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 TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 630,084, filed February 13, 2018, which is incorporated by reference in its entirety.

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

[0003] CD4 + CD25 高Increased levels of regulatory T cells (Tregs) have been shown to be a key regulator of 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), 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 2004,10:942). 2005,92:913), 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 has been shown 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 show 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-infiltrating Tregs and overall survival (Curiel JT et al. Nat Med 2004, 10:942). In these patients, DAB conjugated to recombinant interleukin 2 diphtheria toxin has been shown to 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). 389 Treatment with IL2 (denileukin diftitox, ONTAK) reduced Tregs and improved antitumor responses (Barnett B et al. Am J Reprod Immunol 2005,54:369). 389 IL-2, ONTAK) is a fusion protein of human IL-2 and the enzymatic and translocation domains of diphtheria toxin. It binds CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (γ c ) preferentially binds to cells expressing the high affinity IL-2R. After binding to the IL-2R, denileukin diftitox is internalized by endocytosis and inhibits protein synthesis, ultimately resulting in cell death. Denileukin diftitox has also been shown to significantly reduce the number of Tregs in the peripheral blood of patients with metastatic renal cell carcinoma and abolish Treg-mediated immune suppression 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 can enhance vaccine-induced anti-tumor 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 most frequent causes of death worldwide, with an estimated 20 million new cases expected annually as early as 2025 (Ferlay et al., 2015). Traditional cancer treatments, such as surgery, radiation, and chemotherapy, often induce severe side effects and fail to cure most patients with advanced disease, leading to recurrence (Bodey et al., 1996). Recent therapies have been developed to selectively target cancerous cells while largely sparing normal, healthy tissues. Among them, immunotherapy has become an important treatment option for cancer patients, as it revolutionizes the field of cancer treatment.

[0007] The underlying principle of cancer immunotherapy is known as immunoediting (Mittal et al., 2014), which is an extrinsic mechanism of cancer suppression that initiates only after cellular transformation has occurred and endogenous mechanisms of cancer suppression have failed. The immunoediting process takes place in three phases: efflux, equilibrium, and escape. In the efflux and equilibrium phases, respectively, immune rejection of cancer cells predominates or is in equilibrium with cancer cell proliferation, controlling malignant growth. However, in the efflux phase, cancer cells once suppressed may escape immune recognition due to insensitivity to immune effector mechanisms and / or induction of immune suppression 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 generate and / or amplify antitumor immune responses to suppress tumor growth, delay tumor recurrence, and prolong survival, thereby keeping cancer cells in the clearance and / or equilibrium phase (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 exploit adaptive immunity via T cells. However, innate immunity can also generate and enhance antitumor responses, and tumor-targeted monoclonal antibodies (mAbs) can be used to stimulate innate antitumor immunity (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 components of this vaccine were tumor-associated antigens (TAA) derived from pooled tumor membrane fractions from surgically resected specimens from 79 colon cancer patients (Hollinshead et al., 1985). These membrane fractions were semi-purified, screened for delayed-type hypersensitivity (DTH) in colon cancer patients versus healthy volunteers, and evaluated in clinical trials in refractory colorectal cancer patients (Hollinshead et al., 1985, Hollinshead, US4810781, 1989, Bristol & Kantor, US7829678, 2010). These trials reported clinical benefit, defined by both antitumor responses and significant prolongation of overall survival in patients who developed sustained IgG responses in addition to cellular responses 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 previously described ensituximab (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, specifically cancer-associated variants of CEACAM5 and CEACAM6 (Zeligs et al., 2017).

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

[0010] Monoclonal antibodies (mAbs) consist of a unique antigen-binding region (fragment antigen-binding, Fab) that is specific for a given mAb, and a constant region (fragment crystallizable, Fc) that is common to all mAbs of the same isotype. The Fc region can regulate the activity of immune cells by engaging 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 cellular cytotoxicity (ADCC). ADCC has been shown to be a major mediator of antitumor effects in vivo in many preclinical studies and plays an important role in the mechanism of action of several mAbs used in cancer therapy (Seidel et al., 2013). Examples of clinically approved mAbs capable of mediating 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 cytotoxicity (CDC), a proteolytic cascade leading to the formation of pores in the plasma membrane that cause lysis of the cell targeted by the antibody.Even when antitumor CDC has been demonstrated in vitro, there is controversy as to whether it is critical for the clinical efficacy of mAb therapy in cancer ( Meyer et al., 2014 ).

[0011] Applicant's earlier U.S. Patent 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 by reference in their entirety) disclose various anti-cancer antibodies, cancer antigens, and related technology. Summary of the Invention

[0012] We have previously shown that NEO-201 binds to cancer-associated variants of CEACAM5 and CEACAM6, specifically via cancer-associated glycosylation variants of these proteins. NEO-201 is a humanized IgG1 monoclonal antibody derived from an immunogenic preparation of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. NEO-201 reacts with most tumor tissues from many different carcinomas, but not with most normal tissues. Functional analysis revealed that NEO-201 can mediate both antibody-dependent cellular 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 and demonstrates safety and tolerability in non-human primates with a transient reduction in circulating neutrophils being the only adverse effect observed.

