Anti-tfr1 antibody mab11-22.1 conjugates for cancer treatment

Abstract

This invention provides a therapeutic agent for treating, preventing, and diagnosing 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 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 chain and heavy chain variable regions of an anti-TfR1 monoclonal antibody (mAb) MAb11-22.1. This anti-TfR1 monoclonal antibody exhibits high specificity for tumor cells and exists in ADC form, functionally inhibiting the proliferation of several human cancer cell lines and the growth of AML cell line-derived xenograft tumors in mouse models.

Description

Technical Field

[0001] This invention relates to the use of antibodies to bind to and regulate the endocytosis and activity of transferrin receptor 1 (TfR1) and its isotypes in human cancer cells. The invention also relates to antibodies or their conjugates with small molecules (antibody-drug conjugates, ADCs) for the in vitro, in situ, and / or in vivo diagnosis and / or treatment of mammalian cell or pathological conditions associated with TfR1 and its isotypes. Prior Technology

[0002] Transferrin receptor 1 (TfR1, CD71) is a type II transmembrane glycoprotein composed of 760 amino acids linked by two disulfide bonds to form a 180-kDa homodimer. TfR1 plays a crucial role in the regulation of iron uptake and cell growth (BOMFORD and MUNRO. Hepatology 5:870-875, 1985). When diferritin (Tf) binds to TfR1 on the cell surface, the whole Tf-TfR1 complex is endocytosed through clathrin-coated pits and delivered to acidic endosomes. In these acidic endosomes, the iron-Tf-TfR1 complex undergoes a low-pH-triggered conformational change, and iron is subsequently released and transported into the cytoplasm. Then, the deferrofer Tf / TfR1 complex (apo-Tf / TfR1) is looped back to the cell surface, where the deferrofer Tf (apo-Tf) dissociates from its receptor (WARD. Invest Radiol. 22:74-83, 1987; DANIELS et al. Clin Immunol. 121:144-158, 2006). TfR1 expression is increased in rapidly proliferating cells, such as precursors of hemocytocytes, hepatocytes, and keratinocytes, where iron is highly required for heme synthesis. TfR1 expression is reduced or absent in non-dividing cells. TfR1 is overexpressed in primary and metastatic cancer cells derived from lymphocytes, pancreas, stomach, colon, lung, breast, bladder, and skin (GATTER et al., *Journal of Clinical Pathology* 36:539-545, 1983; FAULK et al., *Lancet* 2:390-392, 1980; SUTHERLAND et al., *Proceedings of the National Academy of Sciences of the United States of America* 78:4515-4519, 1981; DANIELS et al., *Clinical Immunology* 121:144-158, 2006; JEONG et al., *Biochem Biophys Res Commun.* 471:373-379, 2016; PEER et al., *Nature 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 appear to be more sensitive to iron deprivation, targeting Tf or TfR1 by blocking their binding or interfering with the endocytosis of the whole Tf / TfR1 complex can induce iron deprivation and thus kill malignant cells.

[0003] The literature over the past thirty years describes numerous attempts to treat malignant diseases by developing anti-human TfR1 antibodies or TfR1-binding peptides to compete with Tf in receptor binding or endocytosis inhibition (for the review, see TORTORELLA and KARAGIANNIS. J Membr Biol. 247:291-307, 2014; CANDELARIA et al. Frontiers in Immunology, March 17, 2021. doi.org / 10.3389 / fimmu.2021.607692). TROWBRIDGE and LOPEZ (Proceedings of the National Academy of Sciences, 79, 1175-1179, 1982; U.S. Patent No. 4,434,156) reported a mouse anti-TfR1 antibody, named 42 / 6, which blocks the binding of Tf to TfR1 through a non-competitive mechanism and is able to inhibit the growth of human T-cell leukemia cell lines in the S phase of the cell cycle in vitro. Although patients found 42 / 6 to be well tolerated in a phase Ia clinical trial, it induces human anti-mouse antibodies (HSMS) due to its mouse IgA isotype and is rapidly cleared by the kidneys, resulting in insufficient potency (BROOKS et al., Clin Cancer Res. 1:1259-1265, 1995). MOURA et al. (J Exp Med, 194, 417-425, 2001) reported a more potent neutralizing mouse anti-TfR1 IgG2b antibody (A24) that inhibits T cell proliferation by directly competing with TfR1 with high affinity (KD = 2.7 nM), reducing TfR expression and impairing TfR recirculation. Compared to 42 / 6, which exerts its antiproliferative effect by blocking cells in the S phase of the cell cycle, A24 exerts its effect by inducing apoptosis in target cells and blocking the in vitro 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] To overcome the lack of efficacy and HAMA response in humans, several chimeric, humanized, or fully human antibodies have been developed in recent years. As an 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. *Journal of Immunotherapy* 34:500-508, 2011; DANIELS-WELLS. *Journal of Immunotherapy* 38:307-310, 2015.), and its humanized version, hu128.1 (IgG1), has also shown efficacy in the AIDS-NHL CLDX model (DANIELS-WELLS. *Cancer Research* 80(16 Supplement):5655, 2020.). The anticancer activities of ch128.1 and hu128.1 are thought to depend on Fc-mediated antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent phagocytosis (ADCP). Three fully human IgG1 isotype neutralizing antibodies have been reported. PPMX-T003 and H7-IgG1 blocked tumor growth and prolonged mouse survival in various leukemia or lymphoma models (SHIMOSAKI et al., Biochemical & Biophysical Research Communications 485:144-151, 2017; ZHANG et al., Cancer Research 77(13 Supplement):5586, 2017). More notably, 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.), indicating that TfR1 can be used to treat solid tumors in addition to hematopoietic malignancies.

[0005] Due to the broad spectrum of TfR1 expression in dividing cells, anti-TfR1 antibodies may possess generalized antiproliferative activity that cancer patients may not tolerate. Mild to moderate toxicity of anti-TfR1 antibodies to erythrocyte (RBC) progenitors and myeloid progenitors has been reported in animal models (CANDELARIA et al., Frontiers in Immunology, March 17, 2021). Although many anti-human TfR1 antibodies have shown promising preclinical results, only CX-2029 (anti-TfR1 antibody-drug conjugate (ADC)) has entered clinical trials, demonstrating dose-dependent hematologic toxicities (anemia, neutropenia, and leukopenia) that can be controlled by erythrocyte transfusion in a Phase I clinical trial (JOHNSON et al., Clinical Cancer Research 27:4521-4530, 2021). Therefore, there is a need for anti-TfR1 antibodies that specifically target cancer cells and have minimal cross-reactivity with normal cells for cancer therapy. Summary of the Invention

[0006] MAb11-22.1 monoclonal antibody is a monoclonal antibody (IgG1, κ) generated from one of the hybridomas produced after immunizing mice with several live acute myeloid leukemia (AML) cell lines. It has been identified as a specific monoclonal antibody against human TfR1 expressed on AML and other cancer cells. MAb11-22.1 monoclonal antibody has a very high affinity for TfR1 (KD < 1 × 10⁻¹² M). In vitro assays have shown that MAb11-22.1 monoclonal antibody has a slight inhibitory effect on cancer cell proliferation. In an in vivo study, the MAb11-22.1 chimeric antibody (MAb11-22.1-S239C-DM1) in the form of an antibody-drug conjugate (ADC) conjugated with motansine (“DM1”, N2'-deacetylated-N2'-(3-thiol-1-oxypropyl)-matansine 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 shown in SEQ ID NO: 1 below.

[0008] The translated amino acid sequence in the variable region of the light chain is predicted as SEQ ID NO: 2 below, and the three antigenic determinant regions (CDR 1 to CDR 3, from left to right) are highlighted in bold and underlined.

[0009] The encoding sequence of the variable region of the heavy chain of MAb11-22.1 mAb is shown in SEQ ID NO: 3 below.

[0010] The translated amino acid sequence in the variable region of the heavy chain was predicted as SEQ ID NO: 4 below, with the three antigenic determinant regions (CDR 1 to CDR 3, from left to right) highlighted in bold and underlined.

[0011] This invention relates to binders (defined below), including antibodies (defined below), such as monoclonal antibodies, fragments thereof, and derivatives thereof, said antibodies comprising one or more light chain CDR regions having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) of the same sequence as the light chain CDR region of an anti-TfR1 specific monoclonal antibody named MAb11-22.1; and comprising a light chain variable region that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical to the sequence of 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 the sequence of SEQ ID NO: 4. The binder may contain a light chain CDR1 region similar to 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 contain a light chain CDR2 region similar to 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 contain a light chain CDR3 region similar to 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 contain a heavy chain CDR1 region similar to 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 contain a heavy chain CDR2 region similar to 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 binder may contain a heavy chain CDR3 region similar to 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 various cancers that overexpress TfR1 with MAb11-22.1 and MAb11-22.1-related binders (defined below), said binders being derived from or containing a portion of at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) identical in sequence to one or more CDRs in the light and heavy chains of MAb11-22.1. MAb11-22.1 binds specifically to human TfR1 protein with high affinity and induces endocytosis of this protein.

[0013] The binder is preferentially used to treat tumor-related diseases, including primary and metastatic cancers such as leukemia, lymphoma, multiple myeloma, breast cancer, glioblastoma, prostate cancer, urothelial bladder cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, and gastric cancer, including gastric squamous cell carcinoma, gastric adenocarcinoma, small cell carcinoma of the stomach, gastric squamous cell carcinoma, gastric carcinoid tumors, and gastric and duodenal cancers.

[0014] The conjugate of the binder preferably comprises an antitumor agent, such as a cytotoxic agent, including maytansine or its derivatives, auristatin or its derivatives, epothilone or its derivatives, paclitaxel or its derivatives, or vinca alkaloids; or further comprises: compressorin A-4 phosphate, compressorin A-4 and its derivatives, indole-sulfonamide compounds, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, etc. norelbine, vinflunine, vinglycinate, anhydrovinblastine, sylvatin 10 and its analogues, lecithin B and eryribulin, indole-3-oxadiazine, substituted indole-3-oxadiazine, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, and laulimethicone; DNA topoisomerase inhibitors, such as camptothecin and its derivatives, mitoxantron, and mitoguazone; nitrogen mustard analogs, such as chlorambucil, chlormaphazine, cyclophosphamide, estramustine, ifosfamide, mustard, nitromin, and melphalan. Novembichin, phenamet, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas, such as carmustine, streptozotocin, formmustine, lomustine, nimustine, and ranimustine;Antibiotics, such as enediyne antibiotics, dynemicin, esperamicin, neocarzinostatin, aclacinomycin, actinomycin, anthroamycin, azaserine, bleomycin, actinomycin C, carabicin, idarubicin, carzinophilin, and carminomycin. Actinomycin D, Daunorubicin, Doxorubicin, 6-diazo-5-oxo-L-leucine, Adriamycin, Epirubicin, Esorubicin, Idarubicin, Marcellobycin, Mitomycin, Mycophenolic acid, Nogalamycin, Olivomycin, Peplomycin, Bofeimeisu, Purinomycin Puromycin, doxorubicin-Fe, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate, edatrexate; purine analogs Examples of pyrimidine analogues include fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues include ancitabine, gemcitabine, enoxaparin, azacitidine, 6-azouridine, carmofur, cytarabine, dideoxyuridine, deoxy-fluorouridine, and fluorouridine.Androgens, such as Calusterone, Dromostanolone propionate, Epitiostanol, Mepitiostane, and Testolactone; antiadrenergic compounds, such as Aminoglutethimide, Mitotane, and Trilostane; trichothecenes, such as T-2 toxin, verracurin A, Roridin A, and Anguidine; and aziridines, such as Benzodop. a) Carboquone, Meturedopa, and Uredopa; platinum analogs, such as cisplatin, carboplatin, oxaliplatin, miriplatin, and etoposide; antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; protein kinases and proteasome inhibitors.

[0015] Preferably, a linker is used to bind the binder to the antitumor agent. Binding can be performed as described below regarding the linker of Mertansine (DM1), i.e., using site-specific conjugation to conjugate the thiol group in the binder to the antitumor agent. Various other binding methods can also be used.

