depends mainly on the formulation in the container (e.g., glass vial), its size, type, and the amount of liquid, ranging from a few hours to several days (e.g., 40–60 hours). The secondary drying stage may be performed at approximately 0–40°C, depending mainly on the type and size of the container and the type of protein used. For example, the shelf temperature for the entire moisture removal stage of freeze-drying may be about 15–30°C (about 20°C). The time and pressure required for secondary drying are, for example, the time and pressure at which a suitable freeze-dried cake is produced, depending on temperature and other parameters. The secondary drying time depends on the desired moisture level in the product and typically takes at least about 5 hours (e.g., 10–15 hours). The pressure may be the same as that used in the primary drying step. Freeze-drying conditions may vary depending on the formulation and vial size.

[0067] In some cases, it may be desirable to freeze-dry the protein preparation in the container in which the protein is reconstituted to avoid the transfer step. In this case, the container may be, for example, a 3, 5, 10, 20, 50, or 100 cc vial. As a general suggestion, freeze-drying yields a freeze-dried preparation with a moisture content of less than about 5%, preferably less than about 3%.

[0068] At a desired stage, typically at the time when the protein is administered to the patient, the lyophilized formulation may be reconstituted with a diluent, so that the protein concentration in the reconstituted formulation is the same as the protein concentration in the formulation before lyophilization.

[0069] Reconstitution is generally carried out at a temperature of approximately 25°C to ensure complete hydration, but other temperatures may be used if desired. The time required for reconstitution depends, for example, on the type of diluent, excipients, and amount of protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives are those described above, with aromatic alcohols such as benzyl alcohol or phenolic alcohol being preferred preservatives. The amount of preservative used is determined by evaluating different preservative concentrations for compatibility with the protein and testing the effectiveness of the preservative. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it may be present in an amount of approximately 0.1–2.0%, preferably approximately 0.5–1.5%, and most preferably approximately 1.0–1.2%.

[0070] Alternatively, a non-freeze-dried formulation may be used, comprising a binder and any well-known carrier, excipient, buffer, stabilizer, preservative, auxiliary agent, and other additives described herein and well known in the art.

[0071] (Dosage and administration) The above-described formulations may be administered to subjects in need of treatment (e.g., humans) by appropriate routes such as intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intradermal, intra-articular, intrabursal, intrathecal, intradermal, intratumoral, intranodal, intramedullary, oral, inhalation, or local administration; or they may be administered orally, as an inhalation spray, locally, rectally, nasally, buccally, vaginally, or via an implanted reservoir; in any case, they may be administered as a large, instantaneous dose or as a continuous infusion over a period of time; or they may be administered via an injectable depot administration route, such as using depot-injectable or biodegradable materials and methods for 1, 3, or 6 months.

[0072] Commercial nebulizers for liquid formulations, such as jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations may be sprayed directly, and lyophilized powders may be sprayed after reconstitution. Alternatively, antibodies may be aerosolized using a fluorocarbon formulation and a metered-dose inhaler, or inhaled as lyophilized powder and pulverized powder. When CAR is used in the present invention, compositions of immunoeffector cells (e.g., T cells, NK cells) may be injected directly into tumors, lymph nodes, infection sites, or other locations.

[0073] The subjects treated by the methods described herein may be mammals, more preferably humans. Mammals include, but are not limited to, livestock, sport animals, companion animals, primates, horses, dogs, cats, mice, and rats.

[0074] The "effective dose" refers to the amount of active ingredient necessary to produce a therapeutic effect on a target, whether used alone or in combination with one or more other active ingredients. The effective dose varies depending on the specific condition being treated, the severity of the condition, individual patient parameters such as age, physical condition, size, sex, and weight, the duration of treatment, the characteristics of any parallel treatments, the specific route of administration, and similar factors, all of which are well known to those skilled in the art and can be addressed through standard testing. Generally, it is preferable to use the maximum dose of the individual ingredient or its combination, i.e., the maximum safe dose based on reasonable medical judgment. In some cases, a lower dose or tolerable dose may be appropriate for medical, psychological, or other reasons.

[0075] Empirical considerations, such as the half-life of antibodies, generally contribute to dosage determination. For example, antibodies adapted to the human immune system, such as humanized or fully human antibodies, may be used to extend the half-life of the antibody and prevent it from being attacked by the host immune system. The frequency of administration may be determined and adjusted during the course of treatment, and is generally based on the treatment and / or inhibition and / or improvement and / or delay of gastric cancer, but is not always necessary. Alternatively, a sustained-release formulation of the antibody may be appropriate. Various formulations and devices for achieving sustained release are known in this field.

[0076] In one embodiment, the antibody dose described herein may be determined empirically in an individual that has received one or more doses of the antibody. The antibody dose is increased in stages in the individual. To evaluate the effectiveness of the antibody, indicators of disease (e.g., tumor growth) may be tracked according to conventional methods.

[0077] In general, for the administration of any of the antibodies described herein, the initial candidate dose may be estimated from the tests described below. For repeated administrations over several days or longer, depending on the condition, treatment is continued until the desired symptom inhibition occurs or until a sufficient therapeutic level is reached to alleviate the cancer. An exemplary dosing regimen involves administering a high dose initially, followed by a lower maintenance dose. However, other dosing regimens may be useful depending on the pattern of pharmacokinetic decay the physician seeks to achieve. For example, administration once to four times per week is possible. In some embodiments, the dosing frequency is once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks, or once a month, once every two months, or once every three months, or longer intervals. The progress of this therapy can be readily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) may change over time.

[0078] Depending on the treatment goal and the site of the cancer, conventional methods known to those skilled in the medical field may be used to administer the pharmaceutical composition to the target.

[0079] The injectable composition may contain various carriers such as vegetable oil, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.).

[0080] In the case of intravenous injection, water-soluble antibodies may be administered by drip infusion, in which case a pharmaceutical preparation containing the antibody and physiologically acceptable excipients is injected. Examples of physiologically acceptable excipients include 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients.