[0013] Applicants have shown herein that NEO-201 binds to Treg cells and can therefore be used to purify Treg cells, e.g., activated Treg cells, and also to kill Treg cells. These results were particularly unexpected, since it was believed that the NEO-201 antigen (cancer-associated glycosylation variants of CEACAM5 and CEACAM6) is not expressed by Treg cells. Based on these results, the nature of the NEO-201 antigen has been 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 that have glycosylation that is the same as or similar to the cancer-associated glycosylation of CEACAM5 / 6 that constitutes the NEO-201 antigen.

[0014] Infiltration of Treg cells has been associated with many cancer types, and several studies have shown that selective ablation of Treg cells promotes anti-cancer immune responses. The newly described ability of NEO-201 to kill Treg cells supports the use of NEO-201 to enhance anti-cancer immune responses, regardless of NEO-201 antigen expression by the cancer itself. For example, NEO-201 is expected to enhance anti-tumor immune responses for vaccine drugs. Selective ablation of Treg cells using NEO-201 may be beneficial in 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, whether for research or therapy. The reduced suppressive function of human Treg 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 can be transplanted into individuals with autoimmune conditions to treat the condition. In addition, purified autologous or heterologous Tregs can be engineered and introduced into patients for the treatment of autoimmune diseases.

[0016] The worked example describes experiments performed using PBMCs from normal donors for phenotypic and functional analysis. Tregs were isolated from PBMCs using the EasySep™ StemCell Treg Isolation Kit and Anti-Biotin Kit, either with the manufacturer-provided Treg purification reagents or customized using biotin-labeled NEO-201 mAb. Phenotypic analysis was performed by flow cytometry for the following markers: CD4, CD25, CD127, FoxP3, CD15s, CD45RA, CCR4, NEO-201 antigen, CEACAM5, and CEACAM6. The ability of NEO-201-isolated Tregs to suppress the proliferation of autologous CD4+ T-responder cells was assessed using a Treg co-culture suppression assay. The percentage of NEO-201+ cells in the population of CD4+CD25highCD127-FoxP3+CD15s+CCR4+ Tregs ranged from 61.8% to 81.9%. NEO-201+ Tregs were CD45RA-. Isolated CD4+NEO-201+ Tregs were able to suppress the proliferation of CD4+ T-responder cells.

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

[0018] From these results, it was concluded that NEO-201 reacts with human Tregs and can be used as a novel marker for the identification and purification of Tregs. Tregs isolated using NEO-201 mAb can be functionally suppressed and removed by CDC. ADCC-mediated Treg cell death can also occur based on the ability of the antibody to bind to Treg cells. This application is the first to demonstrate 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 with NEO-201. Percentage of functional Tregs isolated using commercial kits (human CD4+CD127lowCD25+) or selection based on NEO-201 expression. EasySep™ human CD4+CD127lowCD25+ regulatory T cell isolation kit produced 67.14% active Treg cells, whereas selection based on NEO-201 positive expression produced 99.12% active Treg cells. [Diagram 2] Regulatory T cells are CEACAM-5 and CEACAM-6 negative as determined by flow cytometry. Phenotypic analysis of isolated T-reg (EasySep™ Human CD4+CD127lowCD25+ 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 CD4+ / CD25high / CD127- / FOXp3+ cells are NEO-201+ / CEACAM5- and CEACAM6- cells. [Diagram 3] NEO-201 mediates CDC on CD4+CD15s+Treg. Treg cells express CD4 and CD15s (sialyl Lewis X glycan). Complement alone did not kill Treg. NEO-201 mediated CDC on Treg cells. CD4+CD15s+Treg population was reduced by 53.4%. Procedure: CD4+ T cells were isolated from PBMC. Cells were treated with - / +NEO-201 mAb (10 μg / mL). Cells were then 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

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

[0022] The method may further comprise administering a cancer vaccine to the patient.Exemplary 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; Litzinger MT et al., Blood 2007,110:3192, each of which is incorporated herein by reference in its entirety.

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

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

[0025] The cancer may not express CEACAM5 or CEACAM6.

[0026] The method may further include determining that the cancer is CEACAM5 and CEACAM6 negative prior to or at the time of administration, which may optionally be determined by testing for expression of CEACAM5 and CEACAM6 proteins, for example, by staining with an antibody specific for CEACAM5 and / or CEACAM6, such as a cross-reactive antibody that specifically binds both CEACAM5 and CEACAM6.

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

[0028] The method may further comprise administering to the patient another therapeutic agent, such as (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, cyclosporine, cyclopentadiene ... Phosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 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-hydroxy-ethoxymethylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors , sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors, and nucleoside analogues 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 YERVOY® ipilimumab.The combination of immune checkpoint blockade (PD-1 blockade and / or CTLA-4 blockade) with 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 their entirety.

[0029] The NEO-201 antibody may induce or enhance an anti-cancer immune response in a patient.

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

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

[0032] The NEO-201 antibody can be conjugated to a cytotoxic moiety.

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

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

[0035] The detection may involve cell sorting, optionally, fluorescence activated cell sorting.

[0036] In another aspect, the disclosure provides a method of detecting Treg cells, comprising contacting a cell with a NEO-201 antibody and detecting a cell that expresses NEO-201. The NEO-201 antibody can be directly or indirectly labeled.

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

[0038] In another aspect, the present disclosure provides a method of isolating Treg cells, comprising isolating cells expressing the NEO-201 antigen. The method may include contacting a sample containing Treg cells with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly labeled. The sample may be or may 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 may 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 comprising the NEO-201 antibody, whereby the Treg cells are retained on the support.

