Monoclonal antibody NEO-201 for treatment of human cancer

Abstract

NEO-201 is a humanized IgG1 monoclonal antibody (mAb) that is highly reactive against the majority of tumor tissues from a number of different cancer tumors, including colon, pancreatic, gastric, lung, breast and uterine cancers, but the majority of normal tissues are not recognized by such antibodies. Functional assays reveal that NEO-201 is capable of mediating both antibody dependent cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) against tumor cells. Furthermore, the growth of human pancreatic xenograft tumors in vivo is greatly weakened by using NEO-201 alone and in combination with human peripheral blood mononuclear cells (PBMCs) that are effector cell sources for ADCC. In-vivo biological distribution research in mice carrying human tumor xenografts reveals that NEO-201 is preferentially accumulated in tumors rather than organ tissues. Single dose toxicity studies in non-human primates demonstrate the safety and tolerance of NEO-201 because the transient reduction of circulating neutrophils is the sole associated side effect observed.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201880085205.0, filed on November 2, 2018, entitled "Monoclonal antibody NEO-201 for the treatment of human cancer".

[0002] The relevant application for disclosure of information

[0003] This application claims the benefits of U.S. Provisional Application Serial No. 62 / 592,778, filed November 30, 2017, and U.S. Provisional Application Serial No. 62 / 581,380, filed November 3, 2017, each of which is hereby incorporated by reference in its entirety.

[0004] Sequence List Information

[0005] This application includes, as part of its disclosure, a biological sequence list in the form of a file named “43282o4402.txt”, which was created on November 2, 2018, and is 32,563 bytes in size, and is hereby incorporated in its entirety by reference. Background Technology

[0006] Cancer represents one of the leading causes of death worldwide, with an estimated 20 million new cases annually projected as early as 2025 (Ferlay et al., 2015). Conventional cancer treatments such as surgery, radiation, and chemotherapy often cause severe side effects and fail to cure most patients with advanced disease, leading to recurrence (Bodey et al., 1996). Recent treatment modalities have evolved to selectively target cancerous cells while largely avoiding normal healthy tissue. Among these, immunotherapy has become a significant treatment option for cancer patients, as it has revolutionized the field of cancer medicine.

[0007] The underlying principle of cancer immunotherapy is known as immune editing (Mittal et al., 2014), which is an external mechanism of cancer suppression that begins only after cellular transformation has occurred and the intrinsic mechanisms of cancer suppression have failed. The immune editing process occurs in three phases: elimination, equilibrium, and escape. In the elimination and equilibrium phases, immune rejection of cancer cells dominates or balances with cancer cell proliferation to control malignant growth, respectively. However, in the escape phase, once kept under surveillance, cancer cells can evade immune recognition due to insensitivity to immune effector mechanisms and / or induced immune suppression in the tumor microenvironment. Cancer cells that escape immune recognition are then able to proliferate more freely and grow into clinically apparent disease (Dunn et al., 2004). The goal of tumor immunotherapy is to keep cancer cells in the elimination and / or equilibrium phases by generating and / or amplifying anti-tumor immune responses to counteract tumor growth, delay tumor recurrence, and prolong survival (Carter, 2001; Hodge et al., 2006; Vergati et al., 2010; Gabitzsch et al., 2015). Treatment options include using checkpoint inhibitory antibodies, antitumor vaccines, and chimeric antigen receptor (CAR)-T cell therapy, all of which utilize adaptive immunity from T cells. However, innate immunity can also generate and enhance antitumor responses, and tumor-targeting monoclonal antibodies (mAbs) can be used to stimulate innate antitumor immunity (Topalian et al., 2011).

[0008] NEO-201 is a novel humanized IgG1 mAb produced for the Hollinshead allogeneic colorectal cancer vaccine platform (Hollinshead et al., 1970; Hollinshead et al., 1972). The immunogenic component of this vaccine is a tumor-associated antigen (TAA) derived from tumor cell membrane fractions collected from surgically removed specimens of 79 colorectal cancer patients (Hollinshead et al., 1985). These cell membrane fractions were semi-purified, screened for delayed-type hypersensitivity (DTH) in colorectal cancer patients compared to healthy volunteers, and evaluated in clinical trials in patients with refractory colorectal cancer (Hollinshead et al., 1985; Hollinshead, US4810781, 1989; Bristol and Kantor, US Patent No. 7829678, 2010). These trials reported clinical benefits, defined as significant prolongation of antitumor response and overall survival, in patients who developed sustained IgG responses in addition to cell-mediated responses to the vaccine, suggesting that the vaccine contains 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 ensciximab (NPC-1C / NEO-102) (Luka et al., 2011; Patel et al., 2013; Beg et al., 2016; Kim et al., 2017) and NEO-201. Preliminary investigations have shown that NEO-201 can bind to tumor-associated variants of CEACAM family members (Zeligs et al., 2017), and efforts are underway to further characterize one or more antigens and one or more specific epitopes recognized by NEO-201.

[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: CEACAM and pregnancy-specific glycoproteins. Proteins encoded by CEACAM include CEA (CEACAM5), CEA-associated cell adhesion molecules (CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, and CEACAM8). The CEACAM family belongs to the Ig superfamily. Structurally, each of the human CEACAMs contains an N-terminal domain comprising 108–110 amino acids and homologous to the Ig variable domain, followed by varying numbers (0–6) of Ig molecules. C2-type constant-like domain. The CEACAM proteins can interact with each other both isotropically and heterotropically. CEACAM1 is a unique protein in this family because it contains an ITIM (anti-receptor tyrosine-based inhibitory motif)-like PD1 in its cytoplasmic domain. This inhibition is triggered by phosphorylation of tyrosine residues with ITIM, leading to the recruitment of tyrosine phosphatases-1 and-2 containing the Src homology 2 domain. The CEACAM1 protein is expressed on a variety of immune cells, including monocytes, granulocytes, activated T cells, B cells, and NK cells. CEACAM1 exists in several isoforms, with two main isoforms being CEACAM1-L and CEACAM1-S, which have long (L) or short (S) cytoplasmic domains, respectively. CEACAM1-S expression is completely absent in human leukocytes. CEACAM1-L is expressed on a subset of activated human NK cells that are CD16-negative but CD56-positive.

[0010] Monoclonal antibodies (mAbs) consist of a unique antigen-binding region (antigen-binding fragment, Fab) specific to a given mAb and a constant region (crystallizable fragment, Fc) common to all mAbs of the same isotype. The Fc region can modulate immune cell activity by binding to members of the Fc receptor (FcR) family expressed on the surface of specific immune cell types. In particular, human IgG1 mAbs can interact with Fcγ receptor IIIa receptors (FcγRIIIa, CD16) expressed on macrophages and NK cells. This interaction can stimulate macrophages to phagocytose cancer cells opsonized by the mAb and can 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 key mediator of in vivo antitumor activity 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 that mediate ADCC include trastuzumab, which targets the HER2 receptor in breast cancer (Seidel et al., 2013;). Seidel et al., 2013; rituximab, which targets the pan-B cell marker CD20 in lymphoma (Seidel et al., 2013; Dall'Ozzo et al., 2004); cetuximab, which targets the epidermal growth factor receptor (EGFR) in colorectal and head and neck cancer (Seidel et al., 2013; Levy et al., 2009; Kawaguchi et al., 2007; Lopez-Albaitero et al., 2009); and avirumab, which targets the immunosuppressive ligand PD-L1 in Merkel cell carcinoma and bladder cancer (Boyerinas et al., 2015). Additionally, the Fc region can potentially interact with the C1 complex to activate complement-dependent cytotoxicity (CDC), where the protein hydrolysis cascade peaks when gaps are formed in the cytoplasmic membrane (causing lysis of the antibody-targeted cells). Even with in vitro evidence demonstrating antitumor CDC, whether antitumor CDC is crucial for the clinical efficacy of mAb therapy in cancer remains controversial (Meyer et al., 2014).