[0081] Intramuscular preparations, such as sterile preparations in the appropriate soluble salt form of an antibody, may be administered dissolved in pharmaceutical excipients such as sterile water for injection, 0.9% saline solution, or 5% glucose solution.

[0082] In one embodiment, the antibody is administered by site-specific or targeted local delivery technology. Examples of site-specific or targeted local delivery technologies include various implantable depot sources or local delivery catheters for antibodies (such as infusion catheters, indwelling catheters, or needle catheters), synthetic grafts, outer membrane wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct use. See, for example, International Patent No. WO00 / 53211 and U.S. Patent No. 5,981,568.

[0083] In another embodiment of the present disclosure, a product is provided that contains any of the pharmaceutical compositions and formulations described herein (e.g., including antibodies or binders) and provides instructions for use and / or reconstitution thereof. The product includes a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., dual-chamber syringes), and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container holds the formulation, and a label on or accompanying the container may indicate instructions for reconstitution and / or use. For example, the label may indicate that the formulation is reconstituted to a specific protein concentration. The container holding the formulation may be a multi-use vial that allows for repeated administration (e.g., 2 to 6 doses) of the reconstituted formulation. The product may further include a second container containing a suitable diluent (e.g., BWFI). When the diluent and the lyophilized formulation are mixed, the final protein concentration of the reconstituted formulation is generally at least 50 mg / mL. The manufactured product may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, and accompanying documentation including instructions for use.

[0084] (Examples) The following examples are not limiting and are merely illustrative. Unless otherwise noted, all cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia), and all secondary antibodies for detection were purchased from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania). Standard procedures described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, were used for DNA and RNA manipulation. Unless otherwise noted, all commercially available reagents and kits were used according to Mecker's instructions.

[0085] To explore target-specific antibodies for cancer treatment, we used live-cell immunization (LCI) and live-cell high-throughput screening (HTS) techniques to generate antibodies that specifically target human tumor surface antigens, and then combined proteomics and molecular biological methods to identify the targets.

[0086] Example 1: Generation and characterization of MAb11-22.1 mAbs specifically targeting AML cell lines. To obtain mAbs against conformational epitopes on cell surface antigens, a mixture of two human AML cell lines, MV4-11 and THP-1, in phosphate-buffered saline (PBS) was used in LCI and LC-HTS, as previously described (see International Patent No. WO2014146487A1, International Patent No. WO2017114204, U.S. Patent Publication No. 20210139602A1; Li et al., PLoS One. 2013, 8:e77398). Using a BD FACSCalibur® flow cytometer and high-throughput sampler (HTS), fluorescence-activated cell sorting (FACS) assays were used to screen hybridoma culture supernatants for interaction with a mixture of these two AML live cell lines. Counterscreening was performed with human peripheral blood mononuclear cells (PBMCs) provided by healthy adults (Stanford Blood Center, Palo Alto, California). Data were analyzed using FlowJo® software (Becton Dickinson, San Jose, California). Hybridoma colonies showing potent and specific binding activity to human AML cell lines but no binding activity to human PBMCs were selected for amplification, detachment from conditional medium, and subcloning following standard procedures (Kohler & Milstein, Nature 1975, 256:495-497; Winter & Milstein, Nature 1991, 349:293-299). For further characterization, monoclonal antibodies (mAbs) produced by these colonies were purified using MabSelect® SuRe® LX Protein A resin (GE Healthcare, Marlborough, Massachusetts).

[0087] The mAb, named MAb11-22.1 mAb, showed high binding affinity to almost all nine human AML cell lines tested, but not to human PBMCs (Figure 1A, left panel). Binding signals were particularly high for NB4 cells, an AML M3 subtype cell line, followed by Kasumi-1 and HL-60 cell lines. To ensure that binding was not mediated by interaction between the mAb and Fc receptors (FcRs) on the surface of AML cells, human TruStain FcX® Fc receptor blocking solution (Biolegend, San Diego, California) was used in the FACS assay. Since the isoform of MAb11-22.1 mAb is IgG1 / κ, determined by the IsoStrip® mouse monoclonal antibody isotyping kit (Roche Molecular Systems, Pleasanton, California), an unrelated IgG1a / κ mouse antibody that does not interact with AML cell lines was used as a negative control (isotype control antibody, Figure 1A, right panel). This confirmed that the interaction between MAb11-22.1 mAb and the AML cell surface is specifically mediated by the variable region of MAb11-22.1 mAb.

[0088] To visualize the binding of MAb11-22.1 mAb to the surface of AML cells, an immunocytochemistry (ICC)-based cell surface antibody binding assay was performed. Cultured AML cells were fixed to glass slides in 4% paraformaldehyde using CytoSpin 4 (Thermo Fisher Scientific, Waltham, Massachusetts). After antigen retrieval by heating, cells were blocked with goat serum (MilliporeSigma, St. Louis, Missouri) and then incubated with MAb11-22.1 mAb or isotype control antibody for 1 hour, followed by incubation with 1:1000 dilution of peroxidase-labeled AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3) and Fcγ fragment-specific antibody (anti-Mo IgG Fc-HRP pAb, Jackson ImmunoResearch Laboratories) for 1 hour. Cells were stained with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. As shown in Figure 1B, ICC assay confirmed that the MAb11-22.1 mAb targets are located on the surface of NB4 and THP-1 cells. The intensity of DAB staining on the surface of NB4 cells was much higher than that on the surface of THP-1 cells and correlated with the mean fluorescence intensity (MFI) in the FACS assay (Figure 1A, left panel).