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

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

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

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

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

[0046] In any of the above or below methods, the NEO-201 antibody can comprise a heavy chain sequence having at least 90% identity to amino acids 20 to 470 of SEQ ID NO:28 and a light chain sequence having at least 90% identity to amino acids 20 to 233 of SEQ ID NO:29.

[0047] In any of the above or below described methods, the NEO-201 antibody may comprise all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

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

[0049] In any of the above or below described methods, the NEO-201 antibody may be humanized.

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

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

[0052] In any 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 any of the above or below methods, the cancer may be 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. The cancer may involve Treg cells.

[0054] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the present invention or testing of the present invention, but suitable methods and materials are described herein. The materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0055] As used in the description and throughout the claims that follow, the meanings of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0056] The term "amino acid" as used herein refers to natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Natural amino acids are those encoded by the genetic code and those amino acids that are 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., an α carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of amino acids, but function in a manner similar to natural amino acids.

[0057] The term "NK depletion" or "natural killer depletion" as used herein refers to patients with low natural killer (NK) cell levels compared to the normal range. NK cells are cytotoxic innate immune lymphocytes. Typically, NK cells comprise 5-20% of peripheral blood mononuclear cells (PBMCs) in healthy individuals. A patient with NK cells comprising less than 5% of PMBCs is referred to as NK depleted. Additionally, if NK cells comprise less than 3% of PMBCs, the patient is referred to as severely NK cell depleted. Additionally, in normal individuals, up to 90% of PBMC NK cells are CD56 dim CD16 + NK cells, which are considered the most cytotoxic subset. Less than 70% of PBMC NK cells express CD56 dim CD16 + If it is NK cells, the patient is said to be NK depleted. In addition, less than 50% of PBMC NK cells express CD56dim CD16 + If it is NK cells, the patient is referred to as severely NK depleted. A given patient may be referred to as NK depleted or severely NK depleted based on meeting either or both of these individual criteria. Generally speaking, the status of a patient as NK depleted or severely NK depleted is determined by testing a sample taken from the patient, for example, a blood sample, for example, a sample obtained and tested one or two weeks ago. The status of a patient as NK depleted or severely NK depleted may also be inferred from the diagnosis of disease and / or course of treatment associated with such depletion of NK cells.

[0058] "Antibody", as used herein, broadly refers to any polypeptide chain-containing molecular structure with 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. A typical antibody molecule is an immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, from all sources, e.g., human, rodent, rabbit, bovine, ovine, porcine, canine, chicken, are considered to be "antibodies". Antibodies include chimeric antibodies, human antibodies, and other non-human mammalian antibodies, humanized antibodies, single chain antibodies (scFv), camelidbodies, nanobodies, IgNAR (single chain antibodies derived from sharks), small modular immunopharmaceuticals (SMIPs), and antibody fragments (e.g., Fab, Fab' 、 F(ab') 2 ), including, but not limited to, the antibody coding sequences described herein. Numerous antibody coding sequences have been described and others 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; 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 NO:28 and SEQ ID NO:29, or the variable regions, optionally together with the constant regions contained therein, as well as fragments and variants thereof. Such variants include sequences comprising one, two, three, four, five or preferably all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29, i.e., 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. Such variants also include antibodies that compete with NEO-201 for binding to the NEO-201 antigen. The antibody may be humanized. The antibody may be expressed comprising one or more leader sequences, which may be removed during expression and / or during antibody processing and secretion. The antibodies may be presented in a monovalent, bivalent, or higher polyvalent format, including, but not limited to, bispecific or multispecific antibodies comprising the NEO-201 antibody sequence and a binding fragment of a different antibody. Typically, the antibodies specifically bind to cancer cells and compete for binding to cancer cells with an antibody comprising a variable heavy chain of SEQ ID NO: 38 and a variable light chain of SEQ ID NO: 39, or comprising a heavy chain of SEQ ID NO: 28 and a light chain of SEQ ID NO: 29. One or more of the CDR sequences contained in SEQ ID NO: 28 and / or SEQ ID NO: 29 may be replaced with a variant sequence, e.g., a light chain CDR1 of SEQ ID NO: 1 or 4, a light chain CDR2 of SEQ ID NO: 2 or 5, a light chain CDR3 of SEQ ID NO: 3 or 6, a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, 10, 30, or 31, a heavy chain CDR3 of SEQ ID NO: 9 or 11, or SEQ ID NO: 30-31. The light chain may comprise the CDRs contained in the light chain sequence of SEQ ID NO: 14, 16, 17, 18, 19, 20, 21, or 29. The heavy chain may comprise the CDRs contained in the heavy chain sequence of SEQ ID NO: 15, 22, 23, 24, 25, 26, 27, or 29.The antibody may comprise a variable heavy chain sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to 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 to SEQ ID NO:39, and optionally the heavy and / or light chain sequences include one, two, three, four, five or preferably all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29, i.e., 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 moiety, such as a cytotoxic moiety, a radioactive moiety, a label, or a purification tag.

[0060] "Antigen" as used herein broadly refers to a molecule or a portion of a molecule to which an antibody can bind, which can further induce an animal to produce an antibody capable of binding to the epitope of that antigen. An antigen may have one epitope or more than one epitope. The specific reaction referred to herein indicates that the antigen reacts in a highly selective manner with its corresponding antibody and not with the multitude of other antibodies that may be elicited by other antigens. An antigen may be tumor-specific (e.g., expressed by neoplastic cells of pancreatic and colon cancer).