[0016] In some embodiments, linkers can be used for conjugation and can consist of flexible residues such as glycine and serine (an example being four repeating glycine and one serine; or simply repeating glycine), allowing adjacent protein domains to move freely relative to each other. Rigid linkers are preferred if it is desirable to maintain distance between domains so that they do not interact; one example is (repeated): glutamic acid, three alanine residues, and lysine. Another rigid linker is (repeated): any amino acid residue and proline. Linkers can be non-cleavable linkers (e.g., thioether, SMCC, PEG linkers) or cleavable linkers such as 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 Conjugators,” discloses multiple acehydrazide thiols linkers and the preparation of conjugators using said linkers. U.S. Patent No. 8,518,891 discloses linkers having an aminoarylmethyl or aminoheteroaryl moiety. Bifunctional linkers are also described. See U.S. Patent No. 11,040,084.

[0017] Appropriate dosage ranges and dosing regimens can be deduced from the in vivo and in vitro experimental results described herein. Binders are preferentially delivered in the form of therapeutic pharmaceutical formulations; and they can also be used for diagnostic, purification, or screening of other binders. Simple Explanation of the Diagram

[0018] Figures 1A to 1C show that MAb11-22.1 mAb binds directly to AML cell lines, but not to peripheral blood mononuclear cells (PBMCs) of healthy individuals.

[0019] Figure 1A shows that MAb11-22.1 mAb interacted with the cell surface of nine human AML cell lines but not with human PBMCs in a fluorescence-activated cell sorting (FACS) assay (left panel). An isotype control antibody was used as a negative control in this assay (right panel). Detection was performed using a fluorescein-labeled AffiniPure goat anti-mouse IgG, Fcγ fragment-specific antibody (anti-Mo IgG Fc-FITC, Jackson ImmunoResearch Laboratories) at a 1:800 dilution. The reaction was performed at 4°C. MFI: mean fluorescence intensity.

[0020] Figure 1B shows an immunocytochemical (ICC) image of MAb11-22.1 mAb bound to the surface of NB4 or THP-1 cells (top panel), while isotype control antibodies did not stain these cells (bottom panel). AML cells in 4% paraformaldehyde were fixed to slides via a centrifuge smear. After antigen retrieval, cells were blocked in PBST containing 1% bovine serum albumin (BSA) and then incubated with primary antibodies for one hour, followed by one hour incubation with 1:1000 diluted peroxidase-labeled AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3), Fcγ fragment specific (Jackson Immunological Research Laboratory). Cells were stained with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin.

[0021] Figure 1C shows the hybridization of MAb11-22.1 mAb with protein bands present in whole-cell lysates of ALL (Raji) and AML cells (NB4, OCI / AML2, and THP-1) using the Western ink dot method. This target protein is 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 detected using a specific mouse monoclonal antibody as a loading control. Detection was performed using AffiniPure goat anti-mouse IgG Fcγ labeled with peroxidase at a 1:10,000 dilution.

[0022] Figures 2A to 2D show the identification and confirmation of transferrin receptor 1 (TfR1) as a target of MAb11-22.1 mAb.

[0023] Figure 2A shows a representative silver-stained SDS-PAGE image of an immunoprecipitation (IP) sample obtained by reacting MAb11-22.1 mAb with AML whole-cell lysate. The IP sample was reduced and loaded equally into lanes 1 and 2, and the target band of approximately 110 kDa was excised as indicated for LC-MS / MS analysis. The reduced MAb11-22.1 mAb was loaded at 8 ng into lane 4 as a control. Protein bands were detected using the ProteoSilver™ Silver Staining Kit (MilliporeSigma) according to the manufacturer's protocol. MW: Molecular weight standard; HC: Heavy chain; LC: Light chain.

[0024] Figure 2B depicts the Western ink dots of non-reducing whole-cell lysate samples of ALL and AML simultaneously detected by MAb11-22.1 mAb and anti-β-actin mAb. The left lane contained 1.2 µg of recombinant TfR1 extracellular domain (rTfR1-ECD, ACRO Biosciences) as a positive control, and the two right lanes contained CHO cell lysates at two concentrations (1x and 2x) as negative controls. Detection was performed using 1:20,000 dilution of peroxidase-labeled AffiniPure goat anti-mouse IgG Fcγ.

[0025] Figure 2C illustrates the direct interaction between MAb11-22.1 mAb and the extracellular domain of TfR1, confirmed by ELISA. ELISA plates were coated with 0.1 µg / mL of purified rTfR1-ECD containing a C-terminal 6xHis tag. Mouse anti-6xHis 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. Detection was performed using peroxidase-labeled AffiniPure goat anti-mouse IgG Fcγ at a 1:6,000 dilution.

[0026] Figure 2D shows the FACS results of CHO cells with or without recombinant expression of human TfR1-GFP fusion protein (rTfR1-GFP) on the cell membrane. CHO cells were not reactive to MAb11-22.1 mAb #1 or #2 (two different purification batches) or a commercially available anti-human TfR1 control mAb (R&D Systems). However, after TfR1 reconstruction on the cell membrane, MAb11-22.1 mAb #1, #2, and the anti-TfR1 control mAb could bind to the cell surface. MAb11-22.1 mAb showed higher affinity than the anti-TfR1 control mAb. Detection was performed using a 1:800 dilution of R-phycoerythrin (R-PE)-labeled AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3), Fcγ fragment-specific secondary antibody (Jackson Immunological Research Laboratory).

[0027] Figures 3A to 3D are data from the ForteBio Octet® QK system output, showing the affinity and kinetics between MAb11-22.1 mAb and rTfR1-ECD.

[0028] Figure 3A shows aligned sensor traces of the binding and dissociation curves of MAb11-22.1 mAb with 20 nM rTfR1-ECD (left panel) or 300 nM goat anti-mouse (H+L) Fab (positive control, right panel), extracted from the raw data using ForteBio software. The ForteBio Octet® QK system is equipped with an anti-mouse Fc capture (AMC) biosensor for immobilizing 10 nM or 50 nM MAb11-22.1 mAb. The durations of the key steps are as follows: 60 seconds (seconds:seconds or sec), baseline in kinetic buffer, 450 seconds immobilization of MAb11-22.1 mAb, 120 seconds baseline / wash in kinetic buffer, 450 seconds antigen binding, followed by 3000 seconds of dissociation in kinetic buffer only. Upper curve: 10 nM mAb; Lower curve: 50 nM mAb.

[0029] Figure 3B shows the kinetics of binding between MAb11-22.1 mAb, starting at 20 nM and serially diluted 2.5 times, and rTfR1-ECD. Binding occurred rapidly during the observation period (3000 seconds), and no dissociation was detected.

[0030] Figure 3C shows the kinetic data of MAb11-22.1 mAb binding to goat anti-mouse IgG (H+L) Fab as a positive control in serial dilutions, used to verify the reliability of the device.

[0031] Figure 4 shows the competitive ELISA results of MAb11-22.1 mAb and transferrin (Tf) binding 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) alone or separately mixed with seven 4-fold serially diluted MAb11-22.1 mAb-HRP conjugates starting at 2 µg / mL. 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 two replicates.

[0032] Figures 5A to 5C show the FACS results of MAb11-22.1 mAb binding to PBMCs and bone marrow (BM) cells in healthy adults and BM cells in AML patients.

[0033] Figure 5A shows that MAb11-22.1 mAb did not bind to normal human PBMCs (Stanford Blood Center) or normal human BM cells (Human Cells, Fremont, CA) regardless of mAb concentration. An isotype control antibody was used as a negative control. Starting at 20 µg / mL, the mAb was serially diluted eightfold and detected using a 1:800 dilution of anti-mouse IgG Fc-FITC.

[0034] Figure 5B shows that MAb11-22.1 mAb binds to BM cells from AML patients in a dose-dependent manner.

[0035] Figure 5C is a scatter plot of PBMCs and BM cells stained with different mAbs. Criteria used to define the positive or negative results for cell surface markers are shown as solid lines, and the plot is subdivided into four quadrants. The presence of stem cells in the BM samples was confirmed by mouse anti-human CD34 mAb (Miltenyi, Cambridge, MA), a FITC-labeled antibody specific for hematopoietic stem cells (lower left panel). Due to the stickiness of BM cells, MAb11-22.1 mAb and isotype control antibodies stained a small subset of BM cells.

[0036] Figure 5D shows that erythrocytes present in PBMC and BM cell clusters stained with anti-human CD233-PE mAb (Mitenia Biosciences) are not reactive to MAb11-22.1 mAb or isotype control antibody.

[0037] Figure 5E shows that erythrocytes and erythroblasts present in PBMC or BM cell clusters stained with anti-human CD235a-PE (Medrin) are not reactive to MAb11-22.1 mAb or isotype control antibody.

[0038] Figures 6A to 6C show the results of the endocytosis assay of MAb11-22.1 mAb on cancer cell lines.

[0039] Figure 6A compares the fluorescence intensity detected by FACS on the cell surfaces of three leukemia cell lines (OCI / AML2, NB4, and Raji) stained alone with MAb11-22.1 mAb, mouse anti-human CD20 mAb, or isotype control antibody under different conditions. Cells were incubated with each mAb at 4°C for one hour, followed by washing with cold PBS and incubation in medium at 4°C or 37°C. Cells were stained with anti-Mo IgG Fc-FITC at 30 min, 1 h, and 2 h, respectively. At 4°C, MAb11-22.1 mAb bound to the surface of the two AML cell lines in a dose-dependent manner but not to Raji cells, and was rapidly internalized after transfer to 37°C (left panel). Anti-CD20 mAb, which interacted with Raji cells but not with AML cells, could be partially internalized by Raji cells at 37°C (middle panel). An isotype control antibody that did not bind to any cell line was used as a negative control (right panel).

[0040] Figure 6B shows a representative fluorescence image of MAb11-22.1 mAb bound to and internalized by the OCI / AML2 cell line. Cells in replicated 96-well plates were incubated with different concentrations of CF488-labeled MAb11-22.1 mAb or CF488-labeled isotype control antibody in DMEM medium at 4°C for one hour, followed by washing with ice-cold PBS to remove the mAb. Cells in one plate were resuspended in ice-cold PBS and photographed using a Keyence fluorescence microscope at 400 × magnification. Cells in replicated plates were resuspended in warm SFM and incubated at 37°C for one hour to allow for mAb internalization. MAb11-22.1 mAb-CF488 bound to the surface of OCI / AML2 cells at 4°C, but was mostly internalized after one hour of incubation at 37°C. As a control, the CF488-labeled isotype control mAb did not stain the cells or cause internalization (right panel). All images were taken using the same settings. The scale bar indicates 100 µm.

[0041] Figure 6C shows a representative image of the dose-related increments in the binding and endocytosis of MAb11-22.1 mAb-CF488 in MDA-MB-231 cells. The assay protocol 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 localized not only on the surface of MDA-MB-231 cells but also present inside the cells, indicating a high tendency for MAb11-22.1 mAb-CF488 to be endocytosed by the MDA-MB-231 cell line. After incubation at 37 °C for one hour, most of the MAb11-22.1 mAb-CF488 was endocytosed. The CF488-labeled isotype control mAb-CF488 did not stain the cells or allow for cell uptake (right panel). These images were taken at 400x magnification with the same exposure time. The scale bar indicates 100 µm.

[0042] Figure 7 illustrates the partial inhibitory effect of MAb11-22.1 mAb on the proliferation of OCI / AML2 cells. Cells (2 × 10⁴ cells / mL) were cultured in a humidified 5% CO₂ incubator at 37°C in the presence of 100 µg / mL MAb11-22.1 mAb or an isotype control antibody. Cell viability was assessed every 24 hours by treating with 10 µL CCK-8 solution for 4 hours using absorbance at 450 nm. Data represent the mean ± SD of three replicates. * P < 0.05.

[0043] Figures 8A and 8B are data outputs from the ForteBio Octet® QK system, showing the affinity and kinetics between the MAb11-22.1 chimeric antibody (cAb) and rTfR1-ECD.

[0044] Figure 8A shows the binding and dissociation curves and a fitted view of the datasheet 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). The Octet QK system is equipped with an anti-human Fc capture (AHC) biosensor for immobilizing 50 nM or 10 nM MAb11-22.1 cAb. The durations of the key steps are as follows: 60 sec baseline in kinetic buffer, 450 sec immobilization of MAb11-22.1 cAb, 120 sec baseline / wash in kinetic buffer, 450 sec binding of antigen to immobilized MAb11-22.1 cAb, followed by 3600 sec dissociation only in kinetic buffer. No decay of the binding signal was detected during the allowed dissociation time, therefore the KD of MAb11-22.1 cAb and rTfR1-ECD is < 1 pM.

