Methods and compositions for targeting TREG cells

Abstract

To provide methods and compositions for targeting TREG cells.SOLUTION: The antibody NEO-201 is shown to bind to Treg cells, and its use in targeting Treg cells is described. NEO-201 may be used for isolation, detection, or purification of active Treg cells and also to kill Treg cells. Therapeutic methods and combination therapies using NEO-201 optionally in combination with another agent are described.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 630,084, filed on 13 February 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence listing information As part of its disclosure, this application includes a biological sequence listing in a file named "43282o4613.txt" with a size of 32,546 bytes, created on 13 February 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0003] CD4 + CD25 高Increased levels of regulatory T cells (Tregs) are associated with hematological malignancies (Beyer M et al. Blood 2005, 106, 2018, Motta M et al. Leukemia 2005, 19:1788, Yang ZZ et al. Blood, 2006, 107:3639), as well as non-small cell lung cancer (Woo et al. Cancer Res 2001, 61:4766), malignant melanoma (Javia et al. J Immunother. 2003, 26:85), gastrointestinal malignancies (Sasada et al. Cancer, 2003:98:1089), ovarian cancer (Curiel JT et al. Nat Med 2004, 10:942), and squamous cell carcinoma of the head and neck (Schaefer et al. Br.J. Cancer). This has been reported in patients with hepatocellular carcinoma (Ormandy LA et al. Cancer Res, 2005:2457), breast cancer (Liyanage UY et al. J Immunol, 2002, 169:2756), pancreatic cancer (Liyanage UY et al. J Immunol, 2002, 169:2756), mesothelioma (Delong P, et al. Cancer Bio Ther 2006, 4:342), metastatic renal cell carcinoma (Dannull J et al. J Clin Invest, 2005, 115:3623), and prostate cancer (Vergati M et al. Cancer Immunol Immunother. 2011, 60:197). This increase is observed in both the tumor microenvironment and peripheral blood. A recent study (Miller AM, et al. J Immunol 2066, 177:7398) reported high levels of Tregs in the peripheral blood of prostate cancer patients after prostatectomy and demonstrated the immunosuppressive function of these Tregs in vitro.

[0004] Clinical studies in melanoma patients have shown that Tregs can inhibit both antigen-specific and non-specific T cell responses (Mukhetji BJ Exp Med. 1989, 169:1961, Chakraborty NG et al. J Immunol 1990, 145:2359). In ovarian cancer patients, a direct correlation has been shown between tumor-invasive Tregs and overall survival (Curiel JT et al. Nat Med 2004, 10:942). In these patients, DAB conjugated with recombinant interleukin-2 diphtheria toxin was used. 389 Treatment with IL2 (denileukin diffitox, ONTAK) reduced Tregs and improved the antitumor response (Barnett B et al. Am J Reprod Immunol 2005, 54:369). Denileukin diffitox (DAB 389 IL-2 (ONTAK) is a fusion protein of human IL-2 and the enzymatic activity and membrane translocation domain of diphtheria toxin. It is represented by CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (γ c It preferentially binds to cells expressing high-affinity IL-2R, which consists of ). After binding to IL-2R, denileukin-diffitox is internalized by endocytosis, inhibiting protein synthesis and ultimately leading to cell death. Denileukin-diffitox has also been shown to significantly reduce the number of Tregs in the peripheral blood of patients with metastatic renal cell carcinoma and to abolish Treg-mediated immunosuppression in vivo (Dannull J et al. J Clin Invest, 2005, 115:3623).

[0005] In summary, CD4 + CD25 高Regulatory T cells reduce the effectiveness of immunotherapy protocols, and depletion of these cells may enhance vaccine-mediated antitumor immune responses and overall survival (Dannull J et al. J Clin Invest, 2005, 115:3623; Vergati M et al. Cancer Immunol Immunother. 2011, 60:197; Antony PA, et al. J Immunother 2002, 25:202).

[0006] Cancer is one of the leading causes of death worldwide, with an estimated 20 million new cases reported annually by 2025 (Ferlay et al., 2015). Traditional cancer treatments, such as surgery, radiation, and chemotherapy, often induce serious side effects and fail to cure the majority of patients with advanced disease, leading to recurrence (Bodey et al., 1996). Recent therapies have been developed to selectively target cancer cells while preserving most of the normal, healthy tissue. Among these, immunotherapy is becoming a crucial treatment option for cancer patients, revolutionizing the field of cancer treatment.

[0007] The fundamental principle of cancer immunotherapy is known as immunoediting (Mittal et al., 2014), which is an exogenous mechanism of tumor suppression that begins only after cell transformation occurs and the endogenous mechanisms of tumor suppression have failed. The immunoediting process takes place in three phases: efflux, equilibrium, and escape. In the efflux and equilibrium phases, immune rejection of cancer cells is dominant or equilibrium with cancer cell proliferation to control malignant growth. However, in the efflux phase, cancer cells that have been suppressed once may escape immune recognition due to insensitivity to immune effector mechanisms and / or induction of immunosuppression in the tumor microenvironment. Cancer cells that escape immune recognition can proliferate more freely and develop into clinically evident disease (Dunn et al., 2004). The goal of cancer immunotherapy is to keep cancer cells in the efflux and / or equilibrium phase by generating and / or amplifying an antitumor immune response to suppress tumor growth, delay tumor recurrence, and extend survival (Carter, 2001; Hodge et al., 2006; Vergati et al., 2010; Gabitzsch et al., 2015). Therapeutic approaches include treating patients with checkpoint inhibitor antibodies, antitumor vaccines, and chimeric antigen receptor (CAR)-T cells, all of which utilize adaptive immunization by T cells. However, innate immunity can also generate and enhance antitumor responses, and innate antitumor immunity can be stimulated using tumor-targeted monoclonal antibodies (mAbs) (Topalian et al., 2011).

[0008] NEO-201 is a novel humanized IgG1 mAb generated against the Hollinshead allogeneic colorectal cancer vaccine platform (Hollinshead et al., 1970; Hollinshead et al., 1972). The immunogenic component of this vaccine was tumor-associated antigen (TAA) derived from a pooled tumor membrane fraction from surgically resected specimens from 79 colorectal cancer patients (Hollinshead et al., 1985). These membrane fractions were semi-purified and screened for delayed-type hypersensitivity (DTH) in colorectal cancer patients versus healthy volunteers, and evaluated in clinical trials in patients with refractory colorectal cancer (Hollinshead et al., 1985; Hollinshead, US4810781, 1989; Bristol & Kantor, U.S. Patent No. 7829678, 2010). These studies reported clinical benefits defined by both an antitumor response and a significant extension of overall survival in patients who exhibited a sustained IgG response in addition to a cellular response to the vaccine, thereby suggesting that the vaccine contained an immunogenic component capable of generating antitumor antibodies (Hollinshead, 1991). This original colorectal cancer vaccine was used to generate monoclonal antibodies in mice, resulting in the aforementioned encituximab (NPC-1C / NEO-102) (Luka et al., 2011, Patel et al., 2013, Beg et al., 2016, Kim et al., 2017) and NEO-201. Previous studies have shown that NEO-201 binds to tumor-associated variants of CEACAM family members, particularly cancer-associated variants of CEACAM5 and CEACAM6 (Zeligs et al., 2017).

[0009] The human carcinoembryonic antigen (CEA) family consists of 29 genes located in tandem on chromosome 19q13.2. Based on nucleotide homology, these genes are classified into two major subfamilies: CEACAM and pregnancy-specific glycoprotein subgroups. Proteins encoding CEACAM include CEA (CEACAM5) and CEACAM-related cell adhesion molecules (CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, CEACAM8). The CEACAM family belongs to the Ig superfamily. Structurally, each human CEACAM contains 108-110 amino acids, an N-terminal domain homologous to the Ig variable domain, and a different number (0-6) of Ig molecules. A constant-like domain of type C2 follows. CEACAM proteins can interact with each other in both homophilic and heterophilic ways. CEACAM1 is a unique protein within this family because its cytoplasmic domain contains an ITIM (immune receptor tyrosine-based inhibitory motif)-like PD1. This inhibitory effect is triggered by phosphorylation of tyrosine residues by ITIM, resulting in the recruitment of tyrosine phosphatases-1 and-2, which contain the Src homology 2 domain. The CEACAM1 protein is expressed in a variety of immune cells, including monocytes, granulocytes, activated T cells, B cells, and NK cells. CEACAM1 arises as several isoforms, the two main ones being CEACAM1-L and CEACAM1-S, which have a long (L) or short (S) cytoplasmic domain, respectively. Expression of CEACAM1-S is completely absent in human leukocytes. CEACAM1-L is expressed in a subpopulation of activated human NK cells that are CD16-negative but CD56-positive.