[0061] "Cancer," as used herein, refers broadly to any neoplastic disease, whether invasive or metastatic, characterized by abnormal and uncontrolled cell division resulting in malignant growth or tumors.

[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, a "chimeric antibody" refers broadly to an antibody molecule in which the constant region or a portion thereof has been altered, replaced or exchanged so that the antigen binding site (variable region) is conjugated to a constant region of a different or altered class, effector function and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or the variable region or a portion thereof has been altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0064] "Conservatively modified variants" as used herein applies to both amino acid and nucleic acid sequences, and refers broadly to conservatively modified variants with respect to a particular nucleic acid sequence, and refers to nucleic acids that code for identical or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, refers to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. Such nucleic acid variations are "silent variations" and are one species of conservatively modified variations. All nucleic acid sequences herein that code for a polypeptide also represent all possible silent variations 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 AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan).

[0065] As used herein, "complementarity determining region", "hypervariable region", or "CDR" refers broadly to one or more hypervariable or complementarity determining regions (CDRs) found in the variable region of an antibody's light or heavy chain. See Kabat, et al. (1987) "Sequences of Proteins of Immunological Interest" National Institutes of Health, Bethesda, MD. These terms include the hypervariable regions 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. The CDRs within each chain are held in close proximity by framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen binding site. Within the CDRs there are select amino acids described as selectivity determining regions (SDRs) which represent essential contact residues used by the CDRs in antibody-antigen interactions. Kashmiri (2005) Methods 36:25-34.

[0066] "Control amount" as used herein broadly refers to a marker that can be any amount or range of amounts compared to the test amount of the marker.For example, the control amount of a marker can be the amount of the marker in a patient with a particular disease or condition or in a person without such disease or condition.The control amount can be either an absolute amount (e.g., micrograms / ml) or a relative amount (e.g., the relative intensity of signals).

[0067] As used herein, "differentially present" broadly refers to the difference in the quantity or quality of a marker present in a sample taken from a patient with a disease or condition compared to a comparable sample taken from a patient without one of the diseases.A nucleic acid fragment can be differentially present between two samples, for example, when the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in another sample, as measured, for example, by hybridization and / or NAT-based assay.A polypeptide is differentially present between two samples, when the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in another sample.It should be noted that if a marker is detectable in one sample but not detectable in the other sample, such a marker can be considered to be differentially present.Optionally, a relatively small amount of upregulation can serve as a marker.

[0068] "Diagnostic" as used herein refers broadly to identifying the presence or nature of a pathology. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and test negative in the assay are referred to as "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0069] As used herein, "diagnosing" broadly refers to classifying a disease or condition, determining the severity of a disease, monitoring the progression of a disease, and predicting the outcome and / or prognosis of recovery from a disease. The term "detecting" may also optionally encompass any of the foregoing. Diagnosis of a disease according to the present invention may, in some embodiments, be effected by determining the level of a polynucleotide or polypeptide of the present invention in a biological sample obtained from a subject, and the determined level may correlate with a predisposition to a disease, or the presence or absence of a disease. It should be noted that a "biological sample obtained from a subject" may also optionally include a sample that has not been physically removed from the subject.

[0070] An "effective amount" as used herein broadly refers to an amount of a compound, antibody, antigen, or cell that achieves a desired result. An "effective amount" is sufficient when administered to a patient to treat a disease to effect such treatment for the disease. An effective amount can be a prophylactically effective amount and / or a preventatively effective amount. An effective amount can be an amount effective to reduce the occurrence of signs / symptoms, an amount effective to prevent, an amount effective to reduce the severity of the occurrence of signs / symptoms, to eliminate the occurrence of signs / symptoms, to delay the development of the occurrence of signs / symptoms, to prevent the development of the occurrence of signs / symptoms, and / or to effect prevention of the occurrence of signs / symptoms. An "effective amount" can 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. The term "effective amount" is synonymous with "therapeutically effective amount" for purposes of this disclosure.

[0071] "Expression vector" as used herein broadly refers to any recombinant expression system intended to constitutively or inducibly express the nucleic acid sequences of the present disclosure in any cell, including prokaryotic, yeast, fungal, plant, insect, or mammalian cells, in vitro or in vivo. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or are integrated into the genome of a host cell. The expression system may or may not have the ability for autonomous replication, i.e., it may drive only transient expression in the cell. The term includes recombinant expression cassettes that contain only the minimum elements required for transcription of the recombinant nucleic acid.

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

[0073] "Heterologous" as used herein refers broadly to a portion of a nucleic acid, indicating that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, a nucleic acid is typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0074] As used herein, "high affinity" refers to an affinity of at least 10 for a target antigen. -8 M, more preferably at least 10 -9 M, and even more preferably at least 10 -10The term "high affinity" broadly refers to an antibody having a KD of at least 10 M. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is at least 10 -7 M, more preferably at least 10 -8 It refers to an antibody having a KD of M.

[0075] "Homology" as used herein 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. Homology can be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequence is identical. A partially homologous nucleic acid or amino acid sequence is one that is 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" can be used interchangeably with "homology".

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

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

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

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

[0080] As used herein, "isolated" refers broadly to a material that is removed from its original environment in which it naturally occurs and is therefore modified by the hand of man from its natural environment. An isolated material can be, for example, an exogenous nucleic acid contained in a vector system, an exogenous nucleic acid contained in a host cell, or any material that is removed from its original environment and is therefore modified by the hand of man (e.g., an "isolated antibody").