[0089] Furthermore, in Western blotting of multiple AML cell lysates simultaneously probed with MAb11-22.1 mAb and mouse anti-human / mouse β-actin mAb (anti-β-actin mAb, Proteintech Group (Rosemont, Illinois)) at 5 μg / mL each, followed by detection with a 1:20,000 dilution of anti-Mo IgG Fc-HRP pAb, a common band of approximately 250 kDa was shown under non-reducing conditions, and a faint band of approximately 110 kDa was shown under reducing conditions (Figure 1C). In whole cell lysates of the acute lymphoblastic leukemia (ALL) cell line Raji, a blot was also shown at the same location, although at a lower intensity. In contrast, MAb11-22.1 mAb did not produce any band in Western blotting of Chinese hamster ovary (CHO) cells (Figure 1C) or human PBMC cell lysates (data not shown). The relative intensities of these hybridized protein bands in AML cells were consistent with the intensities of MFI (Figure 1A) and ICC staining (Figure 1B) in the FACS assay, suggesting that this protein is likely a target of MAb11-22.1 mAb.

[0090] Example 2: Identification and validation of human transferrin receptor 1 (TfR1) as a target for MAb11-22.1 mAbs AML cell lysates were prepared in IP lysis buffer (Thermo Fisher Scientific) according to the manufacturer's instructions. Immunoprecipitation Dynabeads® Protein A (Thermo Fisher Scientific) were incubated at room temperature (RT) for 30 minutes with 50 μg of MAb 11-22.1 mAb or isotype control antibody in PBS. The antibody-bound Dynabeads® were washed and incubated with each cell lysate at RT for 30 minutes, then washed in the same order with 0.5% Triton X-100 / PBS and PBS buffer. Immunoprecipitation (IP) proteins and antibodies were separated from the beads in 4× Laemmli denatured sample buffer containing 2-mercaptoethanol and heated at 95°C for 5 minutes. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with ProteoSilver® silver staining kit (MilliporeSigma) according to the manufacturer's instructions. A single protein band with the same molecular weight as the Western blot was identified in the IP sample by silver staining (Figure 2A). The band was excised from the gel and digested with trypsin. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) performed by BGI Americas Corporation (Cambridge, Massachusetts) identified TfR1 as the most abundant protein in the gel slice (#PSM=32, coverage=29.6%).

[0091] To confirm that TfR1 is a target of MAb11-22.1 mAb, recombinant human TfR1 extracellular domain with an N-terminal His tag (rTfR1-ECD, ACRO Biosystems, Newark, Delaware) was analyzed by SDS-PAGE under non-reducing conditions along with whole cell lysate samples of AML and ALL cells (positive control) and CHO cell lysate (negative control). Subsequently, Western blot probing was performed using MAb11-22.1 mAb and anti-β-actin mAb at 5 μg / mL each, along with 1:20,000 anti-Mo IgG Fc-HRP pAb as primary antibodies (Figure 2B). The results showed that MAb11-22.1 mAb could detect a single band of approximately 80 kDa, which correlates with the calculated molecular weight of rTfR1-ECD (77.0 kDa). As shown in Figure 1C, the Raji, NB4, OCI / AML2, and CHO cell lysates showed consistent results in their reaction with MAb11-22.1 mAb.

[0092] To confirm the interaction between MAb11-22.1 mAb and rTfR1-ECD, a direct ELISA assay was performed. Immulon® microtiter plates (Thermo Fisher) coated with 0.1 μg / mL of rTfR1-ECD were directly reacted with MAb11-22.1 mAb, serial dilutions of 6×His His-Tag monoclonal antibody (anti-His mAb, Proteintech) or isotype control antibody at RT for 1 hour, followed by reaction with 1:2,000 anti-Mo IgG Fc-HRP pAb for 1 hour. A 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added, and absorbance at a wavelength of 450 nm was measured using a SpectraMax microplate reader (Molecular Device, San Jose, California). Figure 2C shows the direct interaction between 6His-tagged rTfR1-ECD and MAb11-22.1 mAb or anti-His mAb, but no interaction with isotype control antibodies, thus demonstrating that TfR1 is a target of MAb11-22.1 mAb.

[0093] To further confirm that MAb11-22.1 mAb recognizes human TfR1, TFRC cDNA encoding the full-length TfR1 protein was amplified from OCI / AML2 and THP-1 cells by reverse transcription polymerase chain reaction (RT-PCR) using primer pairs (SEQ ID NOs: 11 and 12) ordered from Integrated DNA Technologies (Coralville, Iowa). The cDNA was in-frame fused with a GFP-encoding DNA fragment at its C-terminus and then cloned into an in-house expression vector containing the human CMV promoter and the puromycin resistance gene (PuroR) for selection. The TFRC-GFP expression plasmid was transfused into CHO-DG44 cells that were not stained by MAb11-22.1 mAb by FACS and grown in EX-CELL® CD CHO serum-free medium (SFM) supplemented with puromycin. CHO cell colonies stably expressing high levels of EGFP were isolated and subcloned. At 4°C, CHO / rTfR1-GFP cells were stained for 30 minutes with two batches of purified MAb11-22.1 mAb, anti-human TfR1 control mAb (R&D Systems, Minneapolis, Minnesota), and isotype control antibody, followed by detection with R-phycoerythrin (PE)-labeled AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3) and Fcγ fragment-specific pAb (anti-Mo IgG Fc-PE, Jackson ImmunoResearch). The results showed that, in FL1(GFP) gating, CHO / rTfR1-GFP cells were stained by both MAb11-22.1 mAb and anti-TfR1 control mAb, but both antibodies were able to bind to CHO cells lacking rTfR-GFP expression on the cell surface (Figure 2D). IC of MAb11-22.1 mAb 50 The values ​​were 0.03-0.04 μg / mL, and the IC50 was approximately 0.1 μg / mL for the anti-TfR1 control mAb. 50The values ​​were much lower than expected. Staining of CHO / rTfR-GFP cells with various GFP activities using MAb11-22.1 mAb showed a positive correlation between MFI-PE values ​​and MFI-FITC values ​​(data not shown), demonstrating a direct interaction between MAb11-22.1 mAb and the extracellular domain of human TfR1. Forward primer: 5'-GAATGATGGATCAAGCTAGATCAGC-3' (SEQ ID NO: 11) Reverse primer: 5'-CTCATGGAAGCTATGGGTATCAC-3' (SEQ ID NO: 12)