[0011] The applicant’s previous U.S. patents 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 hereby incorporated herein by reference in its entirety) disclose various anticancer antibodies, cancer antigens and related technologies. Summary of the Invention

[0012] The studies described in this embodiment evaluated the in vitro binding characteristics, in vivo activity, and localization of NEO-201 in preclinical models. NEO-201 exhibited broad reactivity against a range of human carcinoma cell lines and tumor tissues, but binding to most healthy tissues was not observed. Furthermore, NEO-201 demonstrated ADCC and CDC activity against human carcinoma cells in vitro and largely attenuated the growth of human pancreatic xenograft tumors in vivo, both alone and in combination with human peripheral blood mononuclear cells (PBMCs) as effector cells for ADCC. Finally, single-dose toxicity studies in non-human primates demonstrated the safety and tolerability of NEO-201, with a transient decrease in circulating neutrophils being the only observed side effect. These studies provide the basic principles for the potential clinical utility of NEO-201 as a novel therapeutic agent for a wide variety of solid tumors. Moreover, the observed CDC activity of the subject antibody opens up opportunities for treating immunocompromised patients in whom ADCC is not expected to be effective, such as those whose immunocompromise is due to their disease or as a result of treatment with radiation, chemotherapy, and other diseases.

[0013] Previous reports have described the preclinical antitumor activity (Patel et al., 2013) and clinical safety and efficacy (Beg et al., 2016; Kim et al., 2017) of a mAb derived from the Hollinshead allogeneic colorectal cancer vaccine platform (called ensiximab (NPC-1C / NEO-102)). This report describes the characterization of a second tumor antigen-targeting mAb (called NEO-201) derived from the same vaccine platform. NEO-201 was shown to be positively stained in vitro in a variety of human cancer cell lines, including cells derived from a variety of tumor types, histological subtypes, and mutation profiles. NEO-201 positivity was more frequently observed in tumor cell lines derived from lung adenocarcinoma versus squamous cell carcinoma and HER2-positive breast cancer cell lines versus triple-negative cancer cell lines. Staining of human tumor samples demonstrated that a wide variety of cancer tissues were positive for NEO-201, including tumors of the colon, pancreas, stomach, lung, breast, and uterus. Expanded investigations using larger sample sizes have revealed that NEO-201 can distinguish between histological and molecular subtypes in various cancers. Interestingly, a higher proportion of tumor tissues responded to NEO-201 compared to cultured cancer cell lines. This observation suggests that targets recognized by NEO-201 are more readily expressed in vivo than in vitro, indicating that target expression depends at least in part on interactions with tumor cells from factors within the local microenvironment. Current experiments are underway to further characterize one or more antigens and one or more epitopes recognized by NEO-201 and to identify known or multiple regulatory mechanisms controlling their expression in tumor tissues rather than normal tissues.

[0014] This study also revealed that NEO-201 is significantly tumor-specific in its staining spectrum, as the vast majority of healthy normal tissues and normal tissues adjacent to tumors were found to be negative for NEO-201. Although NEO-201 positivity was observed in normal tongue and extracervical tissues, the staining intensity was weak, and the microarrays indicated only minimal sample amounts (n=2). Further expanded analysis of NEO-201 staining in normal tissue samples will be conducted to confirm these observations. Furthermore, NEO-201 administration did not induce any observable serious toxicity in mice and was well tolerated when administered to non-human primates. The observed reduction in neutrophils in non-human primates suggests that one or more antigens responding to NEO-201 are expressed on these immune cells, and assessment of the reactivity of NEO-201 with hematopoietic cell types will be performed. These encouraging results indicate that 1) NEO-201 has diagnostic utility in differentiating cancerous from benign tissue obtained from patient biopsies; and 2) NEO-201 effectively targets tumors without causing significant toxicity or off-target effects other than neutropenia. Efforts are underway to further evaluate the safety and tolerability of NEO-201, and clinical trials using NEO-201 to treat cancer are being planned.

[0015] Innate immune effector mechanisms have been shown to play a major role in promoting and enhancing host antitumor immunity. It is well known that the Fc moiety of human IgG1 mAbs activates innate immunity against opsonized targets, potentially mediating ADCC and / or CDC (Strome et al., 2007; Hayes J et al., 2017). In particular, the ability to mediate ADCC is considered a key component of the therapeutic efficacy of various human IgG1 mAbs approved for cancer treatment (Boyerinas et al., 2015; Seidel et al., 2013; Petricevic et al., 2013; Dall'Ozzo et al., 2004; Levy et al., 2009; Kawaguchi et al., 2007; Lopez-Albaitero et al., 2009). Importantly, the V158F polymorphism of the FCGR3A gene (encoding FcγRIIIa) is associated with differential affinity for human IgG1 mAbs (Koene et al., 1997; Wu et al., 1997), where immune cells with a high-affinity V / V genotype from the donor exhibit greater trastuzumab-mediated ADCC activity in vitro (Musolino et al., 2008). This V / V genotype has also shown a significant association with objective response rate and progression-free survival in breast cancer patients treated with trastuzumab (Musolino et al., 2008), thus providing indirect clinical evidence for the role of ADCC in mAb-based therapies. NEO-201 can mediate ADCC in vitro because treatment of tumor cells with NEO-201 enhances the cytotoxic activity of NK cells by 2–5 times, and ADCC activity is retained even at low antibody concentrations (0.1 μg / mL). These data raise the possibility that patients with the V / V genotype may derive increased benefit from NEO-201 treatment. Another promising avenue is the potential to enhance ADCC activity and, possibly, the potential clinical benefits of NEO-201 through enhanced NK cell function and cytokine stimulation. IL-2 is well-known as a potent activator of NK cells (Hank et al., 1990), and IL-21 has been shown to enhance ADCC activity mediated by trastuzumab and cetuximab (Watanabe et al., 2010). Recent preclinical studies of a novel fusion protein hyperagonist of IL-15 signaling (called ALT-803) have demonstrated significantly enhanced proliferation, activation, and lysis of NK cells (and CD8+ T cells), resulting in significant antitumor activity in various animal models of cancer (Han et al., 2011; Gomes-Giacoia et al., 2014; Mathios et al., 2016; Rhode et al., 2016; Kim et al., 2016; Felices et al., 2017).Interestingly, ALT-803 was found to significantly enhance in vitro NK cell degranulation, IFN-γ production, and rituximab-mediated ADCC against B-cell lymphoma cell lines and primary follicular lymphoma cells. Furthermore, in both B-cell lymphoma models, combination therapy with ALT-803 and rituximab produced significantly reduced tumor cell burden and improved survival in vivo (Rosario et al., 2016).

[0016] Another innate immune effector mechanism potentially bound by mAbs is the activation of the complement system to promote CDC, and NEO-201 has been found to mediate CDC to kill tumor cells. The contribution of CDC to the therapeutic efficacy of mAbs is controversial, but it has been suggested to be beneficial for cancer therapy, at least in certain specific cases (Meyer et al., 2014). Furthermore, several different complement regulatory proteins (CRPs) act to inhibit complement activation, and certain membrane-bound CRPs such as CD46, CD55, and CD59 have been reported to be aberrantly expressed in various cancers (Seya et al., 1994; Niehans et al., 1996; Donin et al., 2003), which may confer resistance to CDC. Future investigations will determine whether strategies to block CRPs can enhance NEO-201-mediated CDC in tumor-resistant cells.

[0017] In vivo evaluation of NEO-201 revealed significant antitumor activity when administered in combination with activated human immune effector cells. This combination even resulted in complete remission in some mice (5 / 20, 25%) from both combination groups. Furthermore, NEO-201 was found to preferentially localize to xenograft tumor tissues, but not to various healthy tissues. These data confirm that the mechanism of action of NEO-201 against tumors is ADCC-dependent lysis of tumor cells by innate immune cells. However, it should be noted that antitumor activity was also observed with NEO-201 alone in immunodeficient mice without the addition of human immune cells. This phenomenon may be condition-specific to in vivo conditions, as treatment of CFPAC-1 tumor cells with NEO-201 did not induce significant toxicity in in vitro ADCC assays. One hypothesis for the activity of NEO-201 in the absence of immune effector cells could be CDC induction. The CDC activity of NEO-201 was directly demonstrated in further experiments described in Example 3.

[0018] In summary, this study demonstrates that NEO-201 is a significant tumor-specific antibody capable of engaging both ADCC and CDC mechanisms to kill tumor cells. Furthermore, NEO-201 exhibits safety and antitumor efficacy in an in vivo xenograft model of pancreatic cancer, as well as tolerability in non-human primates. These findings provide supporting fundamental principles for the clinical development of NEO-201 as a diagnostic and therapeutic agent for patients with a wide range of cancers. The results also support the use of NEO-201 in immunocompromised patients (with low NK cell levels), as the antitumor effect can be generated by CDC even in the absence of robust ADCC activity. Attached Figure Description

[0019] Figure 1 A-1D: Flow cytometry analysis of NEO-201 binding to human cancer cell lines. Representative human cancer cell lines with various levels of NEO-201 antigen expression, ( Figure 1 A) Pancreatic CFPAC-1 (high), ( Figure 1 B) NSCLC H441 (Medium), ( Figure 1 C) Breast HCC 1937 (low), and ( Figure 1 D) Colon SW1116 (negative). For each cell line, results are expressed as NEO-201 positivity % and mean fluorescence intensity (MFI). Red, NEO-201 stained cells; black, unstained cells. NEO-201 positivity was defined as positivity ≥10%.