[0081] As used herein, a "label" or a "detectable moiety" refers broadly to a composition detectable by microscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[0082] As used herein, "low stringency," "moderate stringency," "high stringency," or "very stringent conditions" refer broadly to conditions for nucleic acid hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel, et al. (2002) Short Protocols in Molecular Biology (5 thEd.) John Wiley & Sons, NY. Exemplary specific hybridization conditions include: (1) low stringency hybridization conditions: about 45 o C, in 6x sodium / sodium citrate (SSC), then at least 50 o Wash twice in 0.2× SSC, 0.1% SDS at 55°C (low stringency conditions: wash temperature 55°C). o (2) medium stringency hybridization conditions: about 45 C; o 6x SSC, then 60°C o C, washed once or more in 0.2×SSC, 0.1% SDS, (3) high stringency hybridization conditions: about 45 o in 6x SSC at 65°C, then o C, one or more washes in 0.2×SSC, 0.1% SDS, and (4) very high stringency hybridization conditions: 65 o C, 0.5 M sodium phosphate, 7% SDS, then 65 o C, 0.2×SSC, 1% SDS, and one or more washes.

[0083] The term "low level" or "low" as used with respect to a marker such as CD127 is well known in the art and refers to the expression level of a cell marker of interest (e.g., CD127) that is low compared to the expression level of that cell marker in other cells in the cell population being analyzed as a whole. More specifically, the term "low" refers to a distinct cell population that expresses a cell marker at a lower level than one or more other distinct cell populations. Thus, CD127 低 refers to a cell type that stains faintly or dimly when contacted with a labeled CD127 antibody, for example, at a level greater than that of the CD127- subpopulation but less than that of the CD127+ subpopulation.

[0084] "Mammal" as used herein broadly refers to any and all warm-blooded vertebrates of the mammalian class, including humans, characterized by a hair covering on the skin and, in females, milk-producing mammary glands for feeding their young. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cows, 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, bovines, canines, equines, felines, murines, ovines, porcines, primates, and rodents. Mammals also include any and all of those listed in the Mammals of the World maintained by the Smithsonian Museum of Natural History in Washington DC.

[0085] As used herein, "nucleic acid" or "nucleic acid sequence" broadly refers to deoxyribonucleotide or ribonucleotide oligonucleotides in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0086] As used herein, "operably linked" refers broadly to when two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

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

[0088] "Patient" as used herein broadly refers to any animal that requires treatment to either alleviate a disease state or prevent the occurrence or recurrence of a disease state. Also, as used herein, "patient" broadly refers to any animal that has risk factors, medical history, susceptibility, symptoms, signs, and that has been previously diagnosed with a disease, is at risk for a disease, or is a member of a patient population for a disease. A patient may be a clinical patient, such as a human, or a companion animal, livestock animal, farm animal, exotic animal, or zoo animal. The term "subject" may be used interchangeably with the term "patient". In a preferred embodiment of the invention disclosed herein, the patient is a human.

[0089] The terms "polypeptide," "peptide," and "protein" are used interchangeably and refer broadly to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to non-naturally occurring 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] A "promoter" as used herein broadly refers to a set of nucleic acid sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can also optionally include distal enhancer or repressor elements that can be located as far away as several thousand bases from the start site of transcription. 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, a "prophylactically effective amount" refers broadly to an amount of a compound that, when administered to a patient for the prevention of a disease or prevention of recurrence of a disease, is sufficient to effect such prevention of the disease or recurrence. A prophylactically effective amount can be an amount effective to prevent the occurrence of signs and / or symptoms. A "prophylactically effective amount" can vary depending on the disease and its severity, as well as the age, weight, medical history, predisposition to the condition, and pre-existing conditions of the patient being treated.

[0092] "Prevention," as used herein, broadly refers to a course of treatment during which signs and / or symptoms are absent, in remission, or previously present in a patient. Prevention includes preventing a disease from occurring in a patient following treatment of the disease. Additionally, prevention includes treating patients who may potentially develop a disease, particularly those susceptible to a disease (e.g., members of a patient population, those with risk factors, or at risk of developing a disease).

[0093] "Recombinant" as used herein refers broadly to a product, e.g., a cell, or a nucleic acid, protein, or vector, and indicates that the cell, nucleic acid, protein, or vector is derived from the introduction of a heterologous or naturally occurring nucleic acid or protein, or that the cell has been so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses otherwise abnormally expressed, poorly expressed, or not at all native genes.

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

[0095] As used herein, "specifically hybridizable" and "complementary" broadly refer to a nucleic acid being able to form hydrogen bond(s) with another nucleic acid sequence, either by conventional Watson-Crick or other unconventional types. The binding free energy of a nucleic acid molecule and its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, such as RNAi activity. The measurement of binding free energy of a nucleic acid molecule is well known in the art. See, for example, Turner, et al. (1987) CSH Symp. Quant. Biol. LII: 123-33; Frier, et al. (1986) PNAS 83: 9373-77; Turner, et al. (1987) J. Am. Chem. Soc. 109: 3783-85. Percent complementarity refers to the percentage of contiguous 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, 10 out of 10 have at least about 50%, 60%, 70%, 80%, 90%, and 100% complementarity). "Fully complementary" or 100% complementarity refers broadly to all of the contiguous residues of a nucleic acid sequence that hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" refers to polynucleotide strands that exhibit at least about 90% complementarity, except for regions of the polynucleotide strands, such as overhangs, that are selected to be non-complementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under conditions under which the assay is performed in the case of in vitro assays. The non-target sequences typically differ by at least 5 nucleotides.