[0094] Example 3: Measurement of binding affinity between MAb11-22.1 mAb and rTfR1-ECD The binding affinity of MAb11-22.1 mAb to the extracellular domain of TfR1 was determined using the Octet® QK system (Molecular Devices, San Jose, California) with label-free biolayer interferometry (BLI) technology. MAb11-22.1 mAb was immobilized on an anti-mouse IgG Fc capture (AMC) biosensor for 450 seconds, washed with kinetic buffer for 120 seconds, then bound to rTfR1-ECD in serial dilutions for 450 seconds, and subsequently dissociated in kinetic buffer. Goat anti-mouse (H+L)Fab (Jackson ImmunoResearch) was used as a positive control for binding to MAb11-22.1 mAb. When the assay was repeated with serial dilutions of rTfR1-ECD from 300 nM to 0.08 nM, no signal attenuation was observed even when the dissociation time was extended to 60 minutes (Figure 3A, left panel) or the reaction temperature was increased to 37°C (data not shown). Therefore, the interaction affinity between MAb11-22.1 mAb and rTfR1-ECD is very high, and the calculated K D The values ​​were less than 1 pM (Figure 3B). The binding of MAb11-22.1 mAb to anti-mouse (H+L)Fab showed a regular dynamic curve (Figure 3A, right panel) and fairly high affinity (K DThe result (=1.04~1.45 nM) (Figure 3C) indicates that the extremely high affinity between MAb11-22.1 mAb and rTfR1-ECD was not an artificial result.

[0095] Example 4: Competitive ELISA of MAb11-22.1 mAb and transferrin for binding to human TfR1 Competitive ELISA was performed to determine whether MAb11-22.1 mAb binds to human TfR1, which overlaps with the Tf binding site. MAb11-22.1 mAb was directly labeled with HRP using the EZ-Link maleimide-activated HRP kit (Thermo Fisher Scientific) as instructed by the manufacturer. Immunlon microtiter plates were coated with 0.1 μg / mL rTfR1-ECD and incubated for 1 hour with HRP-bound MAb11-22.1 mAb, which was separately mixed with human Tf (MilliporeSigma), which was serially diluted 2.5-fold from 2 μg / mL, and HRP-bound MAb11-22.1 mAb, which was serially diluted 4-fold from 2 μg / mL. Various concentrations of Tf were added individually as negative controls. After thorough washing, TMB was added to detect binding of HRP-bound MAb11-22.1 mAb. After adding stop solution, absorbance at 450 nm was measured using a SpectraMax microplate reader.

[0096] Figure 4 shows that MAb11-22.1 mAb-HRP binds to rTfR1-ECD in a dose-dependent manner, regardless of the presence of Tf at various concentrations. Even with the addition of Tf up to 2 μg / mL (25 μM), the binding of MAb11-22.1 mAb-HRP to rTfR1-ECD was not significantly inhibited, suggesting that the binding site of Tf does not overlap with the binding epitope of MAb11-22.1 mAb.

[0097] Example 5: FACS analysis of the binding of MAb11-22.1 mAb to PBMCs and bone marrow (BM) cells. Other researchers have reported that most anti-TfR1 antibodies interfere with Tf uptake, causing systemic hematological toxicity in patients, including anemia, neutropenia, and leukopenia (CANDELARIA et al. Front.Immunol. 17 March 2021). To address safety concerns regarding MAb11-22.1 mAbs as a treatment candidate, multiple batches of fresh PBMCs from healthy donors were purchased from Stanford Blood Center (Palo Alto, California), and frozen BM cells from healthy donors or AML patients were purchased from HumanCells Bioscience (Fremont, California). The cells were washed with PBS and blocked with ice-cold PBS and 1% bovine serum albumin (BSA). Cells were stained at 4°C with serially diluted MAb11-22.1 mAb and isotype control antibodies starting from 20 μg / mL, followed by detection with 1:800 FITC or PE-conjugated goat anti-Mo IgG Fc pAb (Jackson ImmunoResearch). In addition, anti-human CD34-FITC (REAL487), anti-human CD233-PE (REA368), and anti-human CD235a-PE (REA1092) were purchased from Miltenyi Biotec (San Jose, California), and cells were simultaneously stained with MAb11-22.1 mAb.

[0098] FACS results showed that MAb11-22.1 mAb did not bind to normal PBMCs or normal BM cells, regardless of the mAb concentration used (Figure 5A). Consistent results were obtained in PBMCs and BM cells from different batches (donors) (data not shown). In contrast, BM cells from AML patients reacted with MAb11-22.1 mAb but not with the isotype control antibody (Figure 5B), suggesting that MAb11-22.1 mAb can distinguish between healthy BM and AML BM species. In healthy BM samples, more than 20% of cells were hematopoietic stem cells stainable with anti-human CD34 mAb-FITC (Figure 5C, left panel), but only a small number of cells reacted with MAb11-22.1 mAb and the isotype control antibody (Figure 5C, middle and right panels). Although TfR1 is generally considered a marker for nucleated red blood cells, neither CD233-positive red blood cells nor CD235a-positive red blood cells or erythroblasts reacted with MAb11-22.1 mAb (Figures 5D and 5E, left panel). This suggests that MAb11-22.1 mAb recognizes only TfR1 expressed in malignant cells and has a low risk of causing bone marrow suppression.