[0020] Figure 2A-2C IHC staining of human tumor samples using NEO-201. Figure 2A Representative NEO-201 staining of adjacent normal and malignant tissues from colon, pancreas, stomach, and lung samples. All images were obtained at 100X. Figure 2B Quantification of NEO-201 positive staining in human tumor microarray samples from various cancerous tissues. Figure 2C Quantification of NEO-201 positive staining in human tumor microarray samples from normal tissue adjacent to tumor tissue. n = number of samples.

[0021] Figures 3A-3C NEO-201 mediates ADCC and CDC targeting human tumor cell lines. Figure 3A CFPAC-1 or ASPC-1 cells were used as target cells to measure ADCC activity. Cells were treated with 10 μg / mL NEO-201 or human IgG1 (negative control). Purified NK cells from two healthy donors were used as effector cells at the indicated E:T ratio. *Statistically significant by t-test (p<0.05). Figure 3B ADCC of CFPAC-1 cells treated with an increased dose of NEO-201 was measured using NK cells isolated from healthy donors at an E:T ratio of 12.5:1, which were used as effector cells. The figure depicts the fold increase (%) in specific lysis of tumor cells treated with NEO-201 compared to control cells treated with 10 μg / mL human IgG1. *, statistically significant by t-test (p<0.05). Figure 3C CDC assays were performed using ASPC-1 cells treated with the indicated dose of NEO-201 for the indicated duration. * The results were statistically significant by t-test (p<0.05).

[0022] Figures 4A-4D The antitumor efficacy of NEO-201 in CFPAC-1 tumor xenografts. Figure 4A Tumor volume measurements of CFPAC-1 xenografts from each treatment group at different time points. Mice (n = 10 animals / group) were administered saline solution, human IgG1 (250 μg), or NEO-201 (100 and 250 μg) intraperitoneally on days 13, 17, and 20 after tumor cell engraftment. Approximately 1.0 x 10⁻⁶ g of [agent name missing] was also administered intraperitoneally to mice on days 14, 18, and 21. 7 One IL-2 activated PBMC. Figure 4B ) Quantification of the number of mice still carrying palpable tumors on day 36. Figure 4C Representative images of tumor-bearing mice treated with NEO-201 compared to those treated with saline. Figure 4D The weight measurements of mice carrying tumors at different time points during the study.

[0023] Figures 5A-5B Biodistribution of NEO-201 in mice carrying CFPAC-1 xenografts. Female mice with CFPAC-1-carrying tumors, derived from intravenously administered radiolabeled NEO-201. Figure 5A ) and males ( Figure 5B Standardized radiometric measurements of indicated tissues in mice. n = 4 animals / time point. Days 1, 2, 4, and 7 represent the time between the injection of radiolabeled antibodies and the autopsy.

[0024] Figures 6A-6C Body weight and neutrophil counts from cynomolgus monkeys treated with NEO-201. Figure 6A The percentage change in body weight of monkeys relative to baseline (day -1) was measured 7 and 14 days after receiving a single dose of NEO-201 at the indicated dose level. n = 4 animals / group (2 females, 2 males). Figure 6BThe percentage change in neutrophil levels from baseline (day -7) in the blood of monkeys treated with a single dose of NEO-201 at the indicated dose level. n = 4 animals / group (2 females, 2 males). Figure 6C For each dose and time point, the p-value of neutrophil levels compared to the 0 mg / kg control. *Statistically significant by t-test (p < 0.05).

[0025] Figures 7A-7C HaNK ADCC assay was performed using NEO-201 (4 hours). Target cells = 3000 cells / well. Figures 6A-6B H520 lung cancer tumors treated with NEO-201 (top line, square symbol) or IgG1 negative control (bottom line, circle symbol) for 4 hours ( Figure 6A ) or OV90 ovarian cancer ( Figure 6B The percentage of cells specifically dissolved as a function of the effector:target (E:T) ratio. E:T ratios were 6.25:1, 12.5:1, or 25:1. mAb concentration was 10 μg / mL. Values ​​shown are the mean of three replicates + / - SD. An asterisk (*) indicates statistical significance compared to the IgG negative control (p < 0.01, two-tailed t-test). Figure 6C The percentage of lung (H520, HCC827), breast (ZR-75-1), and ovarian (OV90) cancer cells specifically lysed at a constant E:T ratio of 25:1 over four hours with NEO-201 (right, light gray bar) or negative control IgG (left, solid black bar). mAb concentration was 10 μg / mL. Values ​​shown are the mean of three replicates + / - SD. An asterisk (*) indicates statistical significance compared to the IgG negative control (p < 0.01, two-tailed t-test).

[0026] Figure 8 Treatment with ALT-803 enhanced NEO-201-mediated ADCC activity. NK cells isolated from two normal donors were treated with ALT-803 (25 ng / ml) or a culture medium control for 48 hours and used as effector cells in a 4-hour non-radioactive ADCC assay using a Celigo imaging hematology counter. CF-PAC1 (human pancreatic cancer cell line) cells were stained with calcein AM and used as targets at 3,000 cells / well. Results are expressed as specific lysis % (SE).

[0027] Figure 9Treatment with ALT-803 enhanced the expression of TIM-3 and NKG2D on human NK cells. Purified human NK cells from normal donors were cultured for 48 hours with or without ALT-803 (25 ng / ml). Results are expressed as the percentage of positive cells (MFI).

[0028] Figure 10 Treatment with ALT-803 enhanced the expression of TIM-3 and NKG2D on human NK cells. Purified human NK cells from another normal donor were cultured for 48 hours with or without ALT-803 (25 ng / ml). Results are expressed as the percentage of positive cells (MFI).

[0029] Figure 11 Treatment with ALT-803 enhanced ADCC activity mediated by low concentrations of NEO-201. NK cells isolated from normal donors (ND#6) were treated with ALT-803 (25 ng / ml) or a culture medium control for 48 h and used as effector cells in a 4-hour non-radioactive ADCC assay using a Celigo imaging hematology counter. NEO-201 was used at three different concentrations (10 μg / ml, 1 μg / ml, and 0.1 μg / ml). CF-PAC1 (human pancreatic cancer cell line) cells were stained with calcein AM and used as targets at 3,000 cells / well. E:T = 25:1. Results are expressed as specific lysis % (SE). *Statistically significant (p < 0.01).

[0030] Figure 12 Treatment with ALT-803 enhanced ADCC activity in the normal donor (ND#8) with minimal ADCC activity mediated by NEO-201, and this activity could be blocked by anti-CD16 and anti-TIM-3 antibodies. NK cells isolated from the normal donor with minimal ADCC activity were treated with ALT-803 (25 ng / ml) or a culture medium control for 48 h and used as effector cells in a 4-hour non-radioactive ADCC assay using a Celigo imaging hematology counter. Anti-CD16 and anti-TIM-3 were used at concentrations of 30 μg / ml and 15 μg / ml, respectively. NK cells were pretreated with anti-CD16 or anti-TIM-3 for 2 h before the addition of NEO-201 and effector cells. CF-PAC1 (human pancreatic cancer cell line) cells were stained with calcein AM and used as targets at 3,000 cells / well. NEO-201 was used at a concentration of 10 μg / ml. E:T = 25:1. Results are expressed as specific solubility % (SE). *Statistically significant compared to no ALT-803 treatment (p<0.01). #Statistically significant compared to no anti-CD16 and anti-TIM-3 treatment (p<0.01).