[0045] Figure 8B is a fitted view of the kinetic analysis of the positive control goat anti-human (H+L) Fab in the presence of three-fold serial dilutions (300, 100, 33.3, 11.1, and 3.7 nM) bound to 50 nM MAb11-22.1 cAb. The calculated KD was 2.18 nM.

[0046] Figure 9 shows the effect of MAb11-22.1 cAb with and without the S239C mutation, along with the isotype control monoclonal antibody, on the proliferation of various cancer cell lines in vitro. Optimized amounts of OCI / AML2, Raji, HCC38, and MDA-MB-231 cell lines were co-cultured with each antibody for five days, followed by CCK-8 assays. Two doses (100 and 300 µg / mL) of MAb11-22.1 cAb (shaded bars) and MAb11-22.1-S239C cAb (grey bars) were treated with DM1, as indicated by the "+" sign under the X-axis; antibodies not treated with DM1 are indicated by the "-" sign. Unconjugated isotype control antibody (white bars) reflects the baseline proliferation of each cell line. Data represent the mean ± SD of three replicates.

[0047] Figure 10 illustrates the in vivo efficacy of MAb11-22.1-S239C-DM1 against tumor growth in an OCI / AML2 cell line xenograft (CLDX) mouse model. MAb11-22.1-S239C-DM1 was administered intraperitoneally (ip) at 10 mg / kg (low dose, n = 5) or 20 mg / kg (high dose, n = 6) at 4 × 7 time intervals (day 0 to day 24). The negative control group was administered a DM1-treated isotype control antibody (without the S239C mutation) at 20 mg / kg / 4 × 7 / ip (n = 5). Tumor volume was measured every four 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 the groups. From day 16 following the four antibody treatments, the mean tumor volume in the low-dose group was significantly different from that in the isotype control DM1 group (* P < 0.05). Implementation

[0048] It should be understood that, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" include the plural forms. Monoclonal antibodies are sometimes referred to as "mAbs". The terms "binding agent" and "antibody", in their singular and plural forms, are used interchangeably.

[0049] The binding agents of the present invention comprise MAb11-22.1-like or MAb11-22.1-derived antibodies, antibody fragments, fusion proteins, or antibody-derived or modified chimeric antibody receptors (CARs) comprising a single-stranded variable fragment (scFv) fused to an intracellular signaling domain such as the ζ chain of CD3 (CD3ζ). The CAR is preferably expressed in CAR-immunized effector cells comprising, but not limited to, T cells, NK cells, and macrophages.

[0050] The term "binding agent" includes antibodies (the two can be used interchangeably in the plural form), which are immunoglobulin molecules capable of binding to target antigens, such as antigens on gastric cancer cells, through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses not only full-length (i.e., full-length) polyclonal or monoclonal antibodies, but also their antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv, Fd,rIgG, single-chain (scFv) or sc(Fv)2, their mutants, fusion proteins including antibody portions, humanized antibodies, chimeric antibodies, bifunctional antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antigen-binding (SDAB) molecules, VH or VL domains or VHH domains, and any other modified conformations of immunoglobulin molecules including antigen recognition sites of desired specificity, said modified conformations including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibody-drug conjugates are also included.

[0051] Antibodies can be any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and antibodies do not need to belong to any specific class. Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0052] The antibodies described herein may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies, including antibodies as described in U.S. Patent No. 7,317,091,B2, and including such antibodies generated through affinity maturation). In some instances, the antibody includes a modified constant region, such as an immune-inert constant region, for example, that does not trigger complement-mediated cleavage or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity can be evaluated using the method disclosed in U.S. Patent No. 5,500,362.

[0053] Humanized antibodies are non-human (e.g., mouse) antibodies that specifically chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues from the receptor's complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody) with the desired specificity, affinity, and capability. In some cases, Fv frame region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in the receptor antibody or in the introduced CDR or frame sequence but included to further refine and optimize antibody performance. Typically, humanized antibodies will include at least one and typically two variable domains, with all or substantially all CDR regions corresponding to those regions of the non-human immunoglobulin, and all or substantially all FR regions being those regions of the human immunoglobulin common sequence. Humanized antibodies will preferably also include at least a portion of the immunoglobulin constant region or structural domain (Fc), typically at least a portion of the human immunoglobulin constant region. The antibody may have a modified Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, and / or six) with respect to changes in the original antibody. Humanized antibodies may also involve 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).

[0054] In another instance, the antibody described herein is a chimeric antibody, which may comprise a heavy constant region and a light constant region derived from a human antibody. See, for example, Morrison et al. (1984), Proceedings of the National Academy of Sciences (PNAS) 81, 6851; Neuberger et al. (1984), Nature 312, 604; and Takeda et al. (1984), Nature 314:452. A chimeric antibody is an antibody having a variable region or a portion thereof derived from a first species and a constant region derived from a second species. Typically, in these chimeric antibodies, both the variable regions of the light and heavy chains mimic the variable regions of antibodies derived from one species of mammal (e.g., non-human mammals such as mice, rabbits, and rats), while the constant portion is sequence homologous to that in antibodies derived from another mammal, such as humans. In some embodiments, amino acid modifications may be made in the variable and / or constant regions. See U.S. Patent No. 4,816,567.

[0055] For shorter sequences described or claimed herein, the “percentage of identity” between two amino acid sequences can be determined arithmetically by counting and comparison, or by using the algorithm described by Karlin and Altschul in *Proceedings of the National Academy of Sciences* 87:2264-68, 1990, as modified in *Proceedings of the National Academy of Sciences* 90:5873-77, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) in *Journal of Molecular Biology* 215:403-10, 1990, by Altschul et al. The XBLAST program (score = 50, word length = 3) can be used to perform a BLAST protein search to obtain amino acid sequences homologous to the protein molecule of interest. In cases where gaps exist between two sequences, gapped BLAST can be used, as described by Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST programs and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0056] [Preparation of binder]

[0057] Several methods have been developed to prepare chimeric antibodies, humanized antibodies, or human antibodies for therapeutic applications in humans. The most common method is to prepare mouse mAbs using a hybridoma approach, and then humanize the mAb by converting the VH and VL domains, as well as the frame regions of the constant domains, of the mAb into homologous human frame regions of the human VH and VL domains, and the constant regions of the desired human γ-immunoglobulin isotypes and subtypes. See U.S. Patent No. 5,225,539.

[0058] Monoclonal antibodies can be prepared using conventional hybridoma techniques. Kohler et al., *Nature*, 256:495 (1975). In the hybridoma approach, 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 will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0059] To prepare monoclonal antibodies, immune cells are collected from mammals immunized with antigens, and the level of desired antibody increase in serum is examined as described above. The immune cells are then subjected to cell fusion. The immune cells used for cell fusion are preferably obtained from the spleen. Other preferred parental cells to be fused with the aforementioned immune cells include, for example, mammalian myeloma cells, and more preferably myeloma cells that have been obtained through drug-selective fusion.

[0060] Preferred myeloma cells are those that are efficiently fused, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. Among these, preferred myeloma cell lines are mouse myeloma cell lines, such as cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California, USA), and SP-2 cells (available from the American Type Culture Collection, Rockville, Md. USA). Human myeloma and mouse-human heterologous myeloma cell lines have also been described as being used to produce 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).

[0061] The aforementioned immune cells and myeloma cells can be fused using known methods, such as those of Milstein et al. (Galfre et al., Methods Enzymol. 73:3-46, 1981). Lymphocytes are fused with myeloma cells using suitable fusion agents such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). The resulting hybridomas obtained through cell fusion can be selected by culturing in a standard selection medium, such as HAT medium (containing hypoxanthine, aminopterin, and thymidine). Cell culture is typically continued in HAT medium for several days to several weeks, long enough for all other cells except the desired hybridoma (non-fused cells) to die. Standard limiting dilutions are then performed to screen and select hybridoma cells that produce the desired antibodies.

[0062] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells. The production of monoclonal antibodies against the antigen in the culture medium for growing hybridoma cells is measured. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assay. Enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and / or measurement of absorbance in immunofluorescence can be used to measure the antigen-binding activity of the antibodies.

[0063] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the lines can be subselected using 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. The monoclonal antibodies secreted by the subcultures are appropriately separated from the culture medium, ascites, or serum using routine immunoglobulin purification procedures (e.g., protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography). Alternatively, hybridoma cells can be grown as ascites tumors in the peritoneum of animals such as mice.

[0064] The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or affinity column coupled with the protein of the present invention.

[0065] Monoclonal antibodies thus obtained can also be recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck CAK and Larrick JW, *Therapeutic Monoclonal Antibodies*, published by MacMillan Publishers LTD, UK, 1990). The DNA encoding the antibody can be selected from immune cells, such as antibody-producing hybridomas or immune lymphocytes, inserted into a suitable vector, and introduced into host cells to prepare recombinant antibodies. Recombinant techniques are described in the following literature: specifically, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and 3rd edition, 2001; Ausubel et al., *Current Protocols in Molecular Biology*, John Wiley & Sons, New York, 1987 and periodically updated; *The Series Methods in Enzymology*, Academic Press, San Diego; and Kontermann and Dübel, *Antibody Engineering*, Springer Laboratory Manual, Springer-Verlag Berlin Heidelberg, 2001, etc.

[0066] When the obtained antibodies are administered to humans for treatment, human antibodies or humanized antibodies are preferred to reduce immunogenicity. For example, transgenic animals with a human antibody gene library can be immunized with whole cells or antigens selected from human cell proteins or their lysates. Antibody-producing cells are then collected from the animals and fused with myeloma cells to obtain hybridomas, from which human antibodies against the antigens can be prepared. Alternatively, antibody-producing immune cells, such as immunized lymphocytes, can be immortalized with oncogenes and used to prepare monoclonal antibodies.

[0067] DNA encoding monoclonal antibodies can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies. Hybridoma cells serve as a preferred source of such cDNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells, such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins, thereby generating monoclonal antibodies in the recombinant host cells. Review articles on the recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., *Curr. Opinion in Immunol.*, 5:256-262 (1993) and Pluckthun, *Immunol. Rev.*, 130:151-188 (1992).

[0068] Genetically engineered antibodies can be produced by genetically modifying the DNA encoding antibodies produced by hybridoma cells as described above using conventional techniques. Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, bifunctional antibodies, bispecific antibodies, and multispecific antibodies, can be produced, for example, using conventional recombinant techniques. The DNA can then be modified, for example, by replacing the homologous mouse sequence with the coding sequence that replaces the constant regions of the human heavy and light chains (Morrison et al., (1984) Proceedings of the National Academy of Sciences 81:6851), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. In these ways, genetically engineered antibodies with binding specificity to the target antigen, including chimeric or humanized antibodies, can be prepared.

[0069] Alternatively, certain transgenic animals (e.g., mice) are able to generate a complete human antibody library after immunization in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice has been described as resulting in complete suppression of endogenous antibody production. Transferring human germline immunoglobulin gene arrays into such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, for example, Jakobovits et al., Proceedings of the National Academy of Sciences, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993). Also see the Xenomouse transgenic mouse from Amgen, Inc. (Fremont, CA) and the HuMAb-MouseR™ and TC Mouse™ from Medarex, Inc. (Princeton, NJ). In another alternative, antibodies can be recombinantly prepared via phage display technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) *Annu. Rev. Immunol.* 12:433-455; (Hoogenboom et al., *Journal of Molecular Biology*, 227:381 (1991); Marks et al., *Journal of Molecular Biology*, 222:581-597 (1991). Alternatively, phage display technology (McCafferty et al., (1990) *Nature* 348:552-553) can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene lineages from non-immunized donors.

[0070] F(ab')2 fragments can be produced by pepsin digestion or other proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). Fab fragments can be produced by reducing the disulfide bonds of the F(ab')2 fragment. Such fragments can now also be produced directly from recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, F(ab')2-SH fragments can be recovered directly from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell cultures.

[0071] 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; U.S. Patent No. 8,148,496 discloses a multispecific Fv antibody construct having at least four variable domains that are linked to each other by peptide linkers.