[0099] Example 6: Endomorphism assay of MAb11-22.1 mAb by tumor cells OCI / AML2, NB4, or Raji cells in DMEM medium supplemented with 10% Gibco fetal bovine serum (FBS, Thermo Fisher Scientific) were cultured in two 96-well plates at a rate of 1 × 10⁶ 5Cells were seeded in 100 / wells and incubated at 4°C for 1 hour with MAb11-22.1 mAb, anti-CD20 mAb (R&D Systems), or isotype control mAb, diluted 1:10 times from 10 μg / mL. Cells were centrifuged at 500 × g and washed twice with ice-cold PBS to remove unbound mAbs. Cells from one 96-well plate were resuspended in warm medium and incubated at 37°C to allow mAb internalization, and cells from another plate were resuspended in ice-cold medium and incubated at 4°C to prevent antibody internalization. Samples collected at 30 minutes, 1 hour, and 2 hours were washed twice with ice-cold PBS / 1% BSA, and then incubated at 4°C for 30 minutes with anti-Mo IgG Fc-FITC diluted 1:800. After washing three times with ice-cold PBS / 1% BSA, the cells were resuspended in 150 μL of fixative containing 1% paraformaldehyde and stored in the dark in preparation for FACS analysis.

[0100] FACS results showed that MAb11-22.1 mAb bound to the surface of two AML cell lines, OCI-AML2 and NB4, in a dose-dependent manner at 4°C, but was rapidly internalized at 37°C. Raji cells, despite appearing to have a very low TfR1 copy number on their cell surface, were still able to internalize MAb11-22.1 mAb at 37°C (Figure 6A, left panel). In contrast, anti-CD20 mAb bound only to Raji cells and not to AML cells at 4°C, but was partially internalized by Raji cells at 37°C (Figure 6A, middle panel). As expected, the isotype control antibody did not stain any of the three cell lines (Figure 6A, right panel).

[0101] FACS-based internalization assays provided only indirect evidence of antibody-antigen complex endocytosis and could not distinguish between antibody internalization and antibody shedding. To directly observe mAb internalization, fluorescence microscopy-based internalization analysis was performed using fluorescently labeled mAbs. Briefly, mAbs were labeled with CF488 dye using the Mix-n-Stain® antibody labeling kit (MilliporeSigma) according to the manufacturer's instructions. Cell binding was performed in 96-well culture plates with different concentrations of mAbs in ice-cold DMEM medium at 4°C for 1 hour. After washing the cells twice with ice-cold PBS, one plate was incubated with warm DMEM at 37°C for 1 hour, and the cells from the other plate were resuspended in cold PBS for fluorescence imaging using a Keyence BZ-X800 all-in-one fluorescence microscope (Keyence, Itasca, Illinois). Isotype control antibodies were labeled with CF488 and incubated in parallel with the cells as a negative control.

[0102] After incubation at 4°C for 1 hour, MAb11-22.1 mAb-CF488 bound to the surface of OCI / AML2 cells, forming a high-intensity fluorescent ring that outlined the cells (Figure 6B, top panel). The fluorescence intensity was proportional to the concentration of the mAb used. After incubation at 37°C for 1 hour, the majority of the fluorescent signal moved from the cell surface to the internal space of the cells, suggesting that the surface-bound MAb11-22.1 mAb-CF488 molecule was primarily internalized by OCI / AML2 cells (Figure 6B, bottom panel). This phenomenon was consistently observed in other AML cell lines, including NB4 and THP-1, at different concentrations of MAb11-22.1 mAb-CF488 (1, 0.5, 0.1, and 0.05 μg / mL) (data not shown). The CF488-labeled isotype control antibody did not bind to any of the test cells (Figure 6B, right panel).

[0103] Similar dose-dependent binding and internalization results were observed in the triple-negative breast cancer (TNBC) cell line MDA-MB-231, which reacted strongly with MAb11-22.1 mAb at 4°C, as revealed by FACS analysis. However, the fluorescence signal of MDA-MB-231 cells was, as expected, much stronger than the fluorescence signal on AML cells (Figure 6C). Furthermore, partial internalization occurred even at 4°C, and the majority of the MAb11-22.1 mAb-CF488 molecules were internalized after incubation at 37°C for 1 hour, suggesting that the TfR1 protein expressed on the MDA-MB-231 cell membrane is highly likely to be internalized upon binding to MAb11-22.1 mAb.

[0104] Example 7: In vitro effects of MAb11-22.1 mAb on the proliferation of AML cell lines The effect of MAb11-22.1 mAb on the proliferation of AML cell lines was evaluated using cell-based in vitro assays. Cell viability was determined using the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Rockville, Maryland) according to the manufacturer's instructions. Briefly, AML cells grown in DMEM / 10% FBS and 100 μg / mL of MAb11-22.1 mAb or isotype control antibody were placed in 5 × 10⁶ wells of seven 96-well cell culture plates. 3 Cells were seeded in three different ways at 1 cell / well and incubated at 37°C under a humidified atmosphere of 5% CO2. Every 24 hours, the cells were incubated with 10 μL / well of CCK-8 solution for 4 hours, followed by OD (Oral Discharge). 450 Cell viability was evaluated by measuring [a specific factor].

[0105] As shown in Figure 7, both groups of OCI / AML2 cells continued to grow, but the proliferation rate of MAb11-22.1 mAb-treated cells was much lower than that of cells treated with isotype control antibodies (P<0.05). Similar data were obtained for other AML cell lines (data not shown). Therefore, while MAb11-22.1 mAb partially inhibited the proliferation of OCI / AML2 cells, the mAb itself was not a potent therapeutic candidate. In fact, MAb11-22.1 mAb showed no effect in preliminary in vivo studies using the CLDX mouse model. MAb11-22.1 mAb showed extremely high affinity for human TfR1 and a strong tendency to be internalized by cancer cells, indicating great potential for development as an ADC for cancer therapy.