[0010] Monoclonal antibodies (mAbs) consist of a specific antigen-binding region (fragment antigen binding, Fab) that is specific to a given mAb, and a constant region (fragment crystallizable, Fc) common to all mAbs of the same isotype. The Fc region can modulate the activity of immune cells by engaging with members of the Fc receptor (FcR) family expressed on the surface of specific immune cell types. In particular, human IgG1 mAbs can interact with Fc gamma receptor IIIa (FcγRIIIa, CD16) expressed on macrophages and NK cells. This interaction can stimulate macrophages to phagocytose mAb-opsonized cancer cells and activate NK cells to degranulate and lyse cancer cells through a mechanism known as antibody-dependent cytotoxicity (ADCC). ADCC has been shown to be a major mediator of in vivo antitumor effects in many preclinical studies and plays a crucial role in the mechanism of action of several mAbs used in cancer treatment (Seidel et al., 2013). Clinically approved mAbs that can mediate ADCC include trastuzumab, which targets the HER2 receptor for breast cancer (Seidel et al., 2013, Petricevic et al., 2013); rituximab, which targets the pan B-cell marker CD20 for lymphoma (Seidel et al., 2013, Dall'Ozzo et al., 2004); cetuximab, which targets the epidermal growth factor receptor (EGFR) for colorectal and head and neck cancers (Seidel et al., 2013, Levy et al., 2009, Kawaguchi et al., 2007, Lopez-Albaitero et al., 2009); and avelumab, which targets the immunosuppressive ligand PD-L1 for Merkel cell carcinoma and bladder cancer (Boyerinas et al., 2015). Furthermore, the Fc region can potentially interact with the C1 complex to activate complement-dependent cell-mediated cytotoxicity (CDC), leading to the formation of plasma membrane pores that trigger the lysis of antibody-targeted cells through a proteolytic cascade.Even when antitumor CDCs are demonstrated in vitro, there is controversy as to whether they are important for the clinical efficacy of mAb therapy in cancer (Meyer et al., 2014).

[0011] The applicant's previous U.S. Patents Nos. 5,688,657, 7,314,622, 7,491,801, 7,763,720, 7,829,678, 8,470,326, 8,524,456, 8,535,667, 8,802,090, 9,034,588, 9,068,014, 9,371,375, 9,592,290, 9,718,866, and RE39,760 (each of which is incorporated herein in whole by reference) disclose a variety of anti-cancer antibodies, cancer antigens, and related technologies. [Overview of the project]

[0012] We have previously shown that NEO-201 binds to cancer-associated variants of CEACAM5 and CEACAM6, particularly via cancer-associated glycosylated variants of these proteins. NEO-201 is a humanized IgG1 monoclonal antibody derived from immunogenic preparations of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. NEO-201 reacts to most tumor tissues from many different carcinomas but not to most normal tissues. Functional analysis has revealed that NEO-201 can mediate both antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against tumor cells. Previous studies have shown that NEO-201 attenuates the growth of human tumor xenografts in mice, demonstrating safety and tolerability in non-human primates with only one adverse effect observed: a transient decrease in circulating neutrophils.

[0013] The Applicant has shown herein that NEO-201 binds to Treg cells and can thereby also be used for the purification of Treg cells, such as activated Treg cells, and for the killing of Treg cells. These results were particularly unexpected because the NEO-201 antigen (cancer-related glycosylation variants of CEACAM5 and CEACAM6) is thought not to be expressed by Treg cells. Based on these results, the properties of the NEO-201 antigen are being re-evaluated. Without intending to be limited by theory, it is believed that Treg cells may express one or more proteins on the cell surface with glycosylation that is the same as or similar to the cancer-related glycosylation of CEACAM5 / 6 that constitutes the NEO-201 antigen.

[0014] The infiltration of Treg cells is associated with many cancer types, and in some studies, the selective removal of Treg cells has been shown to promote the anti-cancer immune response. The newly described ability of NEO-201 to kill Treg cells supports the use of NEO-201 to enhance the anti-cancer immune response, regardless of NEO-201 antigen expression by the cancer itself. For example, NEO-201 is expected to enhance the anti-tumor immune response for use in vaccine therapy. Selectively excising Treg cells using NEO-201 may be beneficial for other diseases in which Treg cells are thought or believed to play a role, including neurodegenerative conditions (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS)).

[0015] In addition, NEO-201 binding can be used for the purification of Treg cells, either for research or for therapeutic use. A decrease in the suppressive function of human Tregs appears to be a common feature of autoimmune diseases and conditions. The purification of Treg cells can have diagnostic and / or therapeutic utility. For example, purified Treg cells from healthy donors may be transplanted into individuals with autoimmune conditions to treat their condition. In addition, purified autologous or heterologous Tregs may be engineered for the treatment of autoimmune diseases and introduced into patients.

[0016] The working example describes an experiment performed using PBMC from normal donors for phenotypic and functional analysis. The EasySep™ StemCell Treg Isolation Kit and the anti-biotin kit were used either with the Treg purification reagents provided by the manufacturer or customized using biotinylated NEO-201 mAb to isolate Treg from PBMC. Phenotypic analysis was performed by flow cytometry against markers of CD4, CD25, CD127, FoxP3, CD15s, CD45RA, CCR4, NEO-201 antigen, CEACAM5, and CEACAM6. The ability of Treg isolated by NEO-201 to suppress the proliferation of autologous CD4+ T responder cells was evaluated using a Treg co-culture suppression assay. The proportion of NEO-201+ cells in the population of CD4+CD25hiCD127−FoxP3+CD15s+CCR4+ Treg ranged from 61.8% to 81.9%. NEO-201+ Treg were CD45RA−. The isolated CD4+NEO-201+ Treg were able to suppress the proliferation of CD4+ T responder cells.

[0017] In addition, the ability of NEO-201 to mediate the killing of opsonized Treg was evaluated using a CDC assay. It was demonstrated that the NEO-201 mAb was able to mediate CDC activity against Treg.

[0018] From these results, it was concluded that NEO-201 reacts against human Treg and can be used as a novel marker for the identification and purification of Treg. Treg isolated using the NEO-201 mAb are functionally suppressed and can be removed by CDC. Based on the ability of the antibody to bind to Treg cells, death may also occur in ADCC-mediated Treg cells. This application first demonstrates that this anti-cancer agent may also be useful in targeting Treg-mediated immunosuppression of anti-tumor immunity.

Brief Description of the Drawings

[0019] [Figure 1] Isolation of functional Treg cells using NEO-201. Selection based on the percentage of functional Tregs isolated using a commercially available kit (human CD4+CD127 lowCD25+) or NEO-201 expression. The EasySep® Human CD4+CD127 lowCD25+ Regulatory T Cell Isolation Kit produced 67.14% active Treg cells, while selection based on NEO-201 positive expression produced 99.12% active Treg cells. [Figure 2] Regulatory T cells are negative for CEACAM-5 and CEACAM-6, as determined by flow cytometry. Phenotypic analysis of isolated T-reg (EasySep® Human CD4+CD127 Low CD25+ Regulatory T Cell Isolation Kit (HD19)). Cells were stained with PE mouse anti-human CD66 antibody (clone B1.1 / CD66) that recognizes CD66a (CEACAM1), CD66c (CEACAM6), CD66d (CEACAM3), and CD66e (CEACAM5). 44.84% of the CD4+ / CD25 High / CD127- / FOXp3+ cells were NEO-201+ / CEACAM5- cells and CEACAM6- cells. [Figure 3] NEO-201 mediates CDC to CD4+CD15s+Treg cells. Treg cells express CD4 and CD15s (sialyl Lewis X glycan). Complement alone did not kill Treg cells. NEO-201 mediated CDC to Treg cells. The CD4+CD15s+Treg population was reduced by 53.4%. Procedure: CD4+ T cells were isolated from PBMCs. Cells were treated with - / + NEO-201 mAb (10 μg / mL). Then, cells were treated with - / + complement (1:8 dilution, 2 hours). Cells were washed and stained for CD4 and CD15s markers. Marker expression was measured by flow cytometry. [Modes for carrying out the invention]

[0020] In one embodiment, the present disclosure provides a method for killing Treg cells in vivo, comprising administering an effective amount of NEO-201 antibody to a patient.

[0021] In another aspect, the present disclosure provides a method for enhancing an anti-cancer immune response in a patient, comprising administering an effective amount of NEO-201 antibody to the patient.

[0022] The method may further include administering a cancer vaccine to the patient. Examples of cancer vaccines that may be administered are disclosed, for example, in Fisher et al., Immun Inflamm Dis. 2017 Mar;5(1):16-28, Klages et al., Cancer Res October 15 2010(70)(20)7788-7799, Reginato et al., Br J Cancer. 2013 Oct 15;109(8):2167-2174, and Litzinger MT et al., Blood 2007, 110:3192, each of which is incorporated herein by reference in whole.