[0072] U.S. Patent Publication No. 20170335281 describes the preparation of genetically modified T cells expressing a CAR, wherein the CAR includes an antigen-binding domain that binds to a cancer-associated antigen. The same general techniques can be used to modify T cells or other immune effector cells to express one or more of CDR1, CDR2, and CDR3 of MAb11-22.1 as antigen-binding domains for cancer therapy. The antigen-binding domain of the CAR peptide molecule can contain any antibody, antibody fragment, scFv, Fv, Fab, F(ab′) 2, single-domain antibody (SDAB), VH or VL domain, or VHH domain.

[0073] Single-domain antibody (sdAb) molecules are disclosed in WO 9404678 and Hamers-Casterman, C. et al. (1993) *Nature* 363:446-448. An sdAb molecule is a naturally occurring single-domain antigen-binding molecule, referred to as a heavy chain lacking a light chain. In one type of sdAb, the variable domain is derived from a naturally occurring heavy chain molecule lacking a light chain, which can be derived from camelid species such as camels, llamas, dromedaries, alpacas, and guanacos. Other species besides camelids can also produce naturally occurring heavy chain molecules lacking a light chain. sdAb molecules can be recombinant, CDR-transplanted, humanized, camelidized, deimmunized, and / or in vitro generated (e.g., selected via phage display).

[0074] Any of the above-mentioned binders can be used in antibody-drug conjugates (ADCs) to target cancer cells by linking the binder to the cytotoxic agents of cancer cells. See U.S. Patent No. 9,764,041; U.S. Publication No. 20170151343.

[0075] [High-affinity antibody variant]

[0076] Antibodies having the sequence variants described herein are within the scope of this invention. One type of variant is a high-affinity variant, as described below.

[0077] Antibodies should be humanized, retaining high affinity for antigens and other beneficial biological properties. To achieve this, humanized antibodies are prepared using a preferred method, through the analysis of parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are commonly used. Computer programs illustrating and demonstrating the possible three-dimensional conformations of selected candidate immunoglobulin sequences are available. These displays allow for the analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analyzing residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and input sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0078] Examples of frame region residues to be modified include residues that directly and non-covalently bind to the target (Amit et al., Science 233: 747-753 (1986)); residues that interact with / affect the conformation of the CDR (Chothia et al., Journal of Molecular Biology 196: 901-917 (1987)); and / or residues involved in the VL-VH interface (EP 239 400 B1). In some embodiments, modification of one or more such frame region residues results in enhanced antibody binding affinity to the target of interest.

[0079] Nucleic acid molecules encoding amino acid sequence variants can be prepared using a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, polymerase chain reaction (PCR) mutagenesis, and cassette mutagenesis of earlier prepared variant or non-variant forms of species-dependent antibodies. A preferred method for generating variants is oligonucleotide-mediated synthesis. In some embodiments, the antibody variant will have only a single substituted hypervariable residue, for example, about two to about fifteen hypervariable substitutions.

[0080] One method for generating variant libraries is through oligonucleotide-mediated synthesis. Three oligonucleotides, each approximately 100 nucleotides long, spanning the entire variable region of the light or heavy chain, can be synthesized. Each oligonucleotide may include: (1) a segment of 60 amino acids generated by a triplet (NNK) sub.20, where N is any nucleotide and K is G or T; and (2) approximately 15-30 nucleotides overlapping at each end with the next oligonucleotide or vector sequence. After annealing of these three oligonucleotides in a PCR reaction, the polymerase fills in the opposite strands, producing a complete double-stranded heavy or light chain variable region sequence. The number of triplets can be adjusted to repeats of any length, and their position within the oligonucleotide can be selected to substitute only amino acids in a given CDR or frame region. By using (NNK), every position of all twenty amino acids in the encoded variant is possible. Overlapping sequences of 5-10 amino acids (15-30 nucleotides) will not be substituted, but these may be selectively placed within the stacking region of the frame, or may be substituted by a single or subsequent round of synthesis. Methods for synthesizing oligonucleotides are well known in the art and are commercially available. Methods for generating antibody variants from these oligonucleotides are also well known in the art, for example, PCR.

[0081] Libraries of heavy and light chain variants at random positions in their sequences can be constructed in any expression vector such as a phage, each containing DNA encoding a specific heavy and light chain variant.

[0082] Once an antibody variant is generated, its biological activity relative to the parent antibody is determined. As mentioned above, this involves determining the binding affinity of the variant to the target. Numerous high-throughput methods exist for rapidly screening antibody variants for their ability to bind to the target of interest.

[0083] Then, by screening one or more antibody variants selected from this initial screening, an affinity higher than that of the parent antibody can be obtained. A common method for determining binding affinity is to assess the binding rate constant and dissociation rate constant using the BIAcore Surface Plasmon Resonance System (BIAcore Corporation). According to the manufacturer's (BIAcore Corporation) instructions, the biosensor chip is activated to covalently couple the target. The target is then diluted and injected onto the chip to obtain a signal in units of response (RU) of immobilized material. Since the signal in RU units is proportional to the quality of the immobilized material, this represents the range of immobilized target density on the matrix. Dissociation data are fitted to a unit-point model to obtain Koff ± SD (standardized measurement deviation). The pseudo-first-order rate constant (Ks) is calculated for each binding curve and plotted as a function of protein concentration to obtain Kon ± SD (standardized fit error). The equilibrium dissociation constant Kd's for binding is calculated as Koff / Kon based on SPR measurements. Since the equilibrium dissociation constant KD is inversely proportional to Koff, assuming that the binding rate (Kon) of all variants is constant, the increase in affinity can be estimated.

[0084] Optionally, the resulting high-affinity candidates can be subjected to one or more other bioactivity assays to confirm that antibody variants with enhanced binding affinity still retain the desired therapeutic properties, as can be tested in the assays described in the examples below. Optimal antibody variants retain the ability to bind to the target with a binding affinity significantly higher than that of the parent antibody.

[0085] Depending on the intended use of the antibody, the selected antibody variant can often be further modified. Such modifications can involve further alterations to the amino acid sequence, fusion with a heteropeptide, and / or covalent modifications, as described below. For example, any cysteine ​​residues that are not involved in maintaining the proper conformation of the antibody variant can often be substituted with serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, (a) cysteine ​​bonds can be added to the antibody to improve its stability (specifically in the case where the antibody is an antibody fragment such as an Fv fragment).

[0086] [preparation]

[0087] After preparing a suitable antibody, it can be formulated into a preparation for administration to a subject. A lyophilized preparation is preferred, and as a first step, a pre-lyophilized preparation needs to be prepared. The amount of antibody in the pre-lyophilized preparation is determined considering the desired dose volume, administration method, etc. Proteins are typically present in solution. For example, proteins can be present in a pH buffer solution with a pH of about 4-8, and preferably about 5-7. Exemplary buffers contain histidine, phosphate, Tris, citrate, succinate, and other organic acids. Buffer concentrations can be from about 1 mM to about 20 mM, or from about 3 mM to about 15 mM, depending on, for example, the desired isotonicity of the buffer and the preparation (e.g., a reconstituted preparation). A preferred buffer is histidine because of its solubility-protective properties. Succinate is also a useful buffer.

[0088] A lyophilization protectant is added to the pre-lyophilized formulation. In a preferred embodiment, the lyophilization protectant is a non-reducing sugar, such as sucrose or trehalose. The amount of lyophilization protectant in the pre-lyophilized formulation is generally such that, upon reconstitution, the resulting formulation will be isotonic, which is preferred, although hypertonic reconstitution formulations may also be suitable. Furthermore, the amount of lyophilization protectant should not be too low, otherwise unacceptable amounts of protein degradation / aggregation will occur during lyophilization.

[0089] When the lyophilization protectant is a sugar (such as sucrose or trehalose) and the protein is an antibody, the exemplary concentration of the lyophilization protectant in the pre-lyophilized formulation is from about 10 mM to about 400 mM, and preferably from about 30 mM to about 300 mM, and most preferably from about 50 mM to about 100 mM.

[0090] For each combination of protein and lyophilization protectant, a protein-to-lyophilization protectant ratio is selected. In cases where the antibody is chosen as the protein and a sugar (e.g., sucrose or trehalose) as the lyophilization protectant to produce an isotonic reconstitution formulation with a high protein concentration, the mol ratio of lyophilization protectant to antibody can be from about 100 to about 1500 mol lyophilization protectant to 1 mol antibody, and preferably from about 200 to about 1000 mol lyophilization protectant to 1 mol antibody, comprising from about 200 to about 600 mol lyophilization protectant to 1 mol antibody.

[0091] In preferred embodiments, it has been found that there is a need to add surfactants to pre-lyophilized formulations. Alternatively or additionally, surfactants may be added to lyophilized formulations and / or reconstituted formulations. Exemplary surfactants include: nonionic surfactants such as polysorbates (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, linoleic acid, or stearyl-sarcosinate; linolenic acid, myristyl, or decaylyl serotonin. Hexaalkyl-betaine; laurylaminopropyl-, cocamylaminopropyl-, oleylaminopropyl-, myristylaminopropyl-, palmylaminopropyl-, or isostearylaminopropyl-betaine (e.g., laurylaminopropyl); myristylaminopropyl-, palmylaminopropyl-, or isostearylaminopropyl-dimethylamine; sodium methylcocamyl or sodium methyloleyl taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.). The amount of surfactant added is such that it reduces the aggregation of reconstituted proteins and minimizes the formation of particles after reconstitution. For example, surfactants may be present in the pre-lyophilized formulation in an amount of about 0.001-0.5%, and preferably about 0.005-0.05%.

[0092] A mixture of freeze-drying protectants (such as sucrose or trehalose) and fillers (e.g., mannitol or glycine) can be used to prepare pre-freeze-dried formulations. The filler allows for the production of a uniform freeze-dried cake without excessive pits.

[0093] Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (1980), may be included in pre-lyophilized formulations (and / or lyophilized and / or reconstituted formulations), provided they do not adversely affect the desired properties of the formulation. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and contain: additional buffers; preservatives; cosolvents; antioxidants, including ascorbic acid and methionine; chelating agents, such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; and / or salt-forming counterions, such as sodium.

[0094] The pharmaceutical compositions and formulations described herein are preferably stable in order to maintain their physical and chemical stability and integrity during storage. Various analytical techniques for measuring protein stability are available in the prior art and are reviewed in: *Peptide and Protein Drug Delivery*, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. *Advanced Drug Delivery Rev.* 10: 29-90 (1993). Stability can be measured at a selected temperature over a selected time period.

[0095] Formulations intended for in vivo administration must be sterile. This can be easily accomplished by filtration through a sterile filter membrane before or after lyophilization and reconstitution. Alternatively, sterility of the entire mixture can be achieved by autoclaving the components, excluding proteins, at approximately 120°C for approximately 30 minutes.

[0096] After the proteins, lyophilization protectants, and other optional components are mixed together, the formulation is lyophilized. Many different freeze dryers are available for this purpose, such as the Hull 50® (Hull, USA) or GT20® (Leybold-Heraeus, Germany) freeze dryers. Lyophilization is accomplished by freezing the formulation and subsequently sublimating ice from the frozen contents at a temperature suitable for initial drying. Under these conditions, the product temperature is below the eutectic point or collapse temperature of the formulation.

[0097] Typically, under suitable pressure (usually in the range of about 50 to 250 mTorr), the shelf temperature for initial drying will be in the range of about -30 to 25°C (assuming the product remains frozen during initial drying). The formulation, size, and type of the container holding the sample (e.g., glass vials), as well as the volume of liquid, will primarily determine the drying time, which can range from several hours to several days (e.g., 40-60 hours). The secondary drying stage can be carried out at about 0-40°C, depending primarily on the type and size of the container and the type of protein used. For example, the shelf temperature for the entire dehydration stage of lyophilization can be about 15-30°C (e.g., about 20°C). The time and pressure required for secondary drying will be the time and pressure required to produce a suitable lyophilized cake, depending on, for example, temperature and other parameters. The secondary drying time is determined by the desired residual moisture content in the product and typically takes at least about 5 hours (e.g., 10-15 hours). The pressure can be the same as that used in the primary drying step. Lyophilization conditions can vary depending on the formulation and vial size.