[0106] Example 8: Sequencing of the variable region cDNA of MAb11-22.1 hybridoma Using the SMARTer RACE 5' / 3' kit (Takara Bio USA, San Jose, California), 5'-rapid amplification (5'-RACE) technology was used to amplify cDNA encoding the variable regions of the light chain (VL) and heavy chain (VH) of MAb11-22.1 hybridoma cells from total RNA. 5'-RACE primers are shown as SEQ ID NOs. 13 and 14. The cDNA fragments were amplified with Q5® High-Fidelity DNA polymerase and cloned into the pMiniT2.0 vector (New England Biolabs, Ipswich, Massachusetts) according to the manufacturer's instructions. Ten E. coli colonies were randomly selected from each transformant for plasmid purification using the Rapid Miniplasmid Kit (IBI Scientific, Dubuque, Iowa) and Sanger DNA sequencing (Genewiz, South San Francisco, California). All sequences were verified for V-domain uniqueness using the BLAST search at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi and the international ImMunoGeneTics (IMGT) information (registered trademark) (http: / / www.imgt.org). The consensus sequences for the VL and VH of MAb11-22.1 mAb are shown as SEQ ID NO: 1 and SEQ ID NO: 3, respectively, in the disclosure section of the invention. The encoded amino acid sequences are shown as SEQ ID NO: 2 and SEQ ID NO: 4, respectively. Light chain primer: 5'-CTGCTCACTGGATGGTGGGAAGATGG-3' (SEQ ID NO: 13) Heavy chain primer: 5'-AGCTGGGAAGGTGTGCACAC-3' (SEQ ID NO: 14)

[0107] Construction and expression of chimeric MAb11-22.1 Ab(cAb): The VL and VH genes of MAb11-22.1 mAb were PCR amplified from sequenced plasmids using primer pairs shown in SEQ ID NOs. 15-18. The PCR fragments were digested with SpeI-NarI(VL) or XbaI-NheI(VH) restriction enzymes (New England Biolabs) and in-frame ligated to the constant regions of the human κ light chain (CL) or human IgG1 heavy chain (CH1-CH3), respectively, which were transported to in-house expression vectors containing mouse CMV promoters and human CMV IE1 promoters, controlling light chain and heavy chain expression, respectively. LV PCR forward primer: 5'-AGCACTAGTGCCGCCACCATGGAATCACAGACTCAGG-3' (SEQ ID NO: 15) LV PCR reverse primer: 5'-CTGGGCGCCGCTACAGTCCGTTTCAGCTCCAGCTTGG-3' (SEQ ID NO: 16) HV PCR forward primer: 5'-AGCTCTAGAGCCGCCACCATGGAGACAGACACACTCCTG-3' (SEQ ID NO: 17) HC PCR reverse primer: 5'-GCCTTTGGTGCTAGCAGAGACAGTGACCAGAGTC-3' (SEQ ID NO: 18)

[0108] After confirmation by DNA sequencing, the expression plasmid was transfected into Dux-1S-HD cells, an in-house developed CHO-DG44 derivative capable of high-density growth in EX-CELL®CD CHO SFM, using the FectoPro transfection reagent (Polyplus-SA (Il Kirsch, France)) according to the manufacturer's instructions. Transient expression and secretion of MAb11-22.1 cAb in EX-CELL®CD CHO SFM were verified by FACS analysis for binding to AML cell lines, followed by affinity purification using MabSelect®SuRe® Protein A resin (GE Healthcare).

[0109] Example 9: Characterization of MAb11-22.1 cAb The purified cAb exhibited similar characteristics to its mAb counterpart in analyses such as SDS-PAGE, ELISA, and FACS (data not shown). The binding kinetics between MAb11-22.1 cAb and rTfR1-ECD were also determined by the Octet® QK system. MAb11-22.1 cAb was immobilized for 450 seconds using an anti-human IgG Fc capture (AHC) biosensor, and goat anti-human (H+L)Fab (Jackson ImmunoResearch) was used as a system control for binding to MAb11-22.1 cAb. By 2.5-fold serial dilutions, the concentration of rTfR1-ECD ranged from 50 nM to 1.28 nM. For the MAb11-22.1 mAbs shown in Figures 3A to 3C, the dissociation (K) of MAb11-22.1 cAb was determined. off Since it was not detected within 60 minutes after observation, the affinity between MAb11-22.1 cAb and rTfR1-ECD was extremely high (K D <1 pM, Figure 8A). The binding of MAb11-22.1 cAb to anti-human (H+L)Fab showed a regular dynamic curve, as expected, K D The value was 2.18 nM (Figure 8B).

[0110] Example 10: In vitro effects of MAb11-22.1-S239C-DM1 on cancer cell lines Although MAb11-22.1 mAb did not exhibit potent cytotoxic activity, its extremely high affinity for human TfR1 and high tumor specificity made it an excellent ADC candidate for cancer treatment. To investigate its potential as an ADC, cysteine ​​scanning was performed on surface-exposed residues in the light and heavy chain constant regions of MAb11-22.1 cAb to investigate its antitumor effects through site-specific binding of various payloads. Site-directed mutagenesis was performed by replacing the serine 239 codon (TCC) in the heavy chain Fc region of MAb11-22.1 cAb with a cysteine ​​codon (TGC) using the primer pair shown in SEQ ID NOs: 19 and 20. Transient expression of MAb11-22.1-S293C cAb mutants was purified using MabSelect®SuRe® Protein A resin. As described in JUNUTULA et al. (Nat Biotechnol. 26:925-932, 2008), cysteine ​​or glutathione conjugated to Cys239 of MAb11-22.1-S293C cAb was removed from the culture medium by treatment with a 10-fold molar excess of dithiothreitol (DTT) in PBS at room temperature. The antibody was refolded to expose the free thiol group of S239C, and the integrity of the cAb was analyzed by SDS-PAGE. Site-directed binding was performed by reacting DM1 with the free thiol group of Cys239 in PBS by slow stirring at room temperature for 10–60 minutes to generate MAb11-22.1-S293C-DM1, which conferred ADC activity with a theoretical drug-antibody ratio (DAR) of 2. Free DM1 was removed by dialysfiltration three times into 10x volume PBS using an Amicon® agitated cell reservoir equipped with a 30-kDa NMW ultrafiltration disc (MilliporeSigma). MAb11-22.1-S293C-DM1 was then filtered and sterilized using a 0.2 μm syringe filter (VWR International, Radnoll, Pennsylvania). To eliminate the effects of non-site-specific binding, unmutated MAb11-22.1 cAb and isotype control antibodies were also treated with DM1 in parallel as negative controls.The integrity and binding ability of DM1-treated antibodies were analyzed using SDS-PAGE, ELISA, and FACS. Endotoxin levels were analyzed using the Pierce® chromogenic endotoxin quantification kit (Thermo Fisher). No significant differences were observed between MAb11-22.1 cAb-DM1 and MAb11-22.1-S239C-DM1 in terms of binding affinity to TfR1 and cancer cell lines. As expected, MAb11-22.1-S239C-DM1 showed a slight delay in gel migration compared to untreated MAb11-22.1-S239C. S239C Forward Primer: 5'-AACTCCTGGGTGGACCT TGC GTGTTTCTGTTCCCCCCTAAGC-3'(Sequence ID 19) S239C Reverse Primer: 5'-AACAC GCA AGGTCCACCCAGGAGTTCAGGAGCAGGGCAAGG-3'(Sequence ID 20)