[0023] In another aspect, the present disclosure provides a method for reducing Treg cell infiltration of cancer in a patient, comprising administering an effective amount of NEO-201 antibody to the patient.

[0024] In another aspect, the Disclosure provides a method for stimulating cancer regression in a patient, comprising administering an effective amount of NEO-201 antibody to the patient, thereby activating, enhancing, or stimulating anti-cancer immunity in the patient.

[0025] This type of cancer cannot express CEACAM5 or CEACAM6.

[0026] The method may further include determining whether the cancer is CEACAM5 and CEACAM6 negative before or during administration, which may optionally be determined by testing the expression of CEACAM5 and CEACAM6 proteins by staining with antibodies specific to CEACAM5 and / or CEACAM6, such as cross-reactive antibodies that specifically bind to both CEACAM5 and CEACAM6.

[0027] In another aspect, the Disclosure provides a method for treating or preventing cancer, reducing the cancer burden, or slowing the growth or proliferation rate of cancer, comprising administering an effective amount of NEO-201 antibody to a patient in need thereof, wherein the cancer is CEACAM5 and CEACAM6 negative.

[0028] The method may further include administering another therapeutic agent to the patient. Such other agents include (a) microtubule inhibitors, topoisomerase inhibitors, platins, alkylating agents, and antimetabolites; (b) MK-2206, ON 013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platins, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclo Phosphamide, cytarabine, cytoxane, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechloretamine, melpharen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, rituximab, Streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, and / or vinorelbine, (c) 1-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9->2-hydroxyethoxymethylguanine, adamanthanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors (d) a PD-1 inhibitor or an anti-PD-1 antibody such as KEYTRUDA® (pembrolizumab) or OPDIVO® (nivolumab), or (e) a CTLA-4 inhibitor or an anti-CTLA-4 antibody such as YERVOY® ipilimumab.The combination of immune checkpoint inhibition (PD-1 inhibition and / or CTLA-4 inhibition) and Treg ablation is expected to be particularly effective in cancer treatment. See Vargas et al., Immunity. 2017 Apr 18;46(4):577-586 and Taylor et al., J Clin Invest. 2017;127(9):3472-3483, each of which is incorporated herein by reference in whole.

[0029] The NEO-201 antibody may induce or enhance the anti-cancer immune response in patients.

[0030] In another aspect, the disclosure provides a method for killing Treg cells in vitro, comprising contacting the Treg cells with an antibody NEO-201. The method may further comprise contacting the Treg cells with complement. The Treg cells may be killed by CDC. The method may further comprise contacting the Treg cells with effector cells such as natural killer cells. The Treg cells may be killed by ADCC.

[0031] In another aspect, the disclosure provides a method for ex vivo killing of Treg cells, comprising contacting a sample containing Treg cells with an effective amount of NEO-201 antibody, the sample of which may be obtained from a patient.

[0032] The NEO-201 antibody can bind to the cytotoxic portion.

[0033] In another aspect, the Disclosure provides a method for detecting Treg cells, comprising detecting the expression of the NEO-201 antigen by such Treg cells, and optionally, a method in which the level of Treg cells in a patient sample, such as blood or a biopsy sample, is used to diagnose cancer or determine the progression of cancer. Optionally, the method may further include assigning or administering a treatment to a patient based on the detection of Treg cells. For example, a patient may be assigned to administer a treatment if Treg cells are detected in the patient sample, or may be administered NEO-201 in an amount effective to kill the Treg cells.

[0034] The method may include contacting the Treg cells with the NEO-201 antibody.

[0035] This detection may include cell sorting, and optionally, fluorescence-activated cell sorting.

[0036] In another aspect, the disclosure provides a method for detecting Treg cells, comprising contacting the cells with an antibody expressing NEO-201 and detecting cells expressing NEO-201. The NEO-201 antibody may be directly or indirectly labeled.

[0037] In another aspect, the disclosure provides a method for staining Treg cells, comprising contacting the cells with an antibody NEO-201, which may be directly or indirectly labeled.

[0038] In another aspect, the present disclosure provides a method of isolating Treg cells, which includes isolating cells expressing the NEO-201 antigen. The method may include contacting a sample containing Treg cells with an NEO-201 antibody, and optionally, the NEO-201 antibody is labeled directly or indirectly. The sample may be or include blood or bone marrow. The method may include separating NEO-201-positive Treg cells from NEO-201-negative cells. The method may further include genetically modifying the Treg cells and optionally introducing the cells into the patient or another individual.

[0039] The Treg cells can be isolated by cell sorting, optionally by fluorescence-activated cell sorting.

[0040] The Treg cells can be isolated by contacting the sample with a support containing the NEO-201 antibody, whereby the Treg cells are retained on the support.

[0041] The method of staining or detection may further include detecting the expression of a combination of markers where the presence, absence, and / or level of expression of another marker, in combination with NEO-201, indicates Treg cells, such as CD4+, CD15s+, FoxP3+, CD25+, CCR4+ and / or CD127 低 or CD127−, such as CD4+CD15s+, CD4+FoxP3+CD25+, or CD4+FoxP3+CD25+CD127 低 For example, it may further include detecting the expression of a combination of markers such as NEO-201+CD4+CD15s+, NEO-201+CD4+CD15s+FoxP3+CD25+, NEO-201+CD4+CD15s+FoxP3+CD25+CD127 低 , NEO-201+CD4+FoxP3+CD25+, or NEO-201+CD4+FoxP3+CD25+CD127 低 , preferably NEO-201+CD4+CD15s+, or preferably NEO-201+CD4+CD1−27 低CD25+, or preferably CD4+CD25 高 Cells that are CD127-FoxP3+CD15s+CCR4+ may be identified, detected, isolated, and / or purified as Treg cells according to the methods disclosed herein.

[0042] In either of the above or below methods, the NEO-201 antibody may contain at least one, two, three, four, five, or preferably all six of the CDR sequences included in SEQ ID NO: 28 and SEQ ID NO: 29.

[0043] In either of the above or the following methods, the NEO-201 antibody may contain a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38.

[0044] In either of the above or the following methods, the NEO-201 antibody may contain a variable light chain sequence having at least 90% identity with SEQ ID NO: 39.

[0045] In either of the above or the following methods, the NEO-201 antibody may contain a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38 and a variable light chain sequence having at least 90% identity with SEQ ID NO: 39.

[0046] In either of the above or the following methods, the NEO-201 antibody may contain a heavy chain sequence having at least 90% identity with amino acids 20-470 of SEQ ID NO: 28 and a light chain sequence having at least 90% identity with amino acids 20-233 of SEQ ID NO: 29.

[0047] In either of the above or the following methods, the NEO-201 antibody may contain all six CDR sequences included in SEQ ID NO: 28 and SEQ ID NO: 29.

[0048] In either of the above or below methods, the NEO-201 antibody may contain a human IgG1 constant domain. Alternatively, the NEO-201 antibody may contain a human IgG2, human IgG3, or human IgG4 constant domain, or a hybrid or chimeric domain containing two or more human IgG1, IgG2, IgG3, or IgG4.

[0049] The NEO-201 antibody can be humanized by either of the methods described above or below.

[0050] In either of the above or below methods, the NEO-201 antibody may be conjugated to another portion.

[0051] In either of the above or below methods, the NEO-201 antibody may be conjugated to another cytotoxic moiety, label, radioactive moiety, or affinity tag.

[0052] In either of the above or below methods, the NEO-201 antibody may compete with the antibodies contained in SEQ ID NO: 28 and SEQ ID NO: 29 for binding to the NEO-201 antigen.

[0053] In either of the above or below methods, the cancer may be selected from hematological malignancies, lung cancers such as non-small cell lung cancer, melanoma, gastrointestinal cancer, ovarian cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, breast cancer, pancreatic cancer, mesothelioma, metastatic renal cell carcinoma, and prostate cancer. The cancer may include Treg cells.

[0054] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the present invention or in the testing of the present invention, but suitable methods and materials are described herein. Materials, methods, and examples are illustrative and not intended to limit the scope.

[0055] In this specification and as used in the following claims, unless the context explicitly indicates otherwise, the meanings of “a,” “an,” and “the” include multiple referents.

[0056] As used herein, the term “amino acid” refers to natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimes that function in a similar manner to natural amino acids. Natural amino acids are those encoded by the genetic code and those that have been later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as natural amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to natural amino acids.