[0098] In some cases, to avoid transfer steps, it may be necessary to lyophilize the protein formulation in a container used for protein reconstitution. In this case, the container may be, for example, a 3, 5, 10, 20, 50, or 100 cc vial. As a general recommendation, lyophilization will result in a moisture content in the lyophilized formulation of less than about 5%, and preferably less than about 3%.

[0099] At the desired stage, typically when it is necessary to administer protein to a patient, the lyophilized formulation can be reconstituted with a diluent such that the protein concentration in the reconstituted formulation is preferably similar to the concentration of the pre-lyophilized formulation.

[0100] Reconstitution is typically performed at approximately 25°C to ensure complete hydration, although other temperatures may be used as needed. The time required for reconstitution will depend on factors such as the type of diluent, excipients, and amount of protein. Exemplary diluents include sterile water, water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextran solution. The diluent may optionally contain a preservative. Exemplary preservatives have been described above, with aromatic alcohols such as benzyl alcohol or phenolic alcohols being preferred preservatives. The amount of preservative used is determined by evaluating the compatibility of different preservative concentrations with the protein and by preservative efficacy testing. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it may be present in an amount of about 0.1-2.0%, and preferably about 0.5-1.5%, but most preferably about 1.0-1.2%.

[0101] Alternatively, non-lyophilized formulations may be used, comprising binders and any well-known carriers, excipients, buffers, stabilizers, preservatives, adjuvants and other additives described herein and well-known in the art.

[0102] [Dosage and Administration]

[0103] The formulations described above can be administered to subjects requiring treatment (e.g., humans) via appropriate routes, such as intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intradermal, intra-articular, intrasynovial, intrathecal, intradermal, intratumoral, intramedullary, oral, inhalation, or local administration; or the formulations can be administered orally, via inhalation spray, locally, rectal, nasal, oral, vaginal, or via implanted receptacle; and in any case, as a bolus or via continuous infusion over a period of time; or via injectable long-acting routes of administration, such as using long-acting injectable or biodegradable materials and methods for 1 month, 3 months, or 6 months.

[0104] Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, can be used for administration. Liquid formulations can be nebulized directly, and lyophilized powders can be nebulized after reconstitution. Alternatively, antibodies can be nebulized using fluorocarbon formulations and metered-dose inhalers, or inhaled as lyophilized powders and ground powders. In the use of CARs in this invention, compositions of immune effector cells (e.g., T cells, NK cells) can be directly injected into tumors, lymph nodes, infection sites, or other sites.

[0105] Subjects to be treated using the methods described herein may be mammals, more preferably humans. Mammals include, but are not limited to, livestock, sport animals, pets, primates, horses, dogs, cats, mice, and rats.

[0106] "Effective dose" refers to the amount of active agent required, alone or in combination with one or more other active agents, to impart a therapeutic effect to a subject. The effective dose varies depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, body size, sex, and weight, the duration of treatment, the nature of concurrent therapies (if any), the specific route of administration, and similar factors, all of which are well known to those skilled in the art and can be resolved through routine experimentation alone. Generally, the maximum dose of the individual component or its combination is preferred; that is, the highest safe dose based on reasonable medical judgment. Lower or tolerable doses may also be appropriate for medical, psychological, or other reasons.

[0107] Empirical considerations such as antibody half-life often help determine the dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to prolong the antibody's half-life and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted during treatment and is usually, but not necessarily, based on the treatment and / or inhibition and / or improvement and / or delay of gastric cancer. Alternatively, a sustained-release formulation of the antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0108] In one instance, the dosage of the antibody, as described herein, can be determined empirically in individuals who have received one or more doses. Individuals are given escalating doses of the antibody. To assess the efficacy of the antibody, disease markers (e.g., tumor growth) can be tracked according to routine practice.

[0109] Typically, for the administration of any antibody described herein, the initial candidate dose can be deduced from the experiments described below. For repeated administration over several days or longer, treatment is continued according to the symptoms until the desired symptom suppression is achieved or until a therapeutic level sufficient to alleviate cancer is reached. Exemplary dosing regimens include administering a higher initial dose followed by a lower maintenance dose. However, other dosing regimens may be useful depending on the pharmacokinetic decay pattern the practitioner wishes to achieve. For example, dosing once to four times per week is envisioned. In some embodiments, the dosing frequency is once weekly, 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 monthly, once every two months, or once every three months, or longer. Progression of this therapy can be easily monitored using conventional techniques and assays. The dosing regimen (including the antibody used) may vary over time.

[0110] Depending on the treatment goals and cancer sites, the drug composition can be administered to the subject using conventional methods known to ordinary medical professionals.

[0111] Injectable compositions may contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.).

[0112] For intravenous injection, water-soluble antibodies can be administered via infusion, thereby infusing a pharmaceutical preparation containing antibodies and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% glucose, 0.9% saline, Ringer's solution, or other suitable excipients.

[0113] Intramuscular preparations (e.g., sterile preparations in the form of suitable soluble salts of antibodies) can be dissolved and administered in pharmaceutical excipients such as water for injection, 0.9% saline, or 5% glucose solution.

[0114] In one embodiment, the antibody is administered via a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable reservoir sources or local delivery catheters for antibodies (e.g., infusion catheters, indwelling catheters or needle catheters, synthetic grafts, outer membrane wrappings, shunts and stents or other implantable devices); site-specific carriers; direct injection or direct application. See, for example, WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0115] In another embodiment of this disclosure, an article of manufacture is provided containing any pharmaceutical composition and formulation described herein (e.g., including antibodies or conjugates), and is provided with instructions for its use and / or reconstitution. The article of manufacture includes a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., dual-chamber syringes), and test tubes. Containers can be formed from a variety of materials such as glass or plastic. The formulation and label on or associated with the container may indicate the direction of 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 multipurpose vial that allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. The article of manufacture may further include a second container containing a suitable diluent (e.g., BWFI). After mixing the diluent and the lyophilized formulation, the final protein concentration in the reconstituted formulation will typically be at least 50 mg / mL. The product may further include other materials that are commercially available and deemed necessary from the user's perspective, including other buffers, diluents, screening procedures, needles, syringes, and packaging inserts with instructions for use.

[0116] [Example]

[0117] The following examples are not limiting but merely exemplary. Unless otherwise stated, all cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA), and all secondary antibodies used for assays were purchased from the Jackson Institute for Immunology (West Grove, PA). DNA and RNA were manipulated using the following standard method: Sambrook et al., Molecular Selection: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Unless otherwise stated, all commercial reagents and kits were used according to the manufacturer's instructions.

[0118] To explore target-specific antibodies for cancer treatment, we used live-cell immunotherapy (LCI) and live-cell high-throughput screening (HTS) technologies to generate antibodies that specifically target human tumor surface antigens, and then used a combination of proteomics and molecular biology approaches to identify the targets.

[0119] [, Example , ] [, 1 , ]

[0120] [, Specific targeting , ] [, AML , ] [, cell lines , ] [, MAb11-22.1 mAb , ] [, The generation and characterization , ] [, , ]

[0121] To obtain mAbs targeting conformational epitopes of cell surface antigens, as previously described, a mixture of two human AML cell lines, MV4-11 and THP-1, in phosphate-buffered saline (PBS) was used for LCI and LC-HTS (see: WO 2014146487A1, WO 2017114204, US20210139602A1; Li et al., PLoS One 2013, 8:e77398). The interaction between hybridoma culture supernatant and a mixture of two live AML cell lines was screened using a BD FACSCalibur™ flow cytometer and a high-throughput sampler (HTS) via fluorescence activated cell sorting (FACS). Re-selection was performed using human peripheral blood mononuclear cells (PBMCs) donated by healthy adults (Stanford Blood Center, Palo Alto, California), and data were analyzed using FlowJo™ software (Becton Dickinson, San Jose, CA). Hybridoma lines exhibiting strong specific binding activity to human AML cell lines but no binding activity to human PBMCs were selected for amplification, removed from conditioned medium, and subselected according to standard protocols (Kohler and Milstein, Nature 1975, 256:495-497; Winter and Milstein, Nature 1991, 349:293-299). Monoclonal antibodies (mAbs) produced from these hybridoma strains were purified using MabSelect™ SuRe™ LX Protein A resin (GE Healthcare, Marlborough, MA, GE Healthcare, Marlborough, Massachusetts) for further characterization.

[0122] One mAb, named MAb11-22.1 mAb, showed high binding capacity to almost all nine tested human AML cell lines, but no binding capacity to human PBMCs (Figure 1A, left panel). The binding signal was particularly high with NB4 cells (AML M3 subtype cell line), followed by the Kasumi-1 and HL-60 cell lines. Human TruStain FcX™ Fc receptor blocking solution (Biolegend, San Diego, CA) was used in the FACS assay to ensure that binding was not mediated by the interaction between the mAb on the AML cell surface and the Fc receptor (FcR). Since the isotype of MAb11-22.1 mAb was determined using the IsoStrip™ Mouse Monoclonal Antibody Genotyping Kit (Roche Molecular Systems, Pleasanton, CA), an irrelevant mouse IgG1a / κ antibody that does not interact with AML cell lines was used as a negative control (isotype control antibody, Figure 1A, right panel) to confirm that the interaction between MAb11-22.1 mAb and the surface of AML cells is specifically mediated by the variable region of MAb11-22.1 mAb.

[0123] To observe the binding of MAb11-22.1 mAb to the surface of AML cells, an immunocytochemical (ICC)-based assay of cell surface antibody binding was performed. Cultured AML cells were fixed onto slides in 4% paraformaldehyde using a centrifuge smear machine (Thermo Fisher Scientific, Waltham, MA). After antigen retrieval via heating, cells were blocked with goat serum (MilliporeSigma, St. Louis, MO) and incubated for one hour with either MAb11-22.1 mAb or an isotype control antibody, followed by one hour incubation with a 1:1000 dilution of peroxidase-labeled AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3), Fcγ fragment specific (anti-Mo IgG Fc-HRP pAb, Jackson Immunological Research Laboratory). Cells were stained with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. As shown in Figure 1B, ICC assays confirmed that the target localization of MAb11-22.1 mAb was 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, consistent with the mean fluorescence intensity (MFI) in the FACS assay (Figure 1A, left panel).

[0124] Furthermore, Western blot analysis of multiple AML cell lysates was performed simultaneously using 5 µg / mL MAb11-22.1 mAb and mouse anti-human / mouse β-actin mAb (anti-β-actin mAb, Proteintech Group, Rosemont, IL), followed by detection with a 1:20,000 dilution of anti-Mo IgG Fc-HRP pAb, showing a common band of approximately 250 kDa under non-reducing conditions and a faint band of approximately 110 kDa under reducing conditions (Fig. 1C). Whole-cell lysates of Raji (an acute lymphoblastic leukemia (ALL) cell line) also showed blots at the same locations, albeit with lower intensity. In contrast, no bands were detected in Chinese hamster ovary (CHO) cell lysates (Fig. 1C) or human PBMC cell lysates (data not shown) using the Western blot method. The relative intensities of these hybrid protein bands in AML cells were consistent with the intensities of MFI (Fig. 1A) and ICC staining in FACS assays (Fig. 1B), suggesting that this protein may be a target of MAb11-22.1 mAb.

[0125] [, Example , ] [, 2 , ]

[0126] [, human transferrin receptor , ] [, 1 , ] [, ( , ] [, TfR1 , ] [, As , ] [, MAb11-22.1 mAb , ] [, The target , ] [, Identification and verification , ] [, , ]

[0127] AML cell lysates were prepared using IP lysis buffer (Thermo Fisher Scientific) according to the manufacturer's instructions. Dynabeads™ protein A (Thermo Fisher Scientific) for immunoprecipitation was incubated with 50 µg MAb11-22.1 mAb or isotype control antibody in PBS at room temperature (RT) for 30 min. The antibody-bound Dynabeads™ were washed and each cell lysate was incubated at RT for 30 min, followed by washing with 0.5% Triton X-100 / PBS and PBS buffer, respectively. Immunoprecipitated (IP) proteins and antibodies were separated from the magnetic beads using 4 × Laemmli denaturing sample buffer containing 2-thiol ethanol and heated to 95°C for 5 min. Proteins were separated by SDS-PAGE and stained using the ProteoSilver™ silver staining kit (Millibor Sigma) according to the manufacturer's instructions. Silver staining revealed a single protein band in the IP sample with the same molecular weight as that detected by the Western ink dot method (Figure 2A). The band was excised from the gel and digested with trypsin. TfR1 was identified as the most abundant protein in the gel section using liquid chromatography-tandem mass spectrometry (LC-MS / MS) services provided by BGI Americas (Cambridge, Massachusetts) (#PSM = 32, coverage = 29.6%).