[0111] Representative cancer cell lines such as OCI / AML2 (AML subtype M4), Raji (ALL), HCC38 (TNBC), and MDA-MB-231 (TNBC) were cultured in DMEM / 10% FBS medium until the logarithmic growth phase. On day 0, baseline OD was determined by the CCK-8 assay. 450 Optimal amounts of each cell line were mixed with individual antibodies in three different 96-well cell culture plates to achieve a ratio of approximately 0.4. Cells were incubated at 37°C in a 5% CO2 atmosphere. Cell proliferation was monitored on days 3 and 5, respectively, using the CCK-8 assay. The isotype control antibody without DM1 treatment reflected the baseline of cell proliferation. In this study, DM1-treated and untreated MAb11-22.1-S239C and MAb11-22.1 cAb were compared.

[0112] As shown in Figure 9, cell proliferation in all four cell lines was inhibited by MAb11-22.1-S239C-DM1, but not by untreated MAb11-22.1-S239C cAb. Wild-type MAb11-22.1 cAb had no apparent effect on cell proliferation, regardless of DM1 treatment. AML and ALL cell lines were highly sensitive to MAb11-22.1-S239C-DM1, as both 100 μg / mL and 300 μg / mL completely inhibited cell proliferation. The two TNBC cell lines were less sensitive to MAb11-22.1-S239C-DM1, as the 100 μg / mL concentration only partially inhibited them.

[0113] Example 11: In vivo evaluation of MAb11-22.1-S239C-DM1 using an AML xenograft mouse model To evaluate the effect of MAb11-22.1-S239C-DM1 on AML tumor growth, a xenograft model was established using nu / nu mice. 1.5 × 10⁴ oz. dissolved in 0.1 mL of PBS was placed in the subcutaneous space of the right flank of 6-week-old male athymoid nude mice (Charles River Laboratories, Wilmington, Massachusetts). 6 OCI / AML2 cells were injected. Once all tumor nodules were visible, the mice were randomly divided into three groups (n=5 or 6) and treated with isotype control antibodies (without S239C mutation) treated with MAb11-22.1-S239C-DM1 (10 mg / kg b.wt for low dose, 20 mg / kg body weight (b.wt) for high dose) or DM1 (20 mg / kg b.wt) once every four days for seven doses (Q4 × 7) via intraperitoneal (ip) injection. Immediately before drug administration, tumor volume and body weight were measured every four days. Volume = 1 / 2 × (width) 2 The tumor volume was calculated using the formula × length. The tumor volume was 2000 mm³. 3 If the condition exceeded a certain limit, or if the tumor became necrotic or developed into an ulcer through the skin, the animal was euthanized.

[0114] OCI / AML2 xenografting is an invasive CLDX mouse model. Neither MAb11-22.1 mAb nor MAb11-22.1 cAb alone significantly slowed the growth of OCI / AML2 xenografted tumors (P>0.05, data not shown). In the current study, 10 mg / kg b.wt (low dose) of MAb11-22.1-S239C-DM1 resulted in complete regression and a persistent response after one or two doses (Figure 10). Mice treated with 20 mg / kg b.wt (high dose) of MAb11-22.1-S239C-DM1 (n=6) showed a different response. Three mice showed a 100% reduction in tumor volume after day 4 or 8, similar to the low-dose group. Another mouse showed tumor regression on days 4 and 8, but the tumor subsequently grew again. The remaining two mice did not respond to MAb11-22.1-S239C-DM1, and one of them was euthanized on day 24 due to tumor progression. In the isotype control group (n=5), all tumors continued to grow, and two mice had tumors reaching 2000 mm². 3 Euthanasia was performed on day 20 because the dose exceeded the limit. Despite the use of very high doses of ADC, no systemic side effects such as changes in vital signs, decreased exercise capacity, decreased appetite, or loss of total body weight were observed during the study.

[0115] The effect of MAb11-22.1-S239C-DM1 on tumor growth was clearer compared to the isotype control group, confirming that MAb11-22.1 cAb is a good candidate as an ADC for cancer treatment. Due to its high affinity and specificity for human TfR1, circulating MAb11-22.1-S239C accumulates in TfR1-positive tumor cells and delivers DM1 into the cells via endocytosis. When high concentrations of glutathione (GSH) are present in tumor cells, DM1 is released from the ADC, potentially exerting a bystander-killing effect on surrounding cells within xenografted tumors. This suggests that MAb11-22.1 ADCs can be applied not only to hematological malignancies but also to solid tumors.