[0031] Figure 13 NK-92 killing assay using NEO-201 (16 hours). Target tumor cells (ASPC-1, BxPC-3, CFPAC-1, or LS174T) were seeded at 3000 cells / well. Cells were then treated with 10 μg / mL human IgG1 isotype control antibody or NEO-201 and subsequently supplemented with the natural killer (NK) cell line NK-92 at effector-to-target (E:T) ratios of 1.5625:1, 3.125:1, 6.25:1, and 12.5:1. After 16 hours of incubation at 37°C, cell viability was quantified using a Celigo imaging hematology counter and GraphPad Prism 7 software. The number of viable target cells (calcein AM+ / PI-) per well was counted, and specific lysis was calculated. Results are presented graphically in a table below, with further details for each tumor cell type. *Statistically significant (p<0.05). Detailed Implementation

[0032] In one aspect, this disclosure provides a method for killing cancer cells, the method comprising administering an effective amount of NEO-201 antibody to a patient in need.

[0033] In one aspect, this disclosure provides a method for treating cancer, the method comprising administering an effective amount of NEO-201 antibody to a patient in need.

[0034] In one aspect, this disclosure provides a method for preventing cancer recurrence, the method comprising administering an effective amount of NEO-201 antibody to a patient in need.

[0035] In one aspect, this disclosure provides a method for reducing the tumor burden in a patient with cancer, the method comprising administering an effective amount of NEO-201 antibody to the patient in need.

[0036] The antibody can mediate complement-mediated cytotoxicity (CDC), thereby killing cancer cells in the patient's body.

[0037] Prior to or at the time of administration, the patient may be of natural killer ("NK") depletion type. Prior to or at the time of administration, the patient may be of severe NK depletion type. The patient may have NK cell deficiency (NKD), such as CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD1). The patient may be of NK depletion type or severe NK depletion type due to another therapy, such as cancer therapy (e.g., chemotherapy or radiation therapy). The patients may be treated with one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, succinylaniline-hydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, afedipine, mitomycin, methotrexate, etoposide), GSK inhibitors (e.g., LiCl, BIO, SB21), BET inhibitors (e.g., JQ1), HSP90 inhibitors (e.g., rhizobacterin, 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel), and / or immunomodulatory agents (e.g., lenalidomide).

[0038] The method may include determining whether the patient is NK-reducing type before or during the administration.

[0039] The method may include determining whether the patient has severe NK cell reduction before or during the administration.

[0040] In the method, NK cells may constitute less than 5% of peripheral blood mononuclear cells (PBMCs) in the individual before or at the time of administration.

[0041] In the method, NK cells may constitute less than 3% of peripheral blood mononuclear cells (PBMCs) in the individual before or at the time of administration.

[0042] In the method, before or during the administration, less than 70% of the PBMC NK cells in the patient may be CD56dimCD16+ NK cells.

[0043] In the method, before or during the administration, less than 50% of the PBMC NK cells in the patient may be CD56dimCD16+ NK cells.

[0044] The NEO-201 antibody may contain 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.

[0045] The NEO-201 antibody may contain a variable heavy chain sequence having at least 90% identity with SEQ ID NO:38.

[0046] The NEO-201 antibody may contain a variable light chain sequence having at least 90% identity with SEQ ID NO:39.

[0047] 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.

[0048] The NEO-201 antibody may comprise 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.

[0049] The NEO-201 antibody may contain all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0050] The NEO-201 antibody may contain a human IgG1 constant domain.

[0051] The NEO-201 antibody may be a humanized antibody.

[0052] The NEO-201 antibody can be conjugated to another part.

[0053] The NEO-201 antibody can be conjugated to another cytotoxic moiety, label, radioactive moiety, or affinity tag.

[0054] The method may further include administering an effective amount of a cytokine agonist to the patient to enhance or stimulate the killing of cells of the tumor. The cytokine agonist may include interleukin-2 (IL-2), interleukin-21 (IL-21), ALT-803, IL-15 inhibitors, checkpoint inhibitors, anti-PD1, anti-PDL1, anti-CTLA-4, anti-41BB, anti-OX40, anti-Tim-3, or combinations thereof.

[0055] The method may further include administering an effective amount of a complement regulatory protein (CRP) antagonist to the patient to enhance or stimulate the killing of tumor cells. The CRP antagonist may antagonize one or more of CD46, CD55, or CD59. The CRP antagonist may comprise an antibody or an antigen-binding fragment thereof.

[0056] The cytokine agonists may include IL-15 agonists or IL-15 superagonists.

[0057] The cytokine agonist may include a complex consisting of an IL-15 mutant (IL-15N72D) that binds to the IL-15 receptor α / IgG1 Fc fusion protein, such as ALT-803.

[0058] The effective dose of the NEO-201 antibody can be reduced compared to treatment with the NEO-201 antibody alone in the absence of the cytokine agonist.

[0059] The tumor may include colon cancer. The tumor may include pancreatic cancer. The tumor may include ovarian cancer. The tumor may include stomach cancer. The tumor may include lung cancer. The tumor may include breast cancer. The tumor may include uterine cancer.

[0060] In another embodiment, this disclosure provides a method for killing tumor cells, the method comprising administering an effective amount of NEO-201 antibody to a patient in need, wherein the patient is a natural killer (“NK”) depleted type prior to or at the time of administration. The NK depletion may include less than 5% or less than 3% of the peripheral blood mononuclear cells (PBMCs) in a sample derived from the patient, such as a blood sample, being NK cells. Alternatively or additionally, less than 70% (or optionally less than 50%) of the PBMC NK cells in the patient prior to or at the time of administration may be CD56dimCD16+ NK cells.

[0061] In another embodiment, this disclosure provides a method for treating cancer, the method comprising administering an effective amount of NEO-201 antibody to a patient in need, wherein the patient is of natural killer (“NK”) depletion type before or at the time of the administration.

[0062] In another embodiment, this disclosure provides a method for preventing cancer recurrence, the method comprising administering an effective amount of NEO-201 antibody to a patient in need, wherein the patient is a natural killer (“NK”) depleted type before or at the time of the administration.

[0063] In another embodiment, this disclosure provides a method for reducing the tumor burden in a patient with cancer, the method comprising administering an effective amount of NEO-201 antibody to a patient in need, wherein the patient is of natural killer (“NK”) depletion type before or at the time of the administration.

[0064] In the aforementioned method, the antibody can mediate CDC, thereby killing cancer cells in the patient, for example, even though effective ADCC is absent due to the patient being NK-reduced. At the time of administration, the patient may be severely NK-reduced. Optionally, the method further includes determining, for example, whether the patient is NK-reduced or severely NK-reduced within a certain period prior to administration, such as 1 or 2 weeks prior. NK-reduced or severely NK-reduced status can also be inferred from the patient's medical history, such as prior to or concurrent use of another therapy that reduces NK cells. For example, the patient may have undergone or concurrently undergone cancer therapy, such as radiation therapy or chemotherapy. The cancer therapy may include administration of one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, succinylaniline-isohydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, afedipine, mitomycin, methotrexate, etoposide), GSK inhibitors (e.g., LiCl, BIO, SB21), BET inhibitors (e.g., JQ1), HSP90 inhibitors (e.g., radicicola, 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel), and / or immunomodulatory agents (e.g., lenalidomide).

[0065] The patient may have NK cell deficiency (NKD), such as CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD1).

[0066] In a preferred embodiment of the invention, which may be used in conjunction with any of the foregoing or the following embodiments, the NEO-201 antibody may comprise 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.

[0067] In a preferred embodiment of the invention, which can be used with any of the foregoing or following embodiments, the NEO-201 antibody may comprise a variable heavy chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity with SEQ ID NO:38. The variable heavy chain having said percentage of sequence identity may comprise all three of the CDR sequences contained in SEQ ID NO:38.

[0068] In a preferred embodiment of the invention, which can be used with any of the foregoing or following embodiments, the NEO-201 antibody may comprise a variable light chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity with SEQ ID NO:39. The variable light chain may comprise all three of the CDR sequences contained in SEQ ID NO:39.

[0069] In a preferred embodiment of the invention, which can be used with any of the foregoing or following embodiments, the NEO-201 antibody may comprise a variable heavy chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity with SEQ ID NO:38 and a variable light chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity with SEQ ID NO:39. The variable light chain may comprise all three of the CDR sequences contained in SEQ ID NO:39, and the variable heavy chain having the stated sequence identity percentage may comprise all three of the CDR sequences contained in SEQ ID NO:38.

[0070] In a preferred embodiment of the invention, which can be used with any of the foregoing or following embodiments, the NEO-201 antibody may comprise a heavy chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity with amino acids 20-470 of SEQ ID NO:28 and a light chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity with amino acids 20-233 of SEQ ID NO:29. The light chain may comprise all three of the CDR sequences contained in SEQ ID NO:29, and the heavy chain having the stated sequence identity percentage may comprise all three of the CDR sequences contained in SEQ ID NO:28.