[0128] To confirm that TfR1 is the target of MAb11-22.1 mAb, the extracellular domain of recombinant human TfR1 with an N-terminal His tag (rTfR1-ECD, ACRO Biosystems, Newark, DE) was analyzed by SDS-PAGE under non-reducing conditions, along with whole-cell lysates of AML and ALL cells (positive control) and CHO cell lysates (negative control). Subsequently, Western ink dot assay was performed simultaneously using 5 µg / mL MAb11-22.1 mAb and anti-β-actin mAb as primary antibodies and a 1:20,000 diluted anti-Mo IgG Fc-HRP pAb as secondary antibodies (Figure 2B). The results showed that MAb11-22.1 mAb could be detected as a single band of approximately 80 kDa, which is close to the calculated molecular weight of rTfR1-ECD (77.0 kDa). As shown in Figure 1C, Raji, NB4, OCI / AML2, and CHO cell lysates showed consistent results in the reaction with MAb11-22.1 mAb.

[0129] The interaction between MAb11-22.1 mAb and rTfR1-ECD was validated using a direct ELISA assay. MAb11-22.1 mAb, 6*His His-Tag monoclonal antibody (anti-His mAb, Protein Technologies), or isotype control antibody were reacted directly with a 0.1 µg / mL rTfR1-ECD-coated Immulon® microtiter plate (Thermo Fisher Scientific) in serially diluted ELISA buffer at RT for one hour, followed by reaction with a 1:2,000 diluted anti-Mo IgG Fc-HRP pAb for one hour. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added, and absorbance was measured at 450 nm using a SpectraMax microplate reader (Molecular Device, San Jose, CA). Figure 2C shows the direct interaction between rTfR1-ECD with the 6His tag and MAb11-22.1 mAb or anti-His mAb, but not with the isotype control antibody, demonstrating that TfR1 is a target of MAb11-22.1 mAb.

[0130] To further confirm that MAb11-22.1 mAb recognizes human TfR1, a pair of primers (SEQ ID NO: 11 and SEQ ID NO: 12) ordered from Integrated DNA Technologies, Inc. (Clarville, Iowa) were used to amplify TFRC cDNA encoding the full-length TfR1 protein from OCI / AML2 and THP-1 cells via reverse transcription-polymerase chain reaction (RT-PCR). The cDNA was fused within a frame to a GFP-encoded DNA fragment and then selected into an internal expression vector containing a human CMV promoter and a puromycin resistance gene (PuroR) for selection. CHO-DG44 cells unstained with MAb11-22.1 mAb in FACS were transfected with TFRC-GFP expression plastids and grown in puromycin-supplemented EX-CELL® CD CHO serum-free medium (SFM). Stable colonies of CHO cells with high EGFP expression levels were isolated and subselected. 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, MN), and isotype control antibody. They were then detected 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 Institute for Immunology). The results showed that, under FL1(GFP) gating, CHO / rTfR1-GFP cells could be stained by both MAb11-22.1 mAb and the anti-TfR1 control mAb; however, neither antibody bound to CHO cells that did not express rTfR-GFP (Figure 2D). The IC50 value of MAb11-22.1 mAb was 0.03-0.04 µg / mL, significantly lower than that of the anti-TfR1 control mAb (approximately 0.1 µg / mL). Staining of CHO / rTfR-GFP cells with various GFP activities using MAb11-22.1 mAb revealed a positive correlation between MFI-PE and MFI-FITC values ​​(data not shown), demonstrating a direct interaction between MAb11-22.1 mAb and the extracellular domain of human TfR1.

[0131] Forward lead: 5'-GAATGATGGATCAAGCTAGATCAGC-3' (SEQ ID NO: 11)

[0132] Reverse primer: 5'-CTCATGGAAGCTATGGGTATCAC-3' (SEQ ID NO: 12)

[0133] [, Example , ] [, 3 , ]

[0134] [, MAb11-22.1 mAb , ] [, and , ] [, rTfR1-ECD , ] [, Measurement of binding affinity between , ] [, , ]

[0135] Label-free biolayer interferometry (BLI) was used to determine the binding affinity between MAb11-22.1 mAb and the extracellular domain of TfR1 using the Octet® QK system (McGol Molecular Instruments, San Jose, California). MAb11-22.1 mAb was immobilized onto an anti-mouse IgG Fc capture (AMC) biosensor for 450 seconds, washed with kinetic buffer for 120 seconds, and then bound to rTfR1-ECD in serial dilutions for 450 seconds, followed by dissociation in kinetic buffer. Goat anti-mouse (H+L) Fab (Jackson Institute for Immunology) was used as a positive control for binding to MAb11-22.1 mAb. Multiple measurements were repeated using various concentrations of rTfR1-ECD, from 300 nM to 0.08 nM, through serial dilutions; however, no signal attenuation was observed even with extended dissociation times up to 60 minutes (Figure 3A, left) or reaction temperatures increased to 37°C (data not shown). Therefore, the interaction affinity between MAb11-22.1 mAb and rTfR1-ECD is very high, with a calculated KD value < 1 pM (Figure 3B). The binding between MAb11-22.1 mAb and anti-mouse (H+L)Fab exhibits a regular kinetic curve (Figure 3A, right panel) and a fairly high affinity (KD = 1.04-1.45 nM) (Figure 3C), indicating that the extremely high affinity between MAb11-22.1 mAb and rTfR1-ECD is not artificial.

[0136] [, Example , ] [, 4 , ]

[0137] [, MAb11-22.1 mAb , ] [, and transferrin and human , ] [, TfR1 , ] [, Combination competition , ] [, ELISA , ]

[0138] To determine whether the binding of MAb11-22.1 mAb to human TfR1 overlaps with the binding site of Tf, a competitive ELISA assay was performed. Following the manufacturer's instructions, MAb11-22.1 mAb was directly labeled with HRP using the EZ-Link maleimine-activated HRP kit (Thermo Fisher Scientific). Immunlon microtiter plates coated with 0.1 µg / mL rTfR1-ECD were incubated for one hour with a mixture of human Tf (Millibosigma) serially diluted 2.5 times starting at 2 µg / mL and HRP-conjugated MAb11-22.1 mAb serially diluted 4 times starting at 2 µg / mL. Various concentrations of Tf were added separately as negative controls. After thorough washing, TMB was added to detect the binding of HRP-conjugated MAb11-22.1 mAb. After adding stop solution, absorbance was measured at 450 nm using a SpectraMax microplate reader.

[0139] Figure 4 shows the dose-dependent binding of MAb11-22.1 mAb-HRP to rTfR1-ECD at various concentrations, independent of the presence of Tf. Adding up to 2 µg / mL (25 µM) of Tf did not significantly interfere with the binding of MAb11-22.1 mAb-HRP to rTfR1-ECD, indicating that the Tf binding site does not coincide with the binding epitope of MAb11-22.1 mAb.

[0140] [, Example , ] [, 5 , ]

[0141] [, right , ] [, MAb11-22.1 mAb , ] [, and , ] [, PBMC , ] [, and bone marrow ( , ] [, BM , ] [, Cell-bound , ] [, FACS , ] [, analyze , ] [, , ]

[0142] Other researchers have shown that most anti-TfR1 antibodies interfere with Tf uptake and lead to systemic hematologic toxicity in patients, including anemia, neutropenia, and leukopenia (CANDELARIA et al., Frontiers in Immunology, March 17, 2021). To address safety concerns regarding MAb11-22.1 mAb as a therapeutic candidate, multiple batches of fresh PBMCs from healthy donors were purchased from Stanford Blood Center (Palo Alto, CA), while frozen BM cells from healthy donors or AML patients were purchased from HumanCells Bioscience (Fremont, CA). Cells were washed with PBS and blocked with ice-cold PBS and 1% bovine serum albumin (BSA). The cells were stained at 4°C with serially diluted MAb11-22.1 mAb and isotype control antibodies starting at 20 µg / mL, followed by detection with FITC or PE-conjugated goat anti-Mo IgG Fc pAb diluted 1:800 (Jackson Institute for Immunology). In addition, cells were stained simultaneously with MAb11-22.1 mAb using anti-human CD34-FITC (REAL487), anti-human CD233-PE (REA368), and anti-human CD235a-PE (REA1092) purchased from Miltenyi Biotechnology (San Jose, California).

[0143] FACS results showed that MAb11-22.1 mAb did not bind to normal PBMCs or normal BM cells regardless of the mAb concentration used (Fig. 5A). Consistent results were obtained using different batches (donor) of PBMCs and BM cells (data not shown). In contrast, BM cells from AML patients were reactive to MAb11-22.1 mAb but not to the isotype control antibody (Fig. 5B), indicating that MAb11-22.1 mAb can distinguish between healthy BM and AML BM samples. In healthy BM samples, more than 20% of the cells were hematopoietic stem cells that could be stained with anti-human CD34 mAb-FITC (Fig. 5C, left panel), but very few cells reacted with MAb11-22.1 mAb and the isotype control antibody (Fig. 5C, middle and right panels). TfR1 is generally considered a marker of nucleated erythrocytes; however, CD233-positive or CD235a-positive erythrocytes and erythroblasts are not reactive to MAb11-22.1 mAb (Figs. 5D and 5E, left panel), suggesting that MAb11-22.1 mAb may only recognize TfR1 expressed on malignant tumor cells and has a low risk of causing myelosuppression.

[0144] [, Example , ] [, 6 , ]

[0145] [, Tumor cells against , ] [, MAb11-22.1 mAb , ] [, endocytosis experiment , ] [, , ]

[0146] OCI / AML2, NB4, or Raji cells grown in DMEM medium containing 10% Gibco fetal bovine serum (FBS, Thermo Fisher Scientific) were seeded at 1 × 10⁵ cells / well in two 96-well plates and incubated at 4°C for one hour with a 1:10 serial dilution (starting at 10 µg / mL) of MAb11-22.1 mAb, anti-CD20 mAb (R&D Systems), or isotype control mAb. After centrifugation at 500 × g, the cells were washed twice with ice-cold PBS to remove unbound mAbs. Cells in one well were resuspended in warm medium and incubated at 37°C to allow for mAb endocytosis, while cells in the other well were resuspended in ice-cold medium and incubated at 4°C to prevent antibody endocytosis. Samples collected at 30 minutes, 1 hour, and 2 hours were washed twice with ice-cold PBS / 1% BSA, followed by incubation at 4°C for 30 minutes with a 1:800 dilution of anti-Mo IgG Fc-FITC. 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 for FACS analysis.

[0147] FACS results showed that MAb11-22.1 mAb bound to the surfaces of two AML cell lines, OCI-AML2 and NB4, in a dose-dependent manner at 4 °C, but was rapidly internalized at 37 °C. Despite the apparent very low TfR1 copy number on the Raji cells surface, they were still able to internalize MAb11-22.1 mAb at 37 °C (Fig. 6A, left panel). In contrast, anti-CD20 mAb bound only to Raji cells at 4 °C, not to AML cells, and was partially internalized by Raji cells at 37 °C (Fig. 6A, middle panel). As expected, the isotype control antibody did not stain as it did for any of the three cell lines (Fig. 6A, right panel).

[0148] FACS-based endocytosis assays only provide indirect evidence of antibody-antigen complex endocytosis and cannot distinguish between antibody endocytosis and antibody shedding. To directly observe mAb endocytosis, fluorescence microscopy-based endocytosis analysis was performed using fluorescently labeled mAbs. In short, mAbs were labeled with CF488 dye using the Mix-n-Stain™ antibody labeling kit (Millibo Sigma) according to the manufacturer's experimental protocol. Cell binding assays were performed in 96-well plates with ice-cold DMEM medium and different concentrations of mAbs, reacting at 4°C for one hour. After washing cells twice with ice-cold PBS, one plate was added to warm DMEM and incubated at 37°C for another hour, while cells in the other plate were resuspended in cold PBS. Fluorescence imaging was performed using a Keyence BZ-X800 integrated fluorescence microscope (Keyence, Itasca, IL). A CF488-labeled isotype control antibody was incubated in parallel with the cells as a negative control.