[0116] The mechanism behind the variability in efficacy in the high-dose group is unclear. This is thought to be due to the small sample size and individual differences in response to the compound because of varying TfR1 expression levels on the tumor cell surface. High-dose ADCs may be quickly rejected or cause drug resistance, so the dose range of MAb11-22.1-S239C-DM1 needs to be optimized. Nevertheless, MAb11-22.1 is an ideal anti-TfR1 antibody for ADC development due to its high tumor specificity and available antitumor efficacy data. To achieve consistent DAR and an optimal dose range for cancer treatment, robust payload and ideal linker conjugation techniques for MAb11-22.1 ADCs are currently under investigation.

[0117] The specific methods and compositions described herein are representative examples of preferred embodiments and are illustrative; they are not intended to limit the scope of the invention. Those skilled in the art will likely conceive of other purposes, aspects, and embodiments by reviewing this specification, and these fall within the scope of the spirit of the invention as defined by the claims. It will be obvious to those skilled in the art that various substitutions and modifications may be made to the inventions disclosed herein without departing from the scope and spirit of the invention. The inventions described exemplary herein can be adequately implemented even without elements or limitations specifically disclosed herein as essential. Therefore, for example, in each example, embodiment, or example of the invention herein, the terms “include,” “contain,” “contain,” “having,” and “have” should be read as synonymous and broadly and without limitation. The methods and processes described exemplary herein may be adequately implemented in different order of steps and are not necessarily limited to the order of steps shown herein or in the claims.

[0118] In no event shall the invention be construed as being limited to any specific examples, embodiments, or methods specifically disclosed herein. In no event shall the invention be construed as being limited by any statement made by an examiner or other officer or employee of the Patent and Trademark Office, unless such a statement is explicitly adopted in whole and specifically and without limitation in the applicant's written response.

[0119] In this specification, the present invention is described broadly and generally. Each of the narrower species and subgenera classifications included in the general disclosure also constitutes part of the present invention. The terms and expressions used herein are for descriptive purposes only and not limiting purposes, and the use of such terms and expressions is not intended to exclude any equivalents of the features or parts thereof shown and described, however it may be conceivable that various modifications are possible within the scope of the claimed invention. Accordingly, although the present invention is specifically disclosed by preferred embodiments and optional features, it will be understood that those skilled in the art may adopt modifications and changes to the concepts disclosed herein, and such modifications and changes will be considered within the scope of the invention as defined by the appended claims.

[0120] (Integrated by reference) All patents and patent applications cited herein are incorporated herein by reference, as are all other references cited herein.

[0121] The following are some commonly mentioned antibody-related patents: U.S. Patent No. 7317091B2 U.S. Patent No. 5,500,362 U.S. Patent No. 4,816,567 U.S. Patent No. 5,225,539 US Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150 U.S. Patent No. 5,932,448 U.S. Patent No. 7,538,196 U.S. Patent No. 8,148,496 U.S. Patent No. 8,518,891 U.S. Patent No. 9,310,373 U.S. Patent No. 9764041 U.S. Patent No. 11,040,084 European Patent No. EP239400B1 International Patent No. WO00 / 53211 and U.S. Patent No. 5,981,568 International Patent No. WO2014146487A1 U.S. Patent Publication No. 20170151343 U.S. Patent Publication No. 20170335281 International Patent No. WO2017114204 U.S. Patent Publication No. 20210139602A1

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Claims

1. A binder conjugate comprising a light chain variable region and a heavy chain variable region, targeting TfR1, and an antitumor agent bound to the binder, The CDR1 to CDR3 in the light chain variable region each have the same sequence as sequence numbers 5, 6, and 7, respectively, The heavy chain variable regions CDR1 to CDR3 each have the same sequence as sequence number 8, sequence number 9, and sequence number 10, respectively. A binder complex.

2. The binder complex according to claim 1, having at least 70% of the same light chain variable region as SEQ ID NO:

2.

3. The binder complex according to claim 1, having at least 70% of the heavy chain variable region identical to SEQ ID NO:

4.

4. The binder conjugate according to claim 1, wherein the binder is a monoclonal antibody.

5. The conjugate conjugate according to claim 4, wherein the monoclonal antibody is a mouse, human, humanized, chimeric, bispecific, or multispecific antibody.

6. The binder complex according to claim 4, wherein the binder is a diabody, a single-domain antigen-binding (SDAB) molecule, a VH or VL domain, or a VHH domain.

7. The binder complex according to claim 4, wherein the monoclonal antibody has an IgG1 heavy chain and a κ light chain.

8. The aforementioned binder is Fab, Fab', F(ab') ２ The binder complex according to claim 1, wherein the binder is a fragment of rIgG, Fv, or Fd.

9. The binder is scFv or sc(Fv) ２ The binder complex according to claim 1.

10. The binder complex according to claim 4, wherein the monoclonal antibody is class IgD, class IgE, class IgG, class IgA, or class IgM, or one subclass of the said class.

11. The binder conjugate according to claim 10, wherein the subclass of the monoclonal antibody is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

12. The binding agent complex according to claim 10, wherein the monoclonal antibody has a κ light chain.

13. A binder comprising a light chain variable region and a heavy chain variable region, which targets TfR1, The CDR1 to CDR3 in the light chain variable region each have the same sequence as sequence number 5, sequence number 6, and sequence number 7, and A binder targeting TfR1, wherein CDR1 to CDR3 in the heavy chain variable region have the same sequence as SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

14. The light chain variable region has the same arrangement as sequence number 2, Having the heavy chain variable region having the same sequence as sequence number 4, The binder according to claim 13.

15. A separated DNA or RNA molecule encoding the binder described in claim 13.

16. The isolated DNA or RNA molecule according to claim 15, having the nucleotide sequence of SEQ ID NO:

1.

17. The isolated DNA or RNA molecule according to claim 15, wherein the DNA molecule has the nucleotide sequence of Sequence ID No.

3.

18. A vector comprising the isolated DNA or RNA molecule described in claim 15.

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