[0071] In a preferred embodiment of the invention, which can be used with any of the foregoing or the following embodiments, the NEO-201 antibody may comprise the heavy chain variable region sequence contained in SEQ ID NO:28 and the light chain variable region sequence contained in SEQ ID NO:29.

[0072] In a preferred embodiment of the invention, which can be used with any of the foregoing or the following embodiments, the NEO-201 antibody may comprise a heavy chain sequence containing amino acids 20-470 of SEQ ID NO:28 and a light chain sequence containing amino acids 20-233 of SEQ ID NO:29.

[0073] In a preferred embodiment of the invention, which can be used with any of the foregoing or following embodiments, the NEO-201 antibody comprises a human IgG1 constant domain.

[0074] In a preferred embodiment of the invention, which can be used with any of the foregoing or following embodiments, the NEO-201 antibody may be a humanized antibody.

[0075] In a preferred embodiment of the invention, which can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody may be conjugated to another portion, such as another cytotoxic portion, a label, a radioactive portion, or an affinity tag.

[0076] In a preferred embodiment of the invention, which can be used in conjunction with any of the foregoing or following embodiments, the method may further include administering an effective amount of a cytokine agonist to the patient to enhance or stimulate the killing of cells of the tumor. The cytokine agonist may include interleukin-2 (IL-2), interleukin-21 (IL-21), ALT-803, IL-15 inhibitors, checkpoint inhibitors, anti-PD1, anti-PDL1, anti-CTLA-4, anti-41BB, anti-OX40, anti-Tim-3, or combinations thereof.

[0077] In a preferred embodiment of the invention, which can be used in conjunction with any of the foregoing or following embodiments, the method may further include administering an effective amount of a complement regulatory protein (CRP) antagonist to the patient to enhance or stimulate the killing of cells of the tumor. The CRP antagonist may antagonize one or more of CD46, CD55, or CD59. The CRP antagonist may comprise an antibody or an antigen-binding fragment thereof. The cytokine agonist may comprise an IL-15 agonist or an IL-15 hyperagonist. The cytokine agonist may comprise a complex consisting of an IL-15 mutant (IL-15N72D) that binds to the IL-15 receptor α / IgG1 Fc fusion protein. The cytokine agonist may comprise ALT-803.

[0078] In a preferred embodiment of the invention that can be used with any of the foregoing or the following embodiments, the effective dose of the NEO-201 antibody is reduced compared to treatment with the NEO-201 antibody alone in the absence of the cytokine agonist.

[0079] In a preferred embodiment of the invention, which can be used in conjunction with any of the foregoing or following embodiments, the cancer may express the NEO-201 antigen. The expression of the NEO-201 antigen can be determined by detecting the NEO-201 antigen in a sample of the cancer. This detection can be performed using techniques including, but not limited to, histological staining, flow cytometry, RT-PCR, dot blotting, protein blotting, RNA blotting, and other techniques known in the art. In the case of recurrent or metastatic cancer, the expression of the NEO-201 antigen can also be inferred from the expression of NEO-201 in the primary cancer or from the responsiveness of the primary cancer to NEO-201 antibody therapy.

[0080] In a preferred embodiment of the invention, which may be used in conjunction with any of the foregoing or following embodiments, the cancer may include colon cancer.

[0081] In a preferred embodiment of the invention, which may be used in conjunction with any of the foregoing or following embodiments, the cancer may include pancreatic cancer.

[0082] In a preferred embodiment of the invention, which may be used in conjunction with any of the foregoing or the following embodiments, the cancer may include ovarian cancer.

[0083] In a preferred embodiment of the invention, which may be used in conjunction with any of the foregoing or following embodiments, the cancer may include gastric cancer.

[0084] In a preferred embodiment of the invention, which may be used in conjunction with any of the foregoing or following embodiments, the cancer may include lung cancer.

[0085] In a preferred embodiment of the invention, which may be used in conjunction with any of the foregoing or following embodiments, the cancer may include breast cancer.

[0086] In a preferred embodiment of the invention, which may be used in conjunction with any of the foregoing or following embodiments, the cancer may include uterine cancer.

[0087] definition

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used in or in the testing of this invention, suitable methods and materials are described herein. The materials, methods, and examples described are illustrative only and are not intended to be limiting.

[0089] Unless the context clearly indicates otherwise, the meanings of “a / an” and “the” as used herein and throughout the claims include plural references.

[0090] As used herein, “amino acid” broadly refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, namely, the α-carbon, carboxyl, amino, and R groups bound to hydrogen, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. “Amino acid mimics” are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to that of naturally occurring amino acids.

[0091] As used herein, the terms "NK cell depletion" or "natural killer cell depletion" refer to a patient having low levels of natural killer (NK) cells relative 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. Patients with NK cells comprising less than 5% of PMBCs are described as having NK cell depletion. Furthermore, if NK cells comprise less than 3% of PMBCs, the patient is described as having severe NK cell depletion. Additionally, in normal individuals, up to 90% of PBMC NK cells are CD56. dim CD16 + NK cells, and these are considered the most cytotoxic subset. If less than 70% of PBMC NK cells are CD56... dim CD16 + If NK cells are present, the patient is referred to as NK-reduced type. Additionally, if less than 50% of PBMC NK cells are CD56... dim CD16 +If NK cells are absent, the patient is referred to as having severe NK cell depletion. A given patient may be classified as having NK cell depletion or severe NK cell depletion based on meeting one or both of these individual criteria. Generally, a patient's status as having NK cell depletion or severe NK cell depletion is determined by testing samples obtained from the patient, such as blood samples, for example, samples obtained and tested within one or two weeks prior. A patient's status as having NK cell depletion or severe NK cell depletion can also be inferred from the disease diagnosis and / or the course of treatment associated with this reduction in NK cells.

[0092] NK cell depletion also includes subjects with NK cell deficiency (NKD). Exemplary NKD conditions include classical NKD (CNKD), characterized by the absence of NK cells and their function in peripheral blood lymphocytes; and functional NKD (FNKD), characterized by the presence of NK cells in peripheral blood lymphocytes with defective NK cell activity. In both CNKD and FNKD, NK cell abnormality is the primary immune deficiency, leading to inadequate ADCC response. CNKD and FNKD can be further subdivided based on patient characteristics, such as the identity of one or more pathogenic genes and other patient characteristics. CNKD includes CNKD subtype 1 (CNKD1), which is autosomal dominant and associated with a defect in the GATA2 gene; and CNKD subtype 2 (CNKD2), which is autosomal recessive and associated with a defect in the MCM4 gene. FNKD includes FNKD1, which is autosomal recessive and associated with a defect in the FCCR3A gene.

[0093] As used herein, the term "antibody" broadly refers to any polypeptide chain molecular structure having a specific shape that binds to and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. Typical antibody molecules are immunoglobulins, and all types of immunoglobulins IgG, IgM, IgA, IgE, and IgD from all sources (e.g., human, rodent, rabbit, cow, sheep, pig, dog, chicken) are considered "antibodies." Antibodies include, but are not limited to, chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single-chain antibodies (scFv), camel antibodies, nanobodies, IgNAR (single-chain antibodies obtained from sharks), small molecule immunopharmaceuticals (SMIPs), and antibody fragments (e.g., Fab, Fab', F(ab')2). Many antibody-coding sequences have been described; and other antibody-coding sequences can be generated by methods well known in the art. See Streltsov, et al. (2005). ProteinSci. 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.

[0094] "NEO-201 antibody" refers to the heavy and light chains contained in SEQ ID NO:28 and 29, or the variable regions therein optionally together with constant regions, as well as fragments and variants thereof. Such variants include sequences containing one, two, three, four, five, or preferably all six CDR sequences contained 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 a humanized antibody. The antibody may be expressed containing one or more leader sequences, which may be removed during antibody expression and / or processing and secretion. The antibody may be presented in a monovalent, bivalent, or higher multivalent form, including but not limited to bispecific or multispecific antibodies containing the NEO-201 antibody sequence and binding fragments of different antibodies. Typically, the antibody specifically binds to tumor cells and competes 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, for binding to tumor cells. One or more of the CDR sequences contained in SEQ ID NO:28 and / or SEQ ID NO:29 may be replaced by 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 SEQ ID NO:30-31. The light chain may contain CDRs contained in the light chain sequences of SEQ ID NO:14, 16, 17, 18, 19, 20, 21, or 29. The heavy chain may contain CDRs contained in the heavy chain sequences 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 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. Optionally, the heavy chain and / or light chain sequence contains one, two, three, four, five, or preferably all six CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29, namely, 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. The antibody may be conjugated to another part, such as a cytotoxic part, a radioactive part, or a labeling or purification tag.