[0057] As used herein, the terms “NK depletion” or “natural killer depletion” refer to patients with low levels of natural killer (NK) cells compared to the normal range. NK cells are cytotoxic innate immune lymphocytes. Typically, NK cells make up 5–20% of peripheral blood mononuclear cells (PBMCs) in healthy individuals. Patients with NK cells containing less than 5% PMBCs are referred to as NK depleted. In addition, if NK cells contain less than 3% PMBCs, this patient is referred to as severe NK cell depletion. Furthermore, in normal individuals, up to 90% of PBMCs are NK cells containing CD56 dim CD16 + These are NK cells, and they are considered the most cytotoxic subset. Less than 70% of PBMC NK cells are CD56 dim CD16 + If the cells are NK cells, this patient is referred to as having NK depletion. In addition, if less than 50% of PBMC NK cells are CD56dim CD16 + If NK cells are depleted, the patient is referred to as having severe NK depletion. A given patient may be referred to as having NK depletion or severe NK depletion based on meeting one or both of these individual criteria. Generally speaking, a patient's condition as NK depletion or severe NK depletion is determined by testing a sample taken from the patient, e.g., a blood sample, e.g., a sample obtained and tested one or two weeks prior. A patient's condition as NK depletion or severe NK depletion may also be inferred from the diagnosis of the disease and / or a series of treatments associated with such NK cell depletion.

[0058] As used herein, “antibody” broadly refers to any polypeptide chain-containing molecular structure having a specific shape that fits and recognizes an epitope, and one or more non-covalent interactions stabilize the complex between the molecular structure and the epitope. Typical antibody molecules are immunoglobulins, and all kinds of immunoglobulins, IgG, IgM, IgA, IgE, and IgD from all sources, e.g., humans, rodents, rabbits, cattle, sheep, pigs, dogs, and chickens, are considered “antibodies.” Antibodies include chimeric antibodies, human antibodies, and other non-human mammalian antibodies, humanized antibodies, single-chain antibodies (scFv), camel bodies, nanobodies, IgNAR (single-chain antibodies derived from sharks), small modular immunotherapy drugs (SMIP), and antibody fragments (e.g., Fab, Fab'). 、 This includes, but is not limited to, F(ab')2). Numerous antibody coding sequences are described, others of which may be generated by methods well known in the art. See Streltsov, et al. (2005) Protein Sci. 14(11):2901-9, Greenberg, et al. (1995) Nature 374(6518):168-173, Nuttall, et al. (2001) Mol Immunol. 38(4):313-26, Hamers-Casterman, et al. (1993) Nature 363(6428):446-8, and Gill, et al. (2006) Curr Opin Biotechnol. 17(6):653-8.

[0059] "NEO-201 antibody" refers to an antibody comprising the heavy and light chains of SEQ ID NOs. 28 and 29, or optionally, the variable region along with the constant region contained therein, as well as fragments and variants thereof. Such variants include sequences containing one, two, three, four, five, or preferably all six of the CDR sequences contained in SEQ ID NOs. 28 and 29, i.e., heavy chain CDR1 of SEQ ID NOs. 32, heavy chain CDR2 of SEQ ID NOs. 33, heavy chain CDR3 of SEQ ID NOs. 34, light chain CDR1 of SEQ ID NOs. 35, light chain CDR2 of SEQ ID NOs. 36, and light chain CDR3 of SEQ ID NOs. 37. Such variants also include antibodies that compete with NEO-201 for binding to the NEO-201 antigen. Such antibodies may be humanized. Such antibodies may be expressed containing one or more leader sequences and may be removed during expression and / or during antibody processing and secretion. The antibody may include, but is not limited to, a bispecific or multispecific antibody containing the NEO-201 antibody sequence and a binding fragment of a different antibody, and may be presented in monovalent, bivalent, or more polyvalent form. Typically, the antibody specifically binds to cancer cells and competes for binding to cancer cells with antibodies containing the variable heavy chain of SEQ ID NO: 38 and the variable light chain of SEQ ID NO: 39, or the heavy chain of SEQ ID NO: 28 and the light chain of SEQ ID NO: 29. One or more of the CDR sequences in SEQ ID NO: 28 and / or SEQ ID NO: 29 may be substituted with variant sequences, such as light chain CDR1 of SEQ ID NO: 1 or 4, light chain CDR2 of SEQ ID NO: 2 or 5, light chain CDR3 of SEQ ID NO: 3 or 6, heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 8, 10, 30, or 31, heavy chain CDR3 of SEQ ID NO: 9 or 11, or heavy chain CDR3 of SEQ ID NO: 30-31. The light chain may contain CDRs included in the light chain sequence of SEQ ID NOs. 14, 16, 17, 18, 19, 20, 21, or 29. The heavy chain may contain CDRs included in the heavy chain sequence of SEQ ID NOs. 15, 22, 23, 24, 25, 26, 27, or 29.The antibody may include a variable heavy chain sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 38, and / or a variable light chain sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39, and optionally, the heavy chain sequence and / or light chain sequence may include one, two, three, four, five, or preferably all six of the CDR sequences included in SEQ ID NO: 28 and SEQ ID NO: 29, namely, heavy chain CDR1 of SEQ ID NO: 32, heavy chain CDR2 of SEQ ID NO: 33, heavy chain CDR3 of SEQ ID NO: 34, light chain CDR1 of SEQ ID NO: 35, light chain CDR2 of SEQ ID NO: 36, and light chain CDR3 of SEQ ID NO: 37. The antibody may be conjugated to another portion, such as a cytotoxic portion, a radioactive portion, a label, or a purification tag.

[0060] As used herein, “antigen” broadly refers to a molecule or part of a molecule to which an antibody can bind, which can be further induced in an animal to produce an antibody that can bind to the epitope of that antigen. An antigen may have one epitope or more than one epitopes. The specific reactions referred to herein indicate that an antigen reacts with its corresponding antibody in a highly selective manner and does not react with a large number of other antibodies that may be induced by other antigens. Antigens may be tumor-specific (e.g., expressed by neoplastic cells of pancreatic and colon cancer).

[0061] As used herein, “cancer” broadly refers to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division that leads to malignant growth or tumor formation.

[0062] As used herein, "cancer vaccine" refers to an immunogenic composition that induces or is intended to induce an immune response against cancer cells.

[0063] As used herein, "chimeric antibody" broadly refers to an antibody molecule in which the constant region or a portion thereof has been altered, substituted, or replaced, resulting in a different or modified class, effector function, and / or species of constant region, or a completely different molecule that confers new characteristics to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or in which the variable region or a portion thereof has been altered, substituted, or replaced with a variable region having different or modified antigen specificity.

[0064] As used herein, “conservatively modified variant” applies to both amino acids and nucleic acid sequences and broadly refers to a conservatively modified variant with respect to a particular nucleic acid sequence, referring to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, a sequence that is essentially the same. Due to the degeneracy of the genetic code, a number of functionally identical nucleic acids code for any given protein. Such nucleic acid mutations are “silent mutations” and are one type of conservatively modified mutation. All nucleic acid sequences in this specification that code for polypeptides also represent any possible silent variation of the nucleic acid. Those skilled in the art will understand that each codon in a nucleic acid can be modified to produce a functionally identical molecule (except for AUG, which is usually the sole codon of methionine, and TGG, which is usually the sole codon of tryptophan).

[0065] As used herein, “complementarity-determining regions,” “hypervariable regions,” or “CDRs” broadly refer to one or more hypervariable regions or complementary-determining regions (CDRs) found in the variable regions of the light or heavy chains of an antibody. See Kabat, et al. (1987) “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, MD. These terms include hypervariable regions as defined by Kabat, et al. (1983) “Sequences of Proteins of Immunological Interest,” USDept. of Health and Human Services or the hypervariable loops in 3-dimensional structures of antibodies. Chothia and Lesk (1987) J Mol. Biol. 196:901-917. CDRs within each chain are held in close proximity by framework regions and, together with CDRs from other chains, contribute to the formation of antigen-binding sites. Within the CDR, there are selectable amino acids that are described as selectivity-determining regions (SDRs) that represent essential contact residues used by the CDR in antibody-antigen interactions. Kashmiri (2005) Methods 36:25-34.

[0066] As used herein, “control dose” broadly refers to any amount or range of amounts of the marker being compared to the test dose of the marker. For example, the control dose of a marker may be the amount of the marker in a patient with a particular disease or condition or in a person without such a disease or condition. The control dose may be either an absolute amount (e.g., micrograms / ml) or a relative amount (e.g., relative intensity of the signal).