[0149] After incubation at 4°C for one hour, MAb11-22.1 mAb-CF488 was observed to bind to the surface of OCI / AML2 cells, forming a high-intensity fluorescent ring outlining the cell contour (Fig. 6B, top). The fluorescence intensity was directly proportional to the concentration of the mAb used. After incubation at 37°C for one hour, most of the fluorescence signal shifted from the cell surface to the cell interior, indicating that most of the surface-bound MAb11-22.1 mAb-CF488 molecules were internalized by OCI / AML2 cells (Fig. 6B, bottom). This phenomenon was also observed in other AML cell lines, including NB4 and THP-1, using different concentrations (1, 0.5, 0.1, and 0.05 µg / mL) of MAb11-22.1 mAb-CF488 (data not shown). The CF488-labeled isotype control antibody did not bind to any of the tested cell lines (Fig. 6B, right).

[0150] Similar dose-dependent binding and endocytosis results were observed in MDA-MB-231 cells (a triple-negative breast cancer (TNBC) cell line), as revealed by FACS analysis, where these cells responded strongly to MAb11-22.1 mAb at 4 °C. However, as expected, the fluorescence signal on MDA-MB-231 cells was much stronger than that in AML cells (Fig. 6C). Furthermore, the MAb11-22.1 mAb-CF488 molecule underwent partial endocytosis even at 4 °C, and after one hour of incubation at 37 °C, most of the molecule was endocytosed, indicating that the TfR1 protein expressed on the MDA-MB-231 cell membrane has a higher endocytic tendency when binding to MAb11-22.1 mAb.

[0151] [, Example , ] [, 7 , ]

[0152] [, MAb11-22.1 mAb , ] [, right , ] [, AML , ] [, In vitro effects of cell line proliferation , ] [, , ]

[0153] The effect of MAb11-22.1 mAb on the proliferation of AML cell lines was assessed using cell-based in vitro assays. Cell viability was determined using the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Rockville, MD) according to the manufacturer's protocol. Briefly, AML cells in DMEM / 10% FBS medium supplemented with 100 µg / mL MAb11-22.1 mAb or isotype control antibody were seeded in triplicate at 5 × 10³ cells per well in seven 96-well cell culture plates and incubated at 37ºC in a 5% CO₂ incubator. 10 µL / well of CCK-8 solution was added every 24 hours and incubated for four hours, followed by measurement of OD₄50 as cell viability.

[0154] As shown in Figure 7, OCI / AML2 cells in both groups continued to grow, but the proliferation rate of cells treated with MAb11-22.1 mAb was significantly lower than that of cells treated with the isotype control antibody (P < 0.05). Similar data were obtained with other AML cell lines (data not shown). Therefore, although MAb11-22.1 mAb can partially inhibit the proliferation of OCI / AML2 cells, the mAb itself is not a potent therapeutic candidate. In fact, in preliminary in vivo studies involving a CLDX mouse model, MAb11-22.1 mAb did not show any effect. Due to its extremely high affinity for human TfR1 and its strong tendency to be internalized by cancer cells, MAb11-22.1 mAb has great potential for development into an ADC for cancer therapy.

[0155] [ , Example , ] [ , 8 , ]

[0156] [ , right , ] [ , MAb11-22.1 , ] [ , Hybridoma , ] [ , variable region , ] [ , cDNA , ] [ , sequencing , ] [ , , ]

[0157] Using the SMARTer RACE 5' / 3' kit (Takara Bio USA, San Jose, CA), cDNA encoding the light chain (VL) and heavy chain (VH) variable regions of MAb11-22.1 mAb was amplified from total RNA of MAb11-22.1 hybridoma cells via 5'-RACE technology. The primers for 5'-RACE are shown in SEQ ID NO: 13 and SEQ ID NO: 14. The cDNA fragments were amplified with Q5® high-fidelity DNA polymerase and selected into the pMiniT2.0 vector (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. Ten *E. coli* colonies from each transformant were randomly selected for plastid purification using a high-speed microplastics kit (IBI Scientific, Dubuque, Iowa) and Sanger DNA sequencing (Genewiz, South San Francisco, CA). The uniqueness of the V domain was verified by BLAST search at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi and by the international ImMunoGeneTics® (IMGT) information system (http: / / www.imgt.org). The identical sequences of VL and VH for MAb11-22.1 mAb are shown in the Summary of the Invention section as SEQ ID NO: 1 and SEQ ID NO: 3, respectively. The encoded amino acid sequences are shown as SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0158] Light chain initiator: 5'-CTGCTCACTGGATGGTGGGAAGATGG-3' (SEQ ID NO: 13)

[0159] Heavy chain primer: 5'-AGCTGGGAAGGTGTGCACAC-3' (SEQ ID NO: 14)

[0160] [, Inlay , ] [, MAb11-22.1 Ab , ] [,( , ] [, cAb , ] [, Construction and representation of ) , ] [, , ]

[0161] Using primer pairs shown in SEQ ID NO: 15 to 18, the VL and VH genes of MAb11-22.1 mAb were amplified by PCR from sequenced plasmids. The PCR fragments were digested with SpeI-NarI (VL) or XbaI-NheI (VH) restriction enzymes (New England Biolabs) and ligated in-frame to constant regions of the human κ light chain (CL) or human IgG1 heavy chain (CH1-CH3), respectively, carried in internal expression vectors containing mouse CMV promoters and human CMV IE1 promoters, to control the expression of the light and heavy chains, respectively.

[0162] LV PCR forward primer: 5'-AGCACTAGTGCCGCCACCATGGAATCACAGACTCAGG-3' (SEQ ID NO: 15)

[0163] LV PCR reverse primer: 5'-CTGGGCGCCGCTACAGTCCGTTTCAGCTCCAGCTTGG-3' (SEQ ID NO: 16)

[0164] HV PCR forward primer: 5'-AGCTCTAGAGCCGCCACCATGGAGACAGACACACTCCTG-3' (SEQ ID NO: 17)

[0165] HC PCR reverse primer: 5'-GCCTTTGGTGCTAGCAGAGACAGTGACCAGAGTC-3' (SEQ ID NO: 18)

[0166] Following confirmation via DNA sequencing, the epiploids were transfected into Dux-1S-HD cells, an internally developed CHO-DG44 derivative capable of high-density growth in EX-CELL® CD CHO SFM, using FectoPro transfection reagent (Polyplus-transfection® SA from Ilkhich, France), according to the manufacturer's instructions. Binding to AML cell lines was analyzed by FACS, followed by affinity purification using MabSelect™ SuRe™ Protein A resin (GE Healthcare), validating the transient expression and secretion of MAb11-22.1 cAb in EX-CELL® CD CHO SFM.

[0167] [, Example , ] [, 9 , ]

[0168] [, MAb11-22.1 cAb , ] [, The representation , ] [, , ]

[0169] 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 using the Octet® QK system. An anti-human IgG Fc capture (AHC) biosensor was used to immobilize MAb11-22.1 cAb for 450 seconds, and a goat anti-human (H+L) Fab (Jackson Institute for Immunology) was used as a system control for binding to MAb11-22.1 cAb. Through 2.5-fold serial dilutions, rTfR1-ECD concentrations ranged from 50 nM to 1.28 nM. Similar to MAb11-22.1 mAb shown in Figures 3A to 3C, no dissociation (K off) of MAb11-22.1 cAb was detected during the 60-minute observation period. Therefore, the affinity between MAb11-22.1 cAb and rTfR1-ECD is extremely high (KD < 1 pM, Figure 8A). The binding between MAb11-22.1 cAb and anti-human (H+L)Fab exhibits a regular kinetic curve, with a KD of 2.18 nM as expected (Figure 8B).

[0170] [ , Example , ] [ , 10 , ]

[0171] [ , MAb11-22.1-S239C-DM1 , ] [ , In vitro effects on cancer cell lines , ] [ , , ]

[0172] Although MAb11-22.1 mAb does not exhibit strong cytotoxic activity, its extremely high affinity for human TfR1 and high tumor specificity make it a promising ADC candidate for cancer treatment. To investigate its potential use as an ADC drug, cysteine ​​scanning was performed on the surface-exposed residues of the constant regions of the light and heavy chains of MAb11-22.1 cAb to explore site-specific conjugation antitumor activity across various payloads. Site-directed mutagenesis was performed using the primer pairs shown in SEQ ID NO: 19 and 20 below, replacing the serine codon 239 (TCC) with the cysteine ​​codon (TGC) in the Fc region of the heavy chain of MAb11-22.1 cAb. The transiently expressed MAb11-22.1-S293C cAb variant was purified using MabSelect™ SuRe™ Protein A resin. As described by JUNUTULA et al. (Nature Biotechnol. 26:925-932, 2008), cysteine ​​or glutathione bound to the MAb11-22.1-S293C cAb was removed from the culture medium by treating it with 10 moles of dithiothreitol (DTT) in PBS at room temperature. The antibody was refolded to expose the free thiol groups of S239C, and the integrity of the cAb was analyzed by SDS-PAGE. Site-specific binding was achieved by reacting Cys239 and the free thiol groups of DM1 in PBS with slow stirring at room temperature for 10 to 60 minutes to produce MAb11-22.1-S293C-DM1, which confers ADC activity with a theoretical drug-to-antibody ratio (DAR) of 2. Free DM1 was removed three times by percolation with ten times the volume of PBS using an Amicon® stirred tank reservoir equipped with a 30-kDa NMW ultrafiltration membrane (Millibo Sigma). MAb11-22.1-S293C-DM1 was then sterilized by filtration using a 0.2 µm syringe filter (VWR International, Radno, PA). DM1 was also used in parallel to treat unmutated MAb11-22.1 cAb and isotype control antibodies as negative controls to exclude effects caused by non-site-specific conjugation. The integrity and binding capacity of the DM1-treated antibodies were analyzed using SDS-PAGE, ELISA, and FACS. Endotoxin was analyzed using the Pierce™ Chromogenic Endotoxin Quantification Kit (Thermo Fisher Scientific). No significant difference in binding affinity for TfR1 and cancer cell lines was observed between MAb11-22.1 cAb-DM1 and MAb11-22.1-S239C-DM1. As expected, MAb11-22.1-S239C-DM1 showed a slight delay in intragel migration compared to untreated MAb11-22.1-S239C.

[0173] S239C forward lead: 5'-AACTCCTGGGTGGACCT [TGC]GTTGTTTCTGTTCCCCCCTAAGC-3'(SEQ ID NO: 19)

[0174] S239C reverse lead: 5'-AACAC [GCA]AGGTCCACCCAGGAGTTCAGGAGCAGGGCAAGG-3'(SEQ ID NO: 20)

[0175] Representative cancer cell lines, including OCI / AML2 (AML subtype M4), Raji (ALL), HCC38 (TNBC), and MDA-MB-231 (TNBC), were cultured in DMEM / 10% FBS medium to the logarithmic growth phase. On day 0, optimized amounts of each cell line were tripletly mixed with a single antibody in 96-well cell culture plates to achieve a baseline OD 450 of approximately 0.4 in a CCK-8 assay. Cells were incubated at 37°C in a 5% CO2 cell culture incubator. Cell proliferation was monitored using CCK-8 assays on days 3 and 5. An isotype control antibody not treated with DM1 was used to reflect baseline cell proliferation. This study compared MAb11-22.1-S239C and MAb11-22.1 cAb with and without DM1 treatment.

[0176] 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 significant 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. Both TNBC cell lines were less sensitive to MAb11-22.1-S239C-DM1, as a concentration of 100 µg / mL only had a partial inhibitory effect.