[0095] As used herein, "antigen" broadly refers to a molecule or part of a molecule that can be bound by an antibody, and which can also induce an animal to produce antibodies that can bind to an epitope of the antigen. An antigen may have one epitope or more than one epitope. The specificity response referred to herein indicates that the antigen will react with its corresponding antibody in a highly selective manner and will not react with a variety of other antibodies that can be induced by other antigens. Antigens may be tumor-specific (e.g., expressed by neoplastic cells of pancreatic and colon cancers).

[0096] As used in this article, “cancer” in a broad sense refers to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division that leads to malignant growth or a tumor.

[0097] As used herein, "chimeric antibody" broadly refers to an antibody molecule in which a constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site (variable region) is linked to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug) of a different or altered class, effector function, and / or species, or which imparts new properties to the chimeric antibody; or a variable region or a portion thereof is altered, replaced, or exchanged with a variable region having a different or altered antigen specificity.

[0098] As used herein, “conserved variant” applies to both amino acid sequences and nucleic acid sequences, and in a broad sense, with respect to a particular nucleic acid sequence, refers to a conserved variant that encodes the same or substantially the same amino acid sequence, or, when the nucleic acid does not encode an amino acid sequence, substantially the same sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. Such nucleic acid variations are “silent variations,” which constitute a class of conserved variants. Each nucleic acid sequence encoding a polypeptide in this article also illustrates each possible nucleic acid silent variation. Those skilled in the art will recognize that each codon in a nucleic acid (except for the codon-only AUG, usually methionine, and the codon-only TGG, usually tryptophan) can be modified to produce functionally identical molecules.

[0099] As used herein, the terms "complementarity-determining region," "hypervariant region," or "CDR" broadly refer to one or more hypervariable or complementarity-determining regions (CDRs) found in the variable regions of the light or heavy chain of an antibody. See Kabat et al. (1987). Sequences of Proteins of Immunological Interest "National Institutes of Health, Bethesda, MD. These statements include, for example, those by Kabat et al. (1983)." Sequences of Proteins of Immunological Interest "The hypervariable region, or hypervariable loop, in the 3D structure of an antibody is defined by the USDTept. of Health and Human Services. Chothia and Lesk (1987)" J Mol.Biol. 196:901–917. The CDRs in each chain are kept close together by the framework region and, together with CDRs from the other chain, promote the formation of antigen-binding sites. Within the CDRs are selective amino acids described as selection-determining regions (SDRs), which represent key contact residues used by the CDRs in antibody-antigen interactions. Kashmiri (2005) Methods 36:25–34.

[0100] As used herein, "control quantity" broadly refers to any quantity or series of quantities that a biomarker can be compared to a test quantity of the biomarker. For example, a control quantity of a biomarker can be the quantity of the biomarker in a patient with a particular disease or condition or in a person who does not have such a disease or condition. A control quantity can be an absolute quantity (e.g., micrograms per ml) or a relative quantity (e.g., the relative intensity of a signal).

[0101] As used herein, "differential presence" broadly refers to a difference in the quantity or quality of a biomarker present in a sample taken from a patient suffering from one of the diseases or conditions, compared to a comparable sample taken from a patient who does not suffer from one of the diseases or conditions. For example, if the amount of a nucleic acid fragment in one sample differs significantly from the amount of a nucleic acid fragment in another sample, the nucleic acid fragment may optionally be differentially present between the two samples, as measured, for example, by hybridization and / or NAT-based assays. If the amount of a polypeptide in one sample differs significantly from the amount of a polypeptide in another sample, the polypeptide may optionally be differentially present between the two samples. It should be noted that a biomarker may be considered differentially present if it is detectable in one sample and undetectable in another. Optionally, a relatively low amount of upregulation may serve as a biomarker.

[0102] As used herein, “diagnosis” in a broad sense refers to the identification of the presence or nature of a pathological condition. Diagnostic methods vary in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of individuals with the disease who test positive (the “true positive” percentage). Individuals with the disease who fail the assay are “false negatives.” Subjects who are not diseased and test negative in the assay are called “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 who test positive but do not have the disease. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it is qualified if it provides a positive indication that aids in diagnosis.

[0103] As used herein, “diagnosis” in a broad sense refers to the classification of a disease or symptom, the determination of the severity of a disease, the monitoring of disease progression, and the prediction of the consequences and / or prospects of recovery. The term “detection” may also optionally encompass any of the foregoing. In some embodiments, the diagnosis of a disease according to the invention may be influenced by determining the level of the polynucleotides or polypeptides of the invention in a biological sample obtained from a subject, wherein the determined level may be related to susceptibility to said disease, or the presence or absence of said disease. It should be noted that “biological sample obtained from a subject” may also optionally include samples not removed from said subject’s body.

[0104] As used herein, "effective amount" broadly refers to the amount of a compound, antibody, antigen, or cell sufficient to achieve treatment of a disease when administered to a patient for the purpose of treating the disease. An effective amount can be an effective amount for prophylaxis and / or effective amount for prevention. An effective amount can be an effective reduction, effective prevention of the occurrence of signs / symptoms, reduction of the severity of signs / symptoms, elimination of the occurrence of signs / symptoms, slowing the development of signs / symptoms, preventing the development of signs / symptoms, and / or effective prevention of the occurrence of signs / symptoms. "Effective amount" can vary depending on the patient's disease and its severity, as well as age, weight, medical history, susceptibility, and pre-existing conditions. For the purposes of this invention, the term "effective amount" is synonymous with "therapeutic effective amount."

[0105] As used herein, "expression vector" broadly refers to any recombinant expression system intended for the constitutive or inductive expression, in vitro or in vivo, of the nucleic acid sequences of the present invention in any cell, including prokaryotic cells, yeast cells, fungal cells, plant cells, insect cells, or mammalian cells. The term includes linear or circular expression systems. The term includes expression systems that remain free or integrated into the host cell genome. The expression system may have the ability to self-replicate or not (i.e., drive only transient expression in the cell). The term includes recombinant expression cassettes containing only the minimum elements required for transcription of the recombinant nucleic acid.

[0106] As used herein, "frame region" or "FR" broadly refers to one or more frame regions within the variable region of the light or heavy chain of an antibody. See Kabat et al. (1987) Sequences ofProteinsof Immunological Interest "National Institutes of Health, Bethesda, MD. These statements include those amino acid sequence regions inserted between the CDRs within the variable regions of the light and heavy chains of the antibody."

[0107] As used herein, "heterologous" broadly refers to a portion of nucleic acid, indicating that the nucleic acid contains two or more subsequences that are not found to be identical in nature. For example, nucleic acids are often produced through recombination, thereby arranging two or more sequences from unrelated genes to create new functional nucleic acids, such as a promoter from one source and a coding region from another. Similarly, heterologous proteins indicate proteins that contain two or more subsequences that are not found to be identical in nature (e.g., fusion proteins).

[0108] As used in this article, "high affinity" in a broad sense refers to an antibody having at least 10 affinity for the target antigen. –8 M, more preferably at least 10–9 M, and even more preferably at least 10 –10 The KD of M. However, for other antibody isotypes, "high affinity" binding can vary. For example, for the IgM isotype, "high affinity" binding refers to binding with at least 10 KD. –7 M, more preferably at least 10 –8 Antibodies against M's KD.

[0109] As used herein, “homology” broadly refers to the degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence or between two polypeptide sequences. Homology can be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. Partially homologous nucleic acid or amino acid sequences are those that are not identical to a reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The terms “sequence identity” and “homology” are used interchangeably.

[0110] As used herein, “host cell” is broadly defined as a cell that contains an expression vector and supports the replication or expression of said expression vector. Host cells can be prokaryotic cells such as Escherichia coli, or eukaryotic cells such as yeast, insect (e.g., SF9), amphibian or mammalian cells such as CHO, HeLa, HEK-293, for example, cultured cells, explants and in vivo cells.