[0067] As used herein, “differentially present” broadly refers to differences in the quantity or quality of markers present in a sample taken from a patient with a disease or condition compared to an equivalent sample taken from a patient without one of the diseases. For example, if the amount of nucleic acid fragments in one sample differs significantly from the amount in another sample, as measured by hybridization and / or NAT-based assays, then nucleic acid fragments may optionally be differentially present between the two samples. Similarly, if the amount of polypeptides in one sample differs significantly from the amount in another sample, then polypeptides may optionally be differentially present between the two samples. It should be noted that if a marker is detectable in one sample but not in another, such a marker can be considered differentially present. Optionally, relatively small amounts of upregulation may serve as markers.

[0068] As used herein, “diagnostic” broadly refers to identifying the presence or nature of a disease. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of affected individuals who test positive (the percentage of “true positives”). Affected individuals not detected by the assay are “false negatives.” Subjects who are not affected and test negative in the assay are called “true negatives.” The “specificity” of a diagnostic assay is the 1-false positive rate, and the “false positive” rate is defined as the proportion of disease-free individuals who test positive. Certain diagnostic methods may not provide a definitive diagnosis of a condition, but it is sufficient if the method provides a positive indication that aids in diagnosis.

[0069] As used herein, “diagnose” broadly includes classifying a disease or symptom, determining the severity of a disease, monitoring the progression of a disease, and predicting the outcome and / or prospects of recovery from a disease. The term “detect” may also optionally encompass any of the foregoing. In some embodiments, the diagnosis of a disease according to the present invention may be influenced by determining the levels of the polynucleotides or polypeptides of the present invention in a biological sample obtained from a subject, the levels determined may correlate with predisposition to the disease or the presence or absence of the disease. It should be noted that “biological sample obtained from a subject” may optionally include samples that have not been physically removed from the subject.

[0070] As used herein, “effective dose” broadly refers to the amount of compound, antibody, antigen, or cell that achieves a desired result. “Effective dose” is sufficient to perform such treatment for a disease when administered to a patient for that purpose. An effective dose may be an effective dose for prevention and / or prevention. An effective dose may be an effective dose to reduce the occurrence of signs / symptoms, an effective dose for prevention, an effective dose for reducing the severity of a sign / symptom occurrence, an effective dose for eliminating a sign / symptom occurrence, an effective dose for delaying the development of a sign / symptom occurrence, an effective dose for preventing the development of a sign / symptom occurrence, and / or for resulting in prevention of a sign / symptom occurrence. “Effective dose” may vary depending on the disease and its severity, as well as the age, weight, medical history, susceptibility, and pre-existing conditions of the patient being treated. For the purposes of this disclosure, the term “effective dose” is synonymous with “therapeutably effective dose.”

[0071] As used herein, “expression vector” broadly refers to any recombinant expression system intended for constitutive or inductive expression of the nucleic acid sequences of this disclosure in vitro or in vivo in any cell, including prokaryotic, yeast, fungal, plant, insect, or mammalian cells. This term includes linear or cyclic expression systems. This term includes expression systems that remain in the episome or are integrated into the genome of a host cell. This expression system may or may not have self-renewal capabilities, i.e., it may only drive transient expression within the cell. This term includes recombinant expression cassettes containing only the minimum elements necessary for transcription of recombinant nucleic acids.

[0072] As used herein, “framework region” or “FR” broadly refers to one or more framework regions within the variable regions of the light and heavy chains of an antibody. See Kabat, et al. (1987) “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, MD. These terms include amino acid sequence regions inserted between CDRs within the variable regions of the light and heavy chains of an antibody.

[0073] As used herein, “heterogeneous” broadly refers to a portion of a nucleic acid in which the nucleic acid contains two or more subsequences that are not found in nature in an identical relationship to one another. For example, a nucleic acid typically has two or more sequences from unrelated genes that are generated by recombination and arranged to create a new functional nucleic acid, such as a promoter from one source and a coding region from another. Similarly, heterogeneous protein refers to a protein in which the protein contains two or more subsequences that are not found in nature in an identical relationship to one another (e.g., a fusion protein).

[0074] As used herein, "high affinity" means at least 10 for the target antigen. -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10This broadly refers to antibodies with M KD. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding to IgM isotypes is at least 10 -7 M, more preferably at least 10 -8 This refers to antibodies that have a KD of M.

[0075] As used herein, “homology” broadly refers to the degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence, or between a polypeptide sequence and a reference polypeptide sequence. Homologousity can be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. Partially homologous nucleic acids or amino acid sequences are not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The term “sequence identity” may be used interchangeably with “homology.”

[0076] As used herein, “host cell” broadly refers to a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be prokaryotic cells, e.g., E. coli, or eukaryotic cells, e.g., yeast, insect (e.g., SF9), amphibian or mammalian cells, e.g., CHO, HeLa, HEK-293, e.g., cultured cells, explants and in vivo cells.

[0077] As used herein, "hybridization" broadly refers to the physical interaction of complementary (including partially complementary) polynucleotide chains through the formation of hydrogen bonds between complementary nucleotides when the chains are arranged antiparallel to each other.

[0078] As used herein, “K-assoc” or “Ka” broadly refers to the association rate of a particular antibody-antigen interaction, while the terms “Kdiss” or “Kd” refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term “KD” refers to the dissociation rate obtained from the Kd to Ka ratio (i.e., Kd / Ka) and expressed as molar concentration (M). The KD value for an antibody can be determined using methods well established in the art.

[0079] As used herein, “immunoassay” broadly refers to an assay that uses an antibody to specifically bind to an antigen. Immunoassays may be characterized by the use of the specific binding properties of a particular antibody to isolate, target, and / or quantify an antigen.

[0080] As used herein, “isolated” broadly refers to material that has been removed from its original environment in which it naturally occurs and thus modified by human intervention from that natural environment. Isolated material may be, for example, exogenous nucleic acids contained in a vector system, exogenous nucleic acids contained within a host cell, or any material that has been removed from its original environment and thus modified by human intervention (e.g., “isolated antibody”).

[0081] As used herein, “label” or “detectable portion” broadly refers to a composition that is detectable by microscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[0082] As used herein, “low stringency,” “medium stringency,” “high stringency,” or “very high stringency conditions” broadly refer to conditions for nucleic acid hybridization and washing. For guidance on performing hybridization reactions, see Ausubel, et al. (2002) Short Protocols in Molecular Biology (5 th(Ed.) John Wiley & Sons, NY. Exemplary specific hybridization conditions include: (1) Low stringency hybridization conditions: approximately 45 o In C, 6 × sodium / sodium citrate (SSC), then at least 50 o Wash twice in 0.2×SSC and 0.1%SDS at C (under low stringency conditions, washing temperature is 55°C). o (2) Can be raised to C), (2) Medium stringency hybridization conditions: approximately 45 o C, 6×SSC, then 60 o (3) High stringency hybridization conditions: approximately 45 o In C, out of 6 × SSC, next is 65 o Washed at least once in C, 0.2 × SSC, 0.1% SDS, and (4) very high stringency hybridization conditions: 65 o C, 0.5M sodium phosphate, 7% SDS, then 65 o This includes, but is not limited to, washing with C, 0.2×SSC, and 1% SDS at least once.

[0083] The terms "low level" or "low" used in relation to markers such as CD127 are well-known in the art and refer to the expression level of the cellular marker in question (e.g., CD127), where the expression level is lower compared to the expression level of that cellular marker in other cells within the overall cell population being analyzed. More specifically, the term "low" refers to a distinct cell population that expresses the cellular marker at a lower level than one or more other distinct cell populations. Therefore, CD127 低 This refers to a type of cell that stains slightly or faintly when in contact with a labeled CD127 antibody, for example, at a level higher than the CD127- subpopulation but lower than the CD127+ subpopulation.

[0084] As used herein, “mammal” broadly refers to any and all warm-blooded vertebrates of the mammalian class, including humans, characterized by a hairy skin and, in females, milk-producing mammary glands for raising offspring. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammals include, but are not limited to, Bovidae, Canidae, Equidae, Felidae, Muridae, Ampidae, Swineidae, Primates, and Rodents. Mammals also include any and all listed in the World Mammals Maintained by the Smithsonian Museum of Natural History in Washington, D.C.

[0085] As used herein, “nucleic acid” or “nucleic acid sequence” broadly refers to deoxyribonucleotides or ribonucleotide oligonucleotides in either single-stranded or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, including known analogues of native nucleotides. The term also encompasses nucleic acid-like structures with synthetic backings. Unless otherwise indicated, a particular nucleic acid sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0086] As used herein, “operably linked” broadly refers to the state in which two DNA fragments are joined such that the amino acid sequence encoded by the two DNA fragments is maintained in frame.