[0096] A "sign" of disease, as used herein, refers broadly to any abnormality detectable on examination of a patient that is indicative of disease, as opposed to a symptom, which is a subjective indication of disease.

[0097] As used herein, "solid support," "support," and "substrate" refer broadly to any material that provides a solid or semi-solid structure to which another material can be attached, 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] "Subject" as used herein refers broadly to any person suitable for treatment according to the invention disclosed herein, including but not limited to avian and mammalian subjects, preferably mammalian. Mammals in the context of the invention disclosed herein include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates, and humans. Any mammalian subject in need of treatment according to the invention disclosed herein is suitable. Human subjects of both genders and at any stage of development (i.e., neonates, infants, juveniles, adolescents, adults) can be treated according to the invention. The invention may be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, cows, goats, sheep, and horses, for veterinary purposes, as well as for drug screening and drug development purposes. "Subject" is used interchangeably with "patient". In a preferred embodiment of the invention disclosed herein, the subject is a human.

[0099] A "symptom" of disease, as used herein, refers broadly to any morbid phenomenon or departure from the normal in structure, function, or sensation experienced by a patient and that is indicative of disease.

[0100] As used herein, "therapeutic", "therapeutic", "treat" or "treatment" broadly refers to treating a disease, arresting or reducing the development of a disease or its clinical symptoms, and / or alleviating a disease, causing regression of a disease or its clinical symptoms. Therapy includes prevention, treatment, repair, reduction, relief, and / or providing relief of a disease, signs and / or symptoms of a disease. Therapy includes the reduction of signs and / or symptoms in patients with ongoing signs and / or symptoms of a disease (e.g., tumor growth, metastasis). Therapy also includes "prevention". The term "reduced" broadly refers to a clinically significant reduction of signs and / or symptoms for purposes of treatment. Therapy includes treating signs of relapsing or recurrent signs and / or symptoms (e.g., tumor growth, metastasis). Therapy 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. Therapy includes treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing the recurrence or recurrence of signs and / or symptoms (eg, tumor growth, metastasis).

[0101] As used herein, "variable region" or "VR" refers broadly to the domains within each pair of light and heavy chains of an antibody that are directly involved in binding the antibody to an antigen. Each heavy chain contains a variable domain (VR) at one end. H ), followed by several constant domains. Each light chain has at one end a variable domain (V L ) at its other end and a constant domain, where 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, a "vector" refers broadly to a plasmid, cosmid, phagemid, phage DNA, or other DNA molecule capable of autonomously replicating in a host cell and characterized by one or a small number of restriction endonuclease recognition sites, such DNA sequences may be cleaved in a determinable manner without loss of the essential biological function of the vector, and into which DNA may be inserted to effect its replication and cloning. A vector may further contain a marker suitable for use in identifying cells transformed with the vector.

[0103] Techniques and procedures are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, see, for example, Sambrook, et al. (2001) Molec. Cloning: Lab. Manual [3 rd [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). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. Nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used in chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. EXAMPLES

[0104] The invention generally described herein will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0105] Example 1 NEO-201 is capable of isolating functional Treg cells.

[0106] This example shows that cell sorting based on NEO-201 binding produces a high percentage of functional Tregs. The percentage of functional Tregs isolated using a commercial kit (human CD4+CD127lowCD25+) or by selection based on NEO-201 expression is shown in Figure 1. The EasySep™ human CD4+CD127lowCD25+ regulatory T cell isolation kit produced 67.14% active Treg cells, whereas selection based on NEO-201 positive expression produced 99.12% active Treg cells.

[0107] 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 basis for NEO-201 binding to Treg cells, cells were tested for CEACAM5 and CEACAM6 expression by flow cytometry. Regulatory T cells are CEACAM-5 and CEACAM-6 negative (Figure 2).

[0108] Methods Binding of human Treg markers to isolated human Tregs was analyzed by flow cytometry. Tregs (1.0 × 10 6) were incubated with 1 μL of LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) per test in 1× phosphate-buffered saline (PBS) for 30 min at 4°C to distinguish between live and dead cells. Cells were then centrifuged, washed twice with cold PBS, and then stained with Pacific Blue-conjugated NEO-201 antibody, anti-CD25-APC-H7, anti-CD15s-Alexa 488, anti-CD127-APC in 1× PBS + 1% BSA (Teknova, Hollister, CA, USA), and intracellularly stained with anti-Foxp3-PerCP-CY5.5. (BioLegend, San Diego, CA) for 30 min at 4°C. After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Fluorescence analysis of cells was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Isolation of T-regs with commercial kits was performed using the EasySep™ Human CD4+CD127lowCD25+ 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 CD4+ / CD25high / CD127- / FOXp3+ cells are NEO-201+ / CEACAM5- and CEACAM6- cells.

[0109] Example 2 NEO-201 can kill Tregs by complement-dependent cytotoxicity (CDC).