[0111] As used herein, “hybridization” in a broad sense refers to the physical interaction between complementary (including partially complementary) polynucleotide chains through the formation of hydrogen bonds between complementary nucleotides when the chains are arranged antiparallel to each other.

[0112] As used herein, the term "K-assoc" or "Ka" broadly refers to the binding rate of a specific antibody-antigen interaction; while the term "Kdiss" or "Kd" refers to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant, derived from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well-established in the art.

[0113] As used herein, “immunoassay” in a broad sense refers to an assay that uses antibodies that specifically bind to an antigen. An immunoassay is characterized by the use of the specific binding properties of a particular antibody to isolate, target, and / or quantify the antigen.

[0114] As used herein, “isolated” in a broad sense refers to material that has been removed from its natural environment and thus artificially altered from its natural environment. Isolated material can be, for example, exogenous nucleic acids contained in a vector system, exogenous nucleic acids contained in a host cell, or any material that has been removed from its natural environment and thus artificially altered (e.g., “isolated antibody”).

[0115] As used herein, “label” or “detectable portion” in a broad sense refers to a composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means.

[0116] As used herein, “low stringency,” “medium stringency,” “high stringency,” or “extremely high stringency” conditions refer broadly to the conditions used for nucleic acid hybridization and washing. Guidelines for performing hybridization reactions can be found in Ausubel et al. (2002). Short Protocols in Molecular Biology (5 th Ed.) John Wiley & Sons, NY. Exemplary specific hybridization conditions include, but are not limited to: (1) low-toughness hybridization conditions, which involve washing twice at about 45°C in 6X sodium chloride / sodium citrate (SSC) followed by washing twice at at least 50°C in 0.2X SSC and 0.1% SDS (for low-toughness conditions, the washing temperature may be increased to 55°C); (2) medium-toughness hybridization conditions, which involve washing once or more at about 45°C in 6X SSC followed by washing once or more at 60°C in 0.2X SSC and 0.1% SDS; (3) high-toughness hybridization conditions, which involve washing once or more at about 45°C in 6X SSC followed by washing once or more at 65°C in 0.2X SSC and 0.1% SDS; and (4) very high-toughness hybridization conditions, which involve washing once or more at 65°C in 0.5M sodium phosphate and 7% SDS followed by washing once or more at 65°C in 0.2X SSC and 1% SDS.

[0117] As used herein, “mammal” in a broad sense refers to any and all warm-blooded vertebrates of the class Mammalia, including humans, characterized by skin covered with hair and, in females, mammary glands for nursing 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, species of bovids, canids, equines, felines, rodents, sheep, suidae, primates, and rodents. Mammals also include any and all mammals listed in the World's Mammal Species Register maintained by the National Museum of Natural History and the Smithsonian Institution in Washington, D.C.

[0118] As used herein, “nucleic acid” or “nucleic acid sequence” refers to deoxyribonucleotides or ribonucleotide oligonucleotides in single-stranded or double-stranded form. The term covers nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also covers nucleic acid-like structures having a synthetic backbone. Unless otherwise indicated, a particular nucleic acid sequence also implicitly covers its conserved 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.

[0119] As used herein, the term “operably ligated” broadly refers to ligating two DNA fragments such that the amino acid sequences encoded by the two DNA fragments remain within the frame.

[0120] As used herein, "complementary site" broadly refers to a portion of an antibody that recognizes an antigen (e.g., the antigen-binding site of the antibody). A complementary site can be a small region (e.g., 15-22 amino acids) of the Fv region of an antibody and can contain portions of both the heavy and light chains of the antibody. See Goldsby et al. Antigens (Chapter 3) Immunology (5th edition), New York: W.H. Freeman and Company, pp. 57–75.

[0121] As used herein, “patient” in a broad sense means any animal that requires treatment to alleviate a disease state or to prevent the onset or recurrence of a disease state. Furthermore, as used herein, “patient” in a broad sense means any animal that has a prior diagnosis of risk factors, medical history, susceptibility, symptoms, signs, or is at risk of disease or is a member of a patient group with disease. A patient can be a clinical patient, such as a human or veterinary patient, such as a companion animal, domestic animal, livestock, exotic animal, or zoo animal. The term “subject” is used interchangeably with the term “patient.”

[0122] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and broadly refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are analogs or simulants of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. The terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical simulants of the corresponding naturally occurring amino acid, as well as naturally occurring and non-naturally occurring amino acid polymers. Polypeptides can be modified, for example, by adding carbohydrate residues to form glycoproteins. The terms “polypeptide,” “peptide,” and “protein” include both glycoproteins and non-glycoproteins.

[0123] As used herein, "promoter" broadly refers to the nucleic acid sequence that directs the transcription of nucleic acids. As used herein, "promoter" includes the essential nucleic acid sequence near the transcription start site, such as the TATA element in the case of a type II polymerase promoter. Promoters may also optionally include distal enhancer or repressor elements, which can be located thousands of base pairs away from the transcription start site. "Constitutive" promoters are promoters active under most environmental and developmental conditions. "Inducible" promoters are promoters active under environmental or developmental regulatory conditions.

[0124] As used herein, "effective preventive dose" broadly refers to the amount of a compound sufficient to provide such prevention against the disease or recurrence when administered to a patient for the prevention or treatment of disease or the prevention of disease recurrence. An effective preventive dose can be an amount that effectively prevents the occurrence of signs and / or symptoms. An "effective preventive dose" can vary depending on the patient's disease and its severity, as well as age, weight, medical history, susceptibility to the disease, and pre-existing conditions.

[0125] As used herein, “prevention and treatment” broadly refers to a treatment process in which signs and / or symptoms are absent in the patient, are in remission, or were previously present in the patient. Prevention and treatment includes preventing disease that develops following treatment of the patient’s disease. Furthermore, prevention includes treating patients who may potentially develop the disease, especially those susceptible to the disease (e.g., members of a patient population, patients with risk factors, or patients at risk of developing the disease).

[0126] As used herein, with respect to products, "recombinant" in a broad sense refers to, for example, cells or nucleic acids, proteins or vectors, indicating that said cells or nucleic acids, proteins or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or indicating that the cells are derived from cells that have been so modified. Thus, for example, recombinant cells express genes not found in the natural (non-recombinant) form of the cell or express natural genes that are otherwise abnormally expressed, poorly expressed or not expressed at all.

[0127] As used herein, "specifically (or selectively) binds" to an antibody, or "specifically (or selectively) immunely reacts with," or "specifically interacts with or binds to," broadly refers to a protein or peptide (or other epitope), and in some embodiments refers to a binding reaction that identifies the presence of a protein in a heterogeneous population of proteins and other biological agents. For example, under specified immunoassay conditions, a specific antibody binds to a particular protein at least twice the background (non-specific signal) and does not substantially bind to other proteins present in the sample in significant amounts. Typically, a specific or selective reaction will be at least twice the background signal or background noise, and more typically greater than about 10 to 100 times the background.

[0128] As used herein, "specific hybridization" and "complementarity" broadly refer to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence via conventional Watson-Crick or other non-conventional methods. The binding free energy of the nucleic acid molecule with its complementary sequence is sufficient to allow for the relevant functions of the nucleic acid, such as RNAi activity. The determination 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) PNAS 83:9373–77; Turner et al. (1987) J.Am.Chem.Soc. 109:3783–85. The percentage of complementarity indicates the percentage 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 are at least about 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Complete complementarity" or 100% complementarity, broadly speaking, means that all consecutive residues in the nucleic acid sequence are hydrogen-bonded to the same number of consecutive residues in the second nucleic acid sequence. "Substantial complementarity" means that, excluding the overhangs, the polynucleotide chain exhibits at least about 90% complementarity in regions selected for non-complementarity, such as the overhangs. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomer to the non-target sequence under conditions requiring specific binding (i.e., physiological conditions in vivo assays or therapeutic treatments, or conditions under which the assay is performed in vitro). Non-target sequences can typically vary by at least 5 nucleotides.

[0129] As used in this article, the “signs” of a disease in a broad sense refer to any abnormalities that can be detected during a patient’s examination; compared with the symptoms that are the subjective indicators of a disease, they are the objective indicators of a disease.

[0130] As used herein, “solid support,” “support,” and “substrate” broadly refer 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 textured and porous materials.