[0087] As used herein, "paratope" broadly refers to a portion of an antibody that recognizes an antigen (e.g., the antigen-binding site of the antibody). A paratope can be a small region of the antibody's Fv region (e.g., 15-22 amino acids) and may include parts of the antibody's heavy and light chains. (Goldsby, et al. Antigens (Chapter 3) Immunology (5)) th See Ed.) New York: WH Freeman and Company, pages 57–75.

[0088] As used herein, “patient” broadly refers to any animal that requires treatment to alleviate a disease condition or to prevent the onset or recurrence of a disease condition. Furthermore, as used herein, “patient” broadly refers to any animal that has risk factors, medical history, susceptibility, symptoms, or signs, and has been previously diagnosed with the disease, is at risk of the disease, or is a member of the patient population of the disease. A patient may be a clinical patient such as a human, or an companion animal, livestock, farm animal, exotic animal, or zoo animal. The term “subject” may be used interchangeably with the term “patient.” In preferred embodiments of the present invention disclosed herein, the patient is a human.

[0089] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably and broadly refer to polymers of amino acid residues. These terms apply to amino acid polymers where one or more amino acid residues are analogs or mimetic versions of corresponding natural amino acids, and to natural amino acid polymers. These terms also apply to amino acid polymers where one or more amino acid residues are artificial chemical mimetic versions of corresponding natural amino acids, and to natural amino acid polymers and non-natural amino acid polymers. Polypeptides can be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms “polypeptide,” “peptide,” and “protein” include glycoproteins, as well as non-glycoproteins.

[0090] As used herein, “promoter” broadly refers to a set of nucleic acid sequences that govern the transcription of a nucleic acid. As used herein, a promoter includes a nucleic acid sequence required near the transcription start site, such as the TATA element in the case of a polymerase type II promoter. Promoters may also optionally include distal enhancer or repressor elements that can be located thousands of base pairs away from the transcription start site. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental and developmental control.

[0091] As used herein, “prophylactic effective dose” broadly refers to the amount of a compound that, when administered to a patient for the prevention of a disease or the prevention of its recurrence, is sufficient to produce such prevention for that disease or its recurrence. A prophylactic effective dose may be an amount that is effective in preventing the onset of signs and / or symptoms. A “prophylactic effective dose” may vary depending on the disease and its severity, as well as the age, weight, medical history, susceptibility to the condition, and pre-existing conditions of the patient being treated.

[0092] As used herein, “prevention” broadly refers to a series of treatments in which the patient is in remission, or in which signs and / or symptoms are absent or previously present in the patient. Prevention includes preventing the disease from occurring in a patient after treatment for the disease. Furthermore, prevention includes treating patients who are potentially at risk of developing the disease, in particular those who are susceptible to the disease (e.g., members of a patient population, those with risk factors, or those at risk of developing the disease).

[0093] As used herein, “recombinant” broadly refers to products, such as cells, or nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors are derived from heterologous nucleic acids or proteins, or from native nucleic acids or proteins, or from cells that have been thus modified. For example, recombinant cells express genes not found in the cell’s native (non-recombinant) form, or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0094] As used herein, “specifically (or selectively) binding” or “specifically (or selectively) immunoreacting” or “specifically interacting with or binding” to an antibody broadly refers to a protein or peptide (or other epitope), and in some embodiments, refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biopharmaceuticals. For example, under specified immunoassay conditions, the identified antibody binds to a specific protein at least twice as much as the background (non-specific signal) and substantially does not bind to other proteins present in the sample in significant amounts. Typically, a specific or selective reaction is at least twice as much background signal or noise, and more typically about 10 to over 100 times as much background.

[0095] As used herein, “specifically hybridizable” and “complementary” broadly refer to the ability of a nucleic acid to form hydrogen bonds (or multiple bonds) with another nucleic acid sequence by either the conventional Watson-Crick method or any other unconventional type. The binding free energy of a nucleic acid molecule and its complementary sequence is sufficient to enable the relevant function of the nucleic acid, such as RNAi activity. The measurement of the binding free energy of nucleic acid molecules is well known in the art. See, for example, Turner, et al. (1987) CSH Symp. Quant. Biol. LII:123-33, Frier, et al. (1986) PNAS83:9373-77, and Turner, et al. (1987) J. Am. Chem. Soc. 109:3783-85. The complementarity percentage indicates the proportion of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., at least about 5, 6, 7, 8, 9, and 10 out of 10 have at least about 50%, 60%, 70%, 80%, 90%, and 100% complementarity). "Fully complementary" or 100% complementarity broadly refers to all consecutive residues in the nucleic acid sequence that hydrogen bond with the same number of consecutive residues in the second nucleic acid sequence. "Substantial complementary" refers to a polynucleotide chain that exhibits at least about 90% complementarity, excluding regions of the polynucleotide chain such as overhangs selected to be non-complementary. Specific binding requires a degree of complementarity sufficient to avoid nonspecific binding of the oligomeric compound to non-target sequences under the conditions under which specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions under which the assay is performed in the case of in vitro assays. Non-target sequences can typically differ by at least 5 nucleotides.

[0096] As used herein, “signs” of a disease broadly refers to the purpose of indicating a disease, as opposed to any abnormality that indicates a disease that can be detected by a patient’s examination, or subjective signs that indicate a disease.

[0097] As used herein, “solid support,” “support,” and “substrate” broadly refer to any material that provides a solid or semi-solid structure to which other materials can be bonded, including, but not limited to, smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces), as well as rough and porous materials. Exemplary solid supports include beads such as activated beads, magnetically responsive beads, or fluorescently labeled beads.

[0098] As used herein, “subject” broadly refers to any person suitable to be treated according to the invention disclosed herein, and includes, but is not limited to, avian and mammalian subjects, preferably mammalian. Mammals in the context of the invention disclosed herein include, but are not limited to, canids, felids, bovids, goats, equids, sheep, pigs, rodents (e.g., rats and mice), rabbits, primates, and humans. Any mammalian subject that requires to be treated according to the invention disclosed herein is suitable. Human subjects of both sexes and any developmental stage (i.e., neonates, infants, juveniles, adolescents, and adults) can be treated according to the invention. The invention may be carried out for veterinary purposes, and for drug screening and drug development purposes, on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, cattle, goats, sheep, and horses. “Subject” is used interchangeably with “patient.” In preferred embodiments of the invention disclosed herein, the subject is human.

[0099] As used herein, “symptoms” of a disease broadly refers to any pathological phenomenon or deviation from normal structure, function, or sensation experienced by a patient that indicates a disease.

[0100] As used herein, “treatment,” “therapeutic,” “to treat,” or “treatment” broadly means treating a disease, stopping or reducing the development of a disease or its clinical symptoms, and / or alleviating a disease and causing regression of a disease or its clinical symptoms. Treatment encompasses providing prevention, treatment, restoration, reduction, mitigation, and / or relief of a disease, its signs, and / or symptoms. Treatment encompasses reducing signs and / or symptoms in patients with signs and / or symptoms of a disease in progress (e.g., tumor growth, metastasis). Treatment also encompasses “prevention.” The term “reduced” broadly means a clinically significant reduction of signs and / or symptoms for therapeutic purposes. Treatment includes treating signs of relapsing or recurrent signs and / or symptoms (e.g., tumor growth, metastasis). Treatment includes, but is not limited to, eliminating signs and / or symptoms at any time, as well as reducing existing signs and / or symptoms, and eliminating existing signs and / or symptoms. Treatment includes treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing the recurrence or relapse of signs and / or symptoms (e.g., tumor growth, metastasis).

[0101] As used herein, “variable region” or “VR” broadly refers to the domains within each pair of light and heavy chains of an antibody that are directly involved in the binding of the antibody to the antigen. Each heavy chain has a variable domain (V) at one end. H It has a variable domain (V) at one end. L It has a constant domain at the other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain.

[0102] As used herein, “vector” broadly refers to plasmids, cosmids, phagemids, phage DNA, or other DNA molecules that can spontaneously replicate in host cells and feature one or more restriction endonuclease recognition sites, wherein such DNA sequences may be cleaved in a deterministic manner without losing the essential biological function of the vector, and DNA may be inserted to cause replication and cloning. The vector may further contain markers suitable for use in identifying cells transformed with the vector.

[0103] The techniques and procedures are generally carried out in accordance with conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, Sambrook, et al. (2001) Molec. Cloning: Lab. Manual [3 rd See Ed. Cold Spring Harbor Laboratory Press. Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques may be performed according to the manufacturer's specifications, as commonly achieved in the art, or as described herein. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein is well known and commonly used in the art. Standard techniques may be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and in the treatment of patients. [Examples]

[0104] The present invention, as generally described herein, will be more readily understood by referring to the following examples, which are included solely for the purpose of describing certain aspects and embodiments of the invention and are not intended to limit the invention.