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

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

[0112] Example 3 Generation of humanized NEO-201 monoclonal antibody

[0113] Hollinshead colon cancer specific vaccine was used as an immunogen to generate monoclonal antibodies in mice. Methods for preparation of tumor associated proteins and peptides have been described previously (Hollinshead, US4810781, 1989). Briefly, cancer tissue was minced and used to generate a single cell suspension, which was then subjected to hypotonic saline membrane extraction, a series of centrifugation steps, followed by low frequency sonication. The resulting membrane extracted proteins were fractionated, concentrated and quantified using Sephadex G-200 resin or by electrophoresis (Hollinshead et al, 1970; Hollinshead et al., 1972; Hollinshead et al., 1985). TAA preparations were mixed with Freund's complete adjuvant and injected subcutaneously into BALB / c mice. This was followed by three booster injections in Freund's incomplete adjuvant at 2-3 week intervals. Mouse sera were tested by ELISA for antibody responses to the immunizing antigen, and mice with strong responses were used to generate immortalized hybridoma cells by fusing mouse B cells from the spleen with the SP2 / 0-Ag14 myeloma cell line to grow and select cells that produced mouse immunoglobulin (IgG). From these mouse IgG, the mouse 16C3 clone (m16C3) was selected based on its reactivity with colon tumor cell membrane extracts from LS174T or HT-29 cells as determined by ELISA. cDNAs encoding the heavy and light IgG1 chains were determined from RNA isolated from the hybridoma clone 16C3 E12 and shown to be unique (Bristol & Kantor, US7829678,2010). The m16C3 protein sequence was humanized as h16C3 and designated NEO-201. Humanization was performed in silico by replacing mouse sequences outside the complementarity determining regions (CDRs) of the Fab regions of both the heavy and light chain proteins with human Fab sequences, retaining the three mouse CDR sequences from each chain. The Fc regions of the heavy and light chains were selected from the human IgG1 isotype used in other humanized approved mAb products.The amino acid sequence was reverse translated into DNA optimized for protein expression in CHO cells. The DNA of heavy and light chain h16C3 was 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 derived and stored. The purified recombinant h16C3 humanized 16C3 antibody had similar characteristics as the original m16C3 antibody (Bristol & Kantor, US7829678,2010).

[0114] The NEO-201 antibody sequence used in these examples is included in the figure below. [ka]

[0115] The boundaries between the expression leader sequence, the variable region, and the constant region are separated by a forward slash (" / ") in each sequence, and the CDR sequences are shown in bold, underlined text. The antibody sequences used contained the variable and constant regions shown. These include a heavy chain CDR1 of SEQ ID NO:32, a heavy chain CDR2 of SEQ ID NO:33, a heavy chain CDR3 of SEQ ID NO:34, a light chain CDR1 of SEQ ID NO:35, a light chain CDR2 of SEQ ID NO:36, and a light chain CDR3 of SEQ ID NO:37. The present invention also includes the following preferred embodiments. (1) A method for killing Treg cells in vivo, comprising administering to a patient an effective amount of a NEO-201 antibody. (2) A method for enhancing an anti-cancer immune response in a patient, the method comprising administering to the patient an effective amount of a NEO-201 antibody. (3) The method according to (2), further comprising administering a cancer vaccine to the patient. (4) A method for reducing Treg cell infiltration in a cancer patient, the method comprising administering to the patient an effective amount of a NEO-201 antibody. (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 CEACAM5 and CEACAM6 negative before or at the time of the administration. (7) A method for treating or preventing cancer, reducing the burden of cancer, or slowing the growth or proliferation rate of cancer, comprising administering an effective amount of a 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 to the patient another therapeutic agent. (9) The other drugs are (a) microtubule inhibitors, topoisomerase inhibitors, platins, alkylating agents, and metabolic antagonists, (b) MK-2206, ON 013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platins, alkylating agents, metabolic antagonists, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide ... amide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, rituximab, strepto 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-hydroxy-ethoxymethylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugar or glycotans. (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 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 a NEO-201 antibody. (12) The method according to (11), further comprising contacting the Treg cells with complement. (13) The method according to (11) or (12), wherein the Treg cells are killed by CDC. (14) The method according to (11), further comprising contacting the Treg cells with effector cells. (15) The method according to (14), wherein the effector cells comprise 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 a cytotoxic moiety. (18) A method for detecting Treg cells, comprising detecting expression of the NEO-201 antigen by the Treg cells, optionally wherein the level of Treg cells in a patient sample, such as a blood or biopsy sample, is used to diagnose or determine the progression of cancer. (19) The method according to (18), comprising contacting the Treg cells with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly bound 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, the method comprising contacting the cells with a NEO-201 antibody. (22) The method according to (21), wherein the NEO-201 antibody is directly or indirectly bound to a label. (23) A method for isolating Treg cells, the method comprising isolating cells that express the NEO-201 antigen. (24) The method according to (23), comprising contacting a sample containing Treg cells with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly labeled. (25) The method according to (24), wherein the sample is or comprises blood or bone marrow. (26) The method according to any one of (23) to (25), comprising separating 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, 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 a sample with a support containing a NEO-201 antibody, thereby retaining the Treg cells on the support. (29) The method described in any 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 of (1) to (29), wherein the NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity to SEQ ID NO:38. (31) The method according to any of (1) to (30), wherein the NEO-201 antibody comprises a variable light chain sequence having at least 90% identity to SEQ ID NO:39. (32) The method according to any of (1) to (31), wherein the NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity to SEQ ID NO: 38 and a variable light chain sequence having at least 90% identity to SEQ ID NO: 39. (33) The method according to any of (1) to (32), wherein the NEO-201 antibody comprises a heavy chain sequence having at least 90% identity to amino acids 20 to 470 of SEQ ID NO: 28 and a light chain sequence having at least 90% identity to amino acids 20 to 233 of SEQ ID NO: 29. (34) The method according to (32) or (33), wherein the NEO-201 antibody comprises all six of the CDR sequences contained 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 comprises 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 moiety. (38) The method according to any one of (1) to (37), wherein the NEO-201 antibody is conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an 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 cellular 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), maximum observed plasma concentration normalized by dose (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 of maximum observed plasma concentration (Tmax), volume of distribution (Vz).