[0177] [, Example , ] [, 11 , ]

[0178] [, use , ] [, AML , ] [, Evaluation of xenotransplantation mouse models , ] [, MAb11-22.1-S239C-DM1 , ] [, its effects in the body , ] [, , ]

[0179] A xenograft model was established using nu / nu mice to evaluate the effect of MAb11-22.1-S239C-DM1 on AML tumor growth. 1.5 × 10⁶ OCI / AML2 cells dissolved in 0.1 ml PBS were injected subcutaneously into the right back of 6-week-old male athymic nude mice (Charles River Laboratories, Wilmington, MA). When all tumor nodules were visible, mice were randomly assigned to three groups (n = 5 or 6) for intraperitoneal (ip) injection of MAb11-22.1-S239C-DM1 (low dose 10 mg / kg b.wt, high dose 20 mg / kg body weight (b.wt)) or an isotype control antibody (without S239C mutation) treated with DM1 (20 mg / kg b.wt), once every four days for seven times (Q4 × 7). Tumor volume and body weight were measured every four days before administration. Tumor volume was calculated using the following formula: Volume = ½ × (width)² × length. The animal was euthanized when the tumor volume exceeded 2000 mm³, or when the tumor became necrotic or ulcerated through the skin.

[0180] The OCI / AML2 xenograft is an invasive CLDX mouse model. Neither MAb11-22.1 mAb nor MAb11-22.1 cAb alone significantly slowed tumor growth in the OCI / AML2 xenograft (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 tumor disappearance and a durable response after one or two administrations (Figure 10). Mice in the high-dose group (n = 6) treated with 20 mg / kg b.wt MAb11-22.1-S239C-DM1 showed varying responses: as in the low-dose group, three mice experienced a 100% reduction in tumor volume from day 4 or 8; another mouse showed tumor regression on days 4 and 8, but the tumor subsequently regrowth; 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 were euthanized on day 20 because their tumors exceeded 2000 mm³. Despite the use of very high doses of ADC, no systemic side effects such as changes in vital signs, loss of activity, appetite, or decreased overall weight were observed during the study.

[0181] Compared with the isotype control group, MAb11-22.1-S239C-DM1 showed a significant effect on tumor growth, confirming that MAb11-22.1 cAb is a promising candidate for an ADC (anti-cancer drug) for cancer treatment. Due to its high affinity and specificity for human TfR1, looping MAb11-22.1-S239C can accumulate on TfR1-positive tumor cells and deliver DM1 intracellularly via endocytosis. In the presence of high concentrations of glutathione (GSH) in tumor cells, DM1 can be released from the ADC and confer an adjuvant killing effect on surrounding cells in xenograft tumors. This means that MAb11-22.1 ADCs can be applied not only to hematologic malignancies but also to solid tumors.

[0182] The mechanisms underlying the changes in efficacy in the high-dose group remain unclear. This may be due to the small sample size and individual variability in response to compounds caused by varying levels of TfR1 expression on the tumor cell surface. Sometimes, high-dose ADCs can be rapidly cleared or induce resistance; therefore, the dose window for MAb11-22.1-S239C-DM1 needs optimization. Nevertheless, based on its high tumor specificity and antitumor potency, MAb11-22.1 is an ideal anti-TfR1 antibody for ADC development. Conjugation techniques with payloads and ideal linkers for MAb11-22.1 ADCs are currently under investigation to achieve consistent DAR and an optimal dose window for cancer treatment.

[0183] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended to limit the scope of the invention. Other objects, aspects, and embodiments will be apparent to those skilled in the art upon consideration of this specification and are covered within the spirit of the invention as defined by the claims. Various substitutions and modifications to the invention disclosed herein without departing from the spirit and scope of the invention will be apparent to those skilled in the art. The invention described herein illustratively may be practiced appropriately without the absence of any one or more elements or limitations not specifically disclosed herein as necessary. Therefore, for example, in each instance herein, in embodiments or examples of the invention, any of the terms “comprising,” “including,” “containing,” “having,” and “have” should be read as synonyms and are read broadly and without limitation. The methods and processes suitably described herein may be implemented in different sequences of steps and are not necessarily limited to the sequence of steps indicated herein or in the claims.

[0184] This patent shall not be construed as limiting itself to the specific embodiments or methods disclosed herein. This patent shall not be construed as being limited by any statement made by any examiner or any other officer or employee of the Patent and Trademark Office, unless such statement is expressly adopted by the applicant in writing in response, without limitation or reservation.

[0185] The invention is described broadly and generally herein. Each of the narrower species and subgenus classifications falling within the general disclosure also forms part of the invention. Terms and expressions used are used as descriptive rather than selective terms, and their use is not intended to exclude any equivalents or portions thereof of the shown and described features, but it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed through preferred embodiments and optional features, modifications and variations of the disclosed concepts herein can be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0186] [Incorporated via reference]

[0187] All patents and patent applications cited in this article are hereby incorporated by way of incorporation, as are all other references cited.

[0188] [, The patents mentioned: , ] [, , ] Overall in the abstract: 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 U.S. Patent 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 EP 239 400 B1 WO 00 / 53211 and U.S. Patent No. 5,981,568 WO 2014146487A1 U.S. Publication No. 20170151343 U.S. Publication No. 20170335281 WO 2017114204 U.S. Publication No. 20210139602 A1 Citations of non-patent literature cited in the specification are listed only by author name: Bomford AB and Munro HN. Hepatology (1985) 5:870-875. Brooks D, Taylor C, Dos Santos B, Linden H, Houghton A, Hecht TT. Clinical Cancer Research (1995) 1:1259-1265. Callens, C, Moura IC, Lepelletier Y, Coulon S, Renand A, Dussiot M, Ghez D, Benhamou M, Monteiro RC, Bazarbachi A, and Hermine O. Leukemia (2008) 22:42-48. Candelaria PV, Leoh LS, Penichet ML, and Daniels-Wells TR. Frontiers in Immunology, March 17, 2021. doi.org / 10.3389 / fimmu.2021.607692 Daniels TR, Delgado T, Rodriguez JA, Helguera G, and Penichet ML. Clinical Immunology (2006) 121:144-158. Daniels TR, Ortiz-Sanchez E, Luria-Perez R, Quintero R, Helgora G, Bonavida B, Martínez-Maza O, and Penichet ML. Journal of Immunotherapy (2011) 34:500-508. Daniels-Wells TR, Candelaria PV, Emiko K, Wen J, Weng L, Kamata M, Almagro JC, Martínez-Maza O and Penichet ML. Cancer Research (2020) 80(16 Supplement):5655. Daniels-Wells TR, Widney DP, Leoh LS, Martinez-Maza O, Penichet ML. Journal of Immunotherapy (2015) 38:307-310. Faulk WP, Hsi BL, Stevens PJ. The Lancet (1980) 2:390-392. Feng R, Wang Y, Ramachandran V, Ma Q, May MM, Li M, Zhou JX, Xu X, Xu K, Fang S, Xia W, Sui D, Liu H, Gao X, Prieto V, Blacklow SC, Lu M, Lee JE. Journal of Experimental and Clinical Cancer Research (J Exp Clin Cancer Res.) (2020) 39:273. Gatter KC, Brown G, Trowbridge IS, Woolston RE, Mason DY. Journal of Clinical Pathology (1983) 36:539-545. Jeong SM, Hwang S, Seong RH. Biochemistry and Biophysics Research Communications (2016) 471:373-379. Johnson M, El-Khoueiry A, Hafez N, Lakhani N, Mamdani H, Rodon J, Sanborn RE, Garcia-Corbacho J, Boni V, Stroh M, Hannah AL, Wang S, Castro H, Spira A. Clinical Cancer Research (2021) 27:4521-4530. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J. Katta V. Kim A. McDorman K. Flagella K. Venook R. Ross S. Spencer SD. Lee Wong W. Lowman HB. Environmental Medicine (2008) 26:925-932. Kohler&Milstein. Chemistry 1975;256:495-497. Lepelletier Y, Camara-Clayette V, Jin H, Hermant A, Coulon S, Dussiot M, Arcos-Fajardo M, Baude C, Canionni D, Delarue R, Brousse N, Benaroch P, Benhamou M, Ribrag V, Monteiro RC, Moura IC, Hermine O. Environmental Medicine (2007) 67:1145–1154. Li M, Gao J, Feng R, Wang Y, Chen X, Sun J, Zhang D, Zhu Z, Ellis LM, Lu M, Lee JE, Feng Z, Liu B. 2013;8:e77398 Moura IC, Centelles MN, Arcos-Fajardo M, Malheiros DM, Collawn JF, Cooper MD, Monteiro RC. International Journal of Veterinary Medicine (2001) 94:417-425. Moura, IC;Y. Lepelletier、B. Arnulf、P. England、C. Baude, C. Chemistry (2004) 103:1838-1845. Nagai K, Nakahata S, Shimosaki S, Tamura T, Kondo Y, Baba T, Taki T, Taniwaki M, Kurosawa G, Sudo Y, Okada S, Sakoda S, Morishita K. Cancer Medicine (2014) 3:1085-1099. Neiveyans M, Melhem R, Arnoult C, Bourquard T, Jarlier M, Busson M, Laroche A, Cerutti M, Pugnière M, Ternant D, Gaborit N, Chardès T, Poupon A, Gouilleux-Gruart V, Pèlegrin A, Poul MA. MAbs. (2019) 11:593-605. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nature Nanotechnology (2007) 2:751-760. Qian Z, LI H, SUN H, HO K. Pharmacology Review (2002) 54:561-587. Richardson DR, Kalinowski DS, Lau S, Jansson PJ, Lovejoy DB. Acta Biochimica & Biophysics - General Subjects (2009) 1790:702-717. Shimosaki S, Nakahata S, Ichikawa T, Kitanaka A, Kameda T, Hidaka T, Kubuki Y, Kurosawa G, Zhang L, Sudo Y, Shimoda K, Morishita K. Biochemical and Biophysical Research Communications (2017) 485:144-151. Sutherland R, Delia D, Schneider C, Newman R, Kemshead J, Greaves M. Proceedings of the National Academy of Sciences (1981) 78:4515-4519. Trowbridge IS and Lopez F. Proceedings of the National Academy of Sciences (1982) 79:1175-1179. Ward JH. Investigative Radiology (1987) 22:74-83. Winter and Milstein. Nature 1991;349:293-299. Zhang L, Nomura F, Aikawa Y, Kurosawa Y, Morishita K, Sudo Y. Cancer Research (2017) 77(13 Supplement):5586.

[0189] TW202342104A_111146642_SEQL.xml

Claims

1. A binding agent targeting TfR1, the binding agent comprising a light chain variable region and a heavy chain variable region, wherein CDR1 to CDR3 of the light chain variable region have sequences identical to those of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and CDR1 to CDR3 of the heavy chain variable region have sequences identical to those of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

2. The TfR1-targeting binder according to claim 1, having a light chain variable region having at least 70% of the same sequence as SEQ ID NO:

2.

3. The TfR1-targeting binder according to claim 1, having a light chain variable region having the same sequence as SEQ ID NO:

2.

4. The TfR1-targeting binder according to claim 1, having a heavy chain variable region having at least 70% of the same sequence as SEQ ID NO:

4.

5. The TfR1-targeting binder according to claim 1, having a heavy chain variable region having the same sequence as SEQ ID NO:

4.

6. An isolated DNA molecule encoding a light chain variable region and a heavy chain variable region of the binding agent as described in claim 1.

7. An isolated RNA molecule encoding a light chain variable region and a heavy chain variable region of a binding agent as described in claim 1.

8. An isolated DNA molecule having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO:

3.

9. The binding agent according to claim 1, wherein the binding agent is a monoclonal antibody.

10. The binding agent according to claim 9, wherein the monoclonal antibody is a mouse, human, humanized, chimeric, bispecific, or multispecific antibody.

11. The binding agent according to claim 9, wherein the binding agent is a bifunctional antibody, a single-domain antigen-binding (SDAB) molecule, a VH or VL domain or a VHH domain.

12. The binding agent according to claim 9, wherein the monoclonal antibody has an IgG1 heavy chain and a κ light chain.

13. The binding agent according to claim 1, wherein the binding agent is a Fab, Fab', F(ab')2, rIgG, Fv, or Fd fragment.

14. The binder according to claim 1, wherein the binder is scFv or sc(Fv) 2.

15. The binding agent according to claim 9, wherein the monoclonal antibody is of the following class: IgD, IgE, IgG, IgA or IgM or a subclass of one of the classes.

16. The binding agent according to claim 15, wherein the monoclonal antibody subclass is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

17. The binding agent according to claim 15, wherein the monoclonal antibody has a κ light chain.

18. A vector comprising the isolated DNA molecule as described in claim 8.

19. The binder according to claim 1, wherein the binder is a binder that targets TfR1 and is conjugated with an antitumor agent.

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