[0105] Example 1 NEO-201 can isolate functional Treg cells.

[0106] This example demonstrates that cell sorting based on NEO-201 binding produces a high percentage of functional Treg cells. Figure 1 shows the percentage of functional Treg cells isolated using a commercially available kit (human CD4+CD127 low CD25+) or by selection based on NEO-201 expression. The EasySep® Human CD4+CD127 Low CD25+ Regulatory T Cell Isolation Kit produced 67.14% active Treg cells, while selection based on NEO-201 positive expression produced 99.12% active Treg cells.

[0107] The applicants have previously shown that NEO-201 binds to cancer-associated glycoprotein variants of CEACAM5 and CEACAM6. Treg cells are not known to express CEACAM5 and CEACAM6. To further determine the criteria for NEO-201 binding to Treg cells, cells were tested for CEACAM5 and CEACAM6 expression by flow cytometry. Regulatory T cells are negative for CEACAM-5 and CEACAM-6 (Figure 2).

[0108] Methods The binding of human Treg markers to isolated human Tregs was analyzed by flow cytometry. Treg(1.0 × 10⁶) 6Live and dead cells were distinguished by incubation at 4°C for 30 minutes in 1 μL of LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) in 1× phosphate-buffered saline (PBS) per test. The cells were then centrifuged, washed twice with cold PBS, and stained with Pacific Blue Conjugate NEO-201 antibody, anti-CD25-APC-H7, anti-CD15s-Alexa 488, and anti-CD127-APC in 1× PBS + 1% BSA (Teknova, Hollister, CA, USA), followed by intracellular staining with anti-Foxp3-PerCP-CY5.5 (BioLegend, San Diego, CA) at 4°C for 30 minutes. After staining, the cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Cell fluorescence analysis was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Isolation of T-regs using a commercially available kit was performed using the EasySep® Human CD4+CD127 Low CD25+ Regulatory T Cell Isolation Kit (HD19) according to the manufacturer's instructions. For analysis of CEACAM expression, cells were stained with PE mouse anti-human CD66 antibody (clone B1.1 / CD66) that recognizes CD66a (CEACAM1), CD66c (CEACAM6), CD66d (CEACAM3), and CD66e (CEACAM5). 44.84% of the CD4+ / CD25 High / CD127- / FOXp3+ cells were NEO-201+ / CEACAM5- and CEACAM6- cells.

[0109] Example 2 NEO-201 can kill Treg cells through complement-dependent cytotoxicity (CDC).

[0110] This example demonstrates that NEO-201 can mediate CDC in CD4+CD15s+Treg cells. Treg cells express CD4 and CD15s (sialyl Lewis X glycan). CD4+ T cells were isolated from PBMCs and treated with - / + NEO-201 mAb (10 μg / mL). The cells were then treated with - / + complement (1:8 dilution, 2 hours). The cells were then washed and stained for CD4 and CD15s markers. Marker expression was measured by flow cytometry. Treg cells were not killed by complement alone. However, when Treg cells were treated with both NEO-201 and complement, the CD4+CD15s+Treg population was reduced by 53.4%, indicating that CDC occurred.

[0111] Complement-dependent cytotoxicity (CDC) assay method. The CDC assay was performed using a modified version of the previously described procedure (Konishi et al., 2008). CD4+ T cells were isolated and then opsonized by treatment with or without 10 μg / mL of NEO-201 at 37°C for 15 minutes. Purified rabbit complement (MP Biomedicals, Santa Ana, CA) was then added to the cells at a 1:8 dilution. After incubation at 37°C for 120 minutes, the cells were washed and stained with fluorescently labeled antibodies against CD4 and CD15s. After incubation for 30 minutes, the cells were washed and fluorescence was measured by flow cytometry using BD FACSVerse. Cell fluorescence analysis was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA).

[0112] Example 3 Production of humanized NEO-201 monoclonal antibodies

[0113] Hollinshead's colon cancer-specific vaccine was used as an immunogenic agent to produce monoclonal antibodies in mice. Methods for preparing tumor-associated proteins and peptides have been previously described (Hollinshead, US4810781, 1989). Briefly, cancer tissue was finely chopped and used to produce a single-cell suspension, which was then subjected to hypotonic saline membrane extraction, a series of centrifugation steps, and subsequent low-frequency sonication. The resulting membrane-extracted proteins were fractionated, concentrated, and quantified by Sephadex G-200 resin or electrophoresis (Hollinshead et al, 1970, Hollinshead et al., 1972, Hollinshead et al., 1985). The TAA preparation was mixed with Freund's complete adjuvant and subcutaneously injected into BALB / c mice. This was followed by three booster injections of Freund's incomplete adjuvant at 2-3 week intervals. Mouse serum was tested by ELISA for antibody response to immunizing antigens. Using mice with a strong response, mouse B cells from the spleen were fused with the SP2 / 0-Ag14 myeloma cell line to grow mouse immunoglobulin (IgG), and immortalized hybridoma cells were generated by selecting the cells that produced the IgG. From these mouse IgGs, mouse 16C3 clones (m16C3) were selected based on their reactivity with colon tumor cell membrane extracts derived from LS174T or HT-29 cells, as determined by ELISA. The cDNA encoding heavy and light chain IgG1 was determined from RNA isolated from hybridoma clone 16C3 E12 and was shown to be unique (Bristol & Kantor, US7829678, 2010). The m16C3 protein sequence was humanized as h16C3 and represented as NEO-201. Humanization was performed computer-aidedly by replacing the mouse sequences outside the complementarity-determining regions (CDRs) of the Fab regions of both the heavy and light chain proteins with human Fab sequences, while retaining three mouse CDR sequences from each chain. The Fc regions of the heavy and light chains were selected from human IgG1 isotypes used in other humanized approved mAb products.The amino acid sequence was back-translated into DNA optimized for protein expression in CHO cells. The heavy and light chains of h16C3 DNA were then chemically synthesized, cloned into mammalian expression plasmids, and transfected into mammalian cell lines (HEK293T and CHO). Several stable CHO cell lines expressing recombinant h16C3 were induced and conserved. The purified recombinant h16C3 exhibited similar characteristics to the original m16C3 antibody (Bristol & Kantor, US7829678, 2010) when used as a humanized 16C3 antibody.

[0114] The NEO-201 antibody sequences used in these examples are shown in the following figure. [ka]