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Claims

1. A method of killing Treg cells in vivo, comprising administering to a patient an effective amount of a NEO-201 antibody.

2. A method of enhancing an anti-cancer immune response in a patient, comprising administering to said patient an effective amount of a NEO-201 antibody.

3. 3. The method of claim 2, further comprising administering a cancer vaccine to the patient.

4. A method of reducing Treg cell infiltration of cancer in a patient, comprising administering to the patient an effective amount of a NEO-201 antibody.

5. The method of any one of claims 2 to 4, wherein the cancer does not express CEACAM5 or CEACAM6.

6. The method of any one of claims 3 to 5, further comprising determining that the cancer is CEACAM5 and CEACAM6 negative prior to or at the time of said administering.

7. A method of treating or preventing cancer, reducing the burden of cancer, or slowing the growth or proliferation rate of cancer, comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof, wherein the cancer is CEACAM5 and CEACAM6 negative.

8. 8. The method of claim 7, further comprising administering to the patient another therapeutic agent.

9. The other therapeutic agent is (a) a microtubule inhibitor, a topoisomerase inhibitor, a platin, an alkylating agent, and an antimetabolite, (b) MK-2206, ON 013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, a microtubule inhibitor, a topoisomerase inhibitor, a platin, an alkylating agent, an antimetabolite, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide ... Famide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, rituximab, streptomycin, tozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, and / or vinorelbine, (c) 1-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9->2-hydroxy-ethoxymethylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugars or sugars.

9. The method of claim 8, wherein the therapeutic agent is selected from: protein synthesis inhibitors, structural protein synthesis inhibitors, adhesion and adsorption inhibitors, and nucleoside analogues 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 YERVOY® ipilimumab.

10. The method of any one of claims 7 to 9, wherein the NEO-201 antibody induces or enhances an anti-cancer immune response in the patient.

11. A method of killing Treg cells in vitro, comprising contacting said Treg cells with a NEO-201 antibody.

12. The method of claim 11, further comprising contacting the Treg cells with complement.

13. The method of claim 11 or 12, wherein the Treg cells are killed by CDC.

14. The method of claim 11, further comprising contacting the Treg cells with effector cells.

15. The method of claim 14, wherein the effector cells comprise natural killer cells.

16. The method of claim 11, 14, or 15, wherein the Treg cells are killed by ADCC.

17. 2. The method of any one of the preceding claims, wherein the NEO-201 antibody is conjugated to a cytotoxic moiety.

18. A method for detecting Treg cells, comprising detecting expression of the NEO-201 antigen by said Treg cells, optionally wherein the level of Treg cells in a patient sample, such as a blood or biopsy sample, is used to diagnose or determine the progression of cancer.

19. 19. The method of claim 18, comprising contacting the Treg cells with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly conjugated to a label.

20. 20. The method of claim 18 or 19, wherein the detection comprises cell sorting, optionally fluorescence activated cell sorting.

21. A method of staining Treg cells, comprising contacting the cells with a NEO-201 antibody.

22. The method of claim 21, wherein the NEO-201 antibody is directly or indirectly conjugated to a label.

23. A method for isolating Treg cells, the method comprising isolating cells that express the NEO-201 antigen.

24. 24. The method of claim 23, comprising contacting a sample containing Treg cells with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly labeled.

25. 25. The method of claim 24, wherein the sample is or comprises blood or bone marrow.

26. The method of any one of claims 23 to 25, comprising separating NEO-201 positive Treg cells from NEO-201 negative cells.

27. The method of any one of claims 23 to 26, wherein the Treg cells are isolated by cell sorting, optionally by fluorescence activated cell sorting.

28. The method of any one of claims 23 to 26, wherein the Treg cells are isolated by contacting the sample with a support comprising a NEO-201 antibody, whereby the Treg cells are retained on the support.

29. The method of any one of the preceding claims, 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 of any one of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity to SEQ ID NO:

38.

31. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a variable light chain sequence having at least 90% identity to SEQ ID NO:

39.

32. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity to SEQ ID NO:38 and a variable light chain sequence having at least 90% identity to SEQ ID NO:

39.

33. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a heavy chain sequence having at least 90% identity to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence having at least 90% identity to amino acids 20-233 of SEQ ID NO:

29.

34. The method of claim 32 or 33, wherein the NEO-201 antibody comprises all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:

29.

35. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a human IgG1 constant domain.

36. 2. The method of any one of the preceding claims, wherein the NEO-201 antibody is humanized.

37. 2. The method of any one of the preceding claims, wherein the NEO-201 antibody is conjugated to another moiety.

38. 2. The method of any one of the preceding claims, wherein the NEO-201 antibody is conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

39. 11. The method of any one of claims 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 of any one of claims 23 to 28, further comprising genetically modifying said Treg cells and, optionally, introducing said cells into said patient or another individual.

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