[0115] The boundaries between the expression leader sequence, variable region, and constant region are separated by a forward slash (" / ") within each sequence, and the CDR sequences are indicated in bold, underlined text. The antibody sequences used included the variable and constant regions shown. These include the heavy chain CDR1 of SEQ ID NO: 32, the heavy chain CDR2 of SEQ ID NO: 33, the heavy chain CDR3 of SEQ ID NO: 34, the light chain CDR1 of SEQ ID NO: 35, the light chain CDR2 of SEQ ID NO: 36, and the light chain CDR3 of SEQ ID NO: 37. Furthermore, the present invention includes the following preferred embodiments. (1) A method for killing Treg cells in vivo, comprising administering an effective amount of NEO-201 antibody to a patient. (2) A method for enhancing an anti-cancer immune response in a patient, comprising administering an effective amount of NEO-201 antibody to the patient. (3) The method according to (2), further comprising administering a cancer vaccine to the patient. (4) A method for reducing Treg cell infiltration of cancer in a patient, comprising administering an effective amount of NEO-201 antibody to the patient. (5) The method according to any one of (2) to (4), wherein the cancer does not express CEACAM5 or CEACAM6. (6) The method according to any one of (3) to (5), further comprising determining that the cancer is negative for CEACAM5 and CEACAM6 before or during administration. (7) A method for treating or preventing cancer, reducing the cancer burden, or slowing the growth or proliferation rate of cancer, comprising administering an effective amount of NEO-201 antibody to a patient in need thereof, wherein the cancer is CEACAM5 and CEACAM6 negative. (8) The method according to (7), further comprising administering another therapeutic agent to the patient. (9) The other drugs include (a) microtubule inhibitors, topoisomerase inhibitors, platin, alkylating agents, and antimetabolites, and (b) MK-2206, ON 013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platin, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrin, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosph Famide, cytarabine, cytoxane, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechloretamine, melpharen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, rituximab, streptomycin Zosyn, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, and / or vinorelbine, (c) 1-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9->2-hydroxyethoxymethylguanine, adamanthanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugars or sugars The method according to (8), selected from protein synthesis inhibitors, structural protein synthesis inhibitors, adhesion and adsorption inhibitors, and nucleoside analogs such as acyclovir, penciclovir, valacyclovir, and ganciclovir; (d) PD-1 inhibitors, or anti-PD-1 antibodies such as KEYTRUDA® (pembrolizumab) or OPDIVO® (nivolumab); or (e) CTLA-4 inhibitors, or anti-CTLA-4 antibodies such as YLERVOY® ipilimumab. (10) The method according to any one of (7) to (9), wherein the NEO-201 antibody induces or enhances an anti-cancer immune response in the patient. (11) A method for killing Treg cells in vitro, comprising contacting the Treg cells with an antibody NEO-201. (12) The method of (11), further comprising bringing the Treg cells into contact with complement. (13) The method according to (11) or (12), wherein the Treg cells are killed by the CDC. (14) The method according to (11), further comprising bringing the Treg cells into contact with effector cells. (15) The method according to (14), wherein the effector cells include natural killer cells. (16) The method according to (11), (14), or (15), wherein the Treg cells are killed by ADCC. (17) The method according to any one of (1) to (16), wherein the NEO-201 antibody is conjugated to the cytotoxic portion. (18) A method for detecting Treg cells, comprising detecting the expression of the NEO-201 antigen by the Treg cells, wherein the level of Treg cells in a patient sample, such as blood or a biopsy sample, is used to diagnose cancer or determine the progression of cancer. (19) The method of (18), comprising contacting the Treg cells with the NEO-201 antibody, wherein the NEO-201 antibody is optionally conjugated directly or indirectly to a label. (20) The method according to (18) or (19), wherein the detection comprises cell sorting, optionally, fluorescence-activated cell sorting. (21) A method for staining Treg cells, comprising contacting the cells with an antibody NEO-201. (22) The method according to (21), wherein the NEO-201 antibody is conjugated directly or indirectly to the label. (23) A method for isolating Treg cells, comprising isolating cells expressing the NEO-201 antigen. (24) The method of (23), comprising contacting a sample containing Treg cells with an antibody NEO-201, wherein the antibody NEO-201 is optionally labeled directly or indirectly. (25) The method of (24), wherein the sample is blood or bone marrow, or contains thereof. (26) The method according to any one of (23) to (25), comprising isolating NEO-201-positive Treg cells from NEO-201-negative cells. (27) The method according to any one of (23) to (26), wherein the Treg cells are isolated by cell sorting, and optionally by fluorescence-activated cell sorting. (28) The method according to any one of (23) to (26), wherein the Treg cells are isolated by contacting the sample with a support containing NEO-201 antibody, thereby retaining the Treg cells on the support. (29) The method according to any one of (1) to (28), wherein the NEO-201 antibody comprises at least one, two, three, four, five, or all six of the CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO: 29. (30) The method according to any one of (1) to (29), wherein the NEO-201 antibody contains a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38. (31) The method according to any one of (1) to (30), wherein the NEO-201 antibody contains a variable light chain sequence having at least 90% identity with SEQ ID NO: 39. (32) The method according to any one of (1) to (31), wherein the NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38 and a variable light chain sequence having at least 90% identity with SEQ ID NO: 39. (33) The method according to any one of (1) to (32), wherein the NEO-201 antibody comprises a heavy chain sequence having at least 90% identity with amino acids 20 to 470 of SEQ ID NO: 28 and a light chain sequence having at least 90% identity with amino acids 20 to 233 of SEQ ID NO: 29. (34) The method according to (32) or (33), wherein the NEO-201 antibody contains all six CDR sequences included in SEQ ID NO: 28 and SEQ ID NO: 29. (35) The method according to any one of (1) to (34), wherein the NEO-201 antibody contains a human IgG1 constant domain. (36) The method according to any one of (1) to (35), wherein the NEO-201 antibody is humanized. (37) The method according to any one of (1) to (36), wherein the NEO-201 antibody is conjugated to another portion. (38) The method according to any one of (1) to (37), wherein the NEO-201 antibody is conjugated to another cytotoxic moiety, label, radioactive moiety, or affinity tag. (39) The method according to any one of (1) to (10), wherein the cancer is selected from hematological malignancies, lung cancer such as non-small cell lung cancer, melanoma, gastrointestinal cancer, ovarian cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, breast cancer, pancreatic cancer, mesothelioma, metastatic renal cell carcinoma, and prostate cancer. (40) The method according to any one of (23) to (28), further comprising genetically modifying the Treg cells and optionally introducing the cells into the patient or another individual.

[0116] Abbreviation Amyotrophic lateral sclerosis (ALS), antibody-dependent cytotoxicity (ADCC), area under the plasma concentration-time curve from time 0 to infinity (AUCinf), dose-normalized area under the plasma concentration-time curve from time 0 to infinity (AUCinf / D), baseline (BL), complement-dependent cytotoxicity (CDC), clearance (CL), maximum observed plasma concentration (Cmax), dose-normalized measured maximum plasma concentration (Cmax / D), estrogen receptor (ER), half-life (HL), immunohistochemistry (IHC), multiple sclerosis (MS) natural killer (NK), non-small cell lung cancer (NSCLC), peripheral blood mononuclear cells (PBMC), progesterone receptor (PR), tumor-associated antigen (TAA), time to maximum observed plasma concentration (Tmax), volume of distribution (Vz).

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Claims

1. A pharmaceutical composition comprising the NEO-201 antibody for use in vivo to kill Treg cells in patients determined to have immunosuppressive Treg cells prior to use, wherein the NEO-201 antibody comprises a variable heavy chain polypeptide comprising the same CDR as contained in SEQ ID NO: 28 and a variable light chain polypeptide comprising the same CDR as contained in SEQ ID NO:

29.

2. (i) The NEO-201 antibody is bound to the cytotoxic portion; (ii) The NEO-201 antibody is (a) comprising a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38 and a variable light chain sequence having at least 90% identity with SEQ ID NO: 39; or (b) comprising a heavy chain sequence having at least 90% identity with amino acids 20-470 of SEQ ID NO: 28 and a light chain sequence having at least 90% identity with amino acids 20-233 of SEQ ID NO: 29; (iii) The NEO-201 antibody contains a human IgG1 constant domain; (iv) The NEO-201 antibody has been humanized; (v) The NEO-201 antibody is conjugated to another portion; or (vi) The NEO-201 antibody is conjugated to a cytotoxic moiety, label, radioactive moiety, or affinity tag; or (vii) is any combination from (i) to (vi), The pharmaceutical composition according to claim 1.

3. A pharmaceutical composition for use according to claim 1 or 2, wherein the patient has cancer and a cancer vaccine is also used.

4. The pharmaceutical composition according to claim 3, wherein the cancer does not express CEACAM5 or CEACAM6.

5. The pharmaceutical composition according to claim 3, wherein the cancer is determined to be negative for CEACAM5 and CEACAM6 before or during use.

6. A pharmaceutical composition according to any one of claims 1 to 5, further comprising the use of other therapeutic agents.

7. The aforementioned other therapeutic agents, (a) Microtubule inhibitors, topoisomerase inhibitors, platin, alkylating agents, and antimetabolites, (b) MK-2206, ON 013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitor, topoisomerase inhibitor, platin, alkylating agent, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxane, dacarbazine, dactinomycin, daunorubicin Syn, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechloretamine, melpharen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, rituximab, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, and / or vinorelbine, (c) 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9->2-hydroxyethoxymethylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, adhesion and adsorption inhibitors, and nucleoside analogs. (d) PD-1 inhibitors or anti-PD-1 antibodies, (e) The pharmaceutical composition according to claim 6, selected from a CTLA-4 inhibitor or an anti-CTLA-4 antibody.

8. The pharmaceutical composition according to any one of claims 3 to 7, wherein the cancer is selected from the group consisting of hematological malignancies, lung cancer, melanoma, gastrointestinal cancer, ovarian cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, breast cancer, pancreatic cancer, mesothelioma, metastatic renal cell carcinoma, and prostate cancer, and the cancer is CEACAM5 and CEACAM6 negative.

9. The pharmaceutical composition according to any one of claims 3 to 7, wherein the cancer is non-small cell lung cancer.

10. A method for killing Treg cells in vitro in a patient sample, The method comprising contacting the patient sample containing Treg cells with NEO-201 antibody and complement, which contains a variable heavy chain polypeptide containing the same CDR as contained in SEQ ID NO: 28 and a variable light chain polypeptide containing the same CDR as contained in SEQ ID NO:

29.

11. The method according to claim 10, further comprising contacting a patient sample containing the Treg cells with effector cells, wherein the effector cells include natural killer cells.

12. A method for killing Treg cells in vitro in a patient sample, A method comprising contacting a patient sample containing Treg cells with a NEO-201 antibody containing a variable heavy chain polypeptide containing the same CDR as contained in SEQ ID NO: 28 and a variable light chain polypeptide containing the same CDR as contained in SEQ ID NO: 29, wherein the NEO-201 antibody is conjugated to a cytotoxic moiety.

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