[0131] As used herein, “subject” broadly refers to a subject suitable for treatment according to the invention, including but not limited to avian and mammalian subjects, with mammals being preferred. Mammals of the invention include, but are not limited to, dogs, cats, cattle, goats, horses, sheep, pigs, rodents (e.g., rats and mice), rabbits, primates, and humans. Any mammalian subject requiring treatment according to the invention is suitable. Human subjects of both sexes and at any developmental stage (i.e., newborns, infants, adolescents, young adults, and adults) can be treated according to the invention. The invention can also be performed on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, cattle, goats, sheep, and horses, for veterinary purposes and for drug screening and drug development purposes. “Subject” may be used interchangeably with “patient”.

[0132] As used in this article, “symptoms” of a disease are broadly defined as any pathological phenomenon or deviation from normal structure, function, or sensation that a patient experiences and indicates the disease.

[0133] As used herein, “therapy,” “therapeutic,” “treating,” or “treatment” broadly refers to treating a disease, halting or reducing the development of a disease or its clinical symptoms, and / or alleviating a disease, thereby causing the disease or its clinical symptoms to subside. Therapy encompasses prophylaxis, treatment, remedy, reduction, mitigation, and / or relief of a disease, its signs and / or symptoms. Therapy encompasses the reduction of signs and / or symptoms in patients with developing disease (e.g., tumor growth, metastasis). Therapy also encompasses “prevention.” For therapeutic purposes, the term “reduction” broadly refers to a clinically significant reduction in signs and / or symptoms. Therapy includes treating recurrent or relapsing signs and / or symptoms (e.g., tumor growth, metastasis). Therapy encompasses, but is not limited to, excluding the occurrence of signs and / or symptoms at any time, and reducing and eliminating existing signs and / or symptoms. Therapy includes treatment of chronic diseases (“maintenance”) and acute diseases. For example, treatment includes treating or preventing recurrence or recurrence of signs and / or symptoms (e.g., tumor growth, metastasis).

[0134] As used in this article, "variable region" or "VR" broadly refers to the structural domains within each pair of light and heavy chains in an antibody that directly participate in the binding of the antibody to the antigen. Each heavy chain has a variable domain (VR) at one end. H Following this are multiple constant structural domains. Each light chain has a variable structural domain (V0) at one end. L And at its other end it has a constant structural domain; the constant structural domain of the light chain is aligned with the first constant structural domain of the heavy chain, and the variable structural domain of the light chain is aligned with the variable structural domain of the heavy chain.

[0135] As used herein, "vector" in a broad sense refers to a plasmid, granulosome, phage particle, phage DNA, or other DNA molecule capable of autonomous replication in a host cell and characterized by one or more restriction endonuclease recognition sites at which such DNA sequences can be cleaved in a deterministic manner without losing the vector's essential biological function, and where the DNA can be inserted to facilitate its replication and cloning. The vector may also contain markers suitable for use in the identification of cells transformed with the vector.

[0136] The techniques and procedures described herein are generally based on conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. (2001). Molec.Cloning:Lab.Manual [3rd Edition] Cold Spring Harbor Laboratory Press. Standard techniques are available for recombinant DNA, oligonucleotide synthesis and tissue culture, and transformations (e.g., electroporation, lipid transfection). Enzymatic reactions and purification techniques may be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The nomenclature used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, and the laboratory procedures and techniques described herein, are well known and commonly used in the art. Standard techniques are available for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0137] Example

[0138] The invention has now been generally described, and the invention will be more readily understood with reference to the following examples, which are included only for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0139] Example 1

[0140] NEO-201 binds to various human cancer cell lines

[0141] Flow cytometry analysis was used to analyze NEO-201 binding in a group of human cancer cell lines. The staining profiles are summarized in Table 1, and representative histograms from cell lines with high, medium, low, and negative staining are shown in [Table 1]. Figure 1As shown in A-1C. Evaluation of NEO-201 binding activity revealed that 3 / 6 (50%) colon cancer cell lines and 4 / 5 (80%) pancreatic cancer cell lines were highly positive. When profiling non-small cell lung cancer (NSCLC) cell lines of various histological subtypes was performed, 3 / 5 (60%) adenocarcinoma cell lines were identified as reacting to NEO-201, while only 1 / 4 (25%) squamous cell carcinoma cell lines were found to be positive. Screening of breast cancer cell lines was also performed. In cell lines expressing estrogen receptor (ER) or progesterone receptor (PR), 2 / 4 (50%) were positive for NEO-201 staining, whether used alone or in combination with HER2. In HER2+ cell lines, 3 / 4 (75%) were recognized by NEO-201, whether used alone or in combination with ER or PR. However, NEO-201 staining was detected at low levels in only 1 / 4 (25%) of triple-negative breast cancer cell lines. In total, 15 out of 30 (50%) of the tested tumor cell lines were recognized by NEO-201. These data indicate that NEO-201 is responsive to a wide range of in vitro cultured tumor cell lines and demonstrate that significant differences in antibody reactivity can occur based on tumor subtype.

[0142] Example 2

[0143] NEO-201 tissue staining is highly tumor-specific.

[0144] Using tissue microarrays representing numerous samples for each cancer type, immunohistochemical studies were employed to investigate the reactivity of NEO-201 in human tumor samples. Figure 2A As shown, the immunoreactivity with NEO-201 7,829,678 was completely absent in normal colon, pancreas, and lung tissues, but highly positive in tumor tissues from these organs. Remarkably, staining was found only on tumor cells, as the surrounding stromal cells were not stained. Figure 2A IHC staining of microarray samples determined that NEO-201 was highly reactive against colon cancer (72%), pancreatic cancer (80%), gastric cancer (71%), lung cancer (61%), breast cancer (55%), and uterine cancer (54%). Additionally, a significant number of ovarian cancer samples (26%) also showed positive staining, but not in prostate cancer tissues (…). Figure 2B No staining was observed in the samples. Overall, 258 / 345 (74.7%) of the tumor tissue samples were positive for NEO-201 staining. Importantly, NEO-201 reactivity was almost entirely absent from normal healthy tissue (Table 2) and from adjacent tissues of normal tumors (with the exception of some uterine and ovarian samples). Figure 2CHowever, the number of tissues in this group of uterine and ovarian tissues was limited (5 and 9 samples, respectively). In summary, these data indicate that NEO-201 recognizes tumor tissues from a wide variety of cancers and is highly tumor-specific.

[0145] Example 3

[0146] NEO-201 mediates ADCC and CDC to kill tumor cells.

[0147] As a humanized IgG1 antibody, NEO-201 theoretically mediates ADCC to kill tumor cells expressing the NEO-201 antigen. To investigate this potential mechanism of action, ADCC assays were performed on cell lines highly positive for NEO-201 staining using human natural killer (NK) cells (CFPAC-1 and ASPC-1) isolated from PBMCs of two different healthy donors. Treatment with NEO-201 was observed to enhance the killing effect of CFPAC-1 and ASPC-1 cells to a level 2 to 6 times greater than that of tumor cells treated with control IgG1. Figure 3A Titration experiments were also conducted, revealing that NEO-201 retains significant ADCC-inducing ability at doses as low as 0.1 μg / mL. Figure 3B ).

[0148] CDC is a complex cascade of proteolytic lysis that ends with the activation of the membrane attack complex (which lyses antibody-bound target cells). Some human IgG1 antibodies can mediate CDC; however, CDC is antibody-dependent on antigen specificity. CDC assays revealed that NEO-201 induces complement-mediated lysis of ASPC-1 cells in a manner dependent on both mAb dose and incubation time. Figure 3C In summary, these data demonstrate that NEO-201 effectively engages with innate immune effector mechanisms to specifically lyse antibody-bound tumor cells in vitro.

[0149] Example 4

[0150] NEO-201, alone and in combination with human PBMC effector cells, reduces the growth of tumor xenografts.

[0151] To determine the potential antitumor efficacy of NEO-201, CFPAC-1 cells were grown as tumor xenografts in immunocompromised NU / NU nude mice. These cells were selected based on their high NEO-201 antigen expression levels and high sensitivity to NEO-201-mediated ADCC. Once the CFPAC-1 tumor grew to approximately 100 mm... 3To determine the size of the tumor, mice carrying tumors were injected three times with saline, 250 μg human IgG1, 100 μg NEO-201, or 250 μg NEO-201, followed by three injections of 1.0 × 10⁻⁶ NEO-201. 7 IL-2-activated (200 U / mL) human PBMCs were used to act as effector cells mediating ADCC. Figure 4A As shown, compared with the saline + PBMC or human IgG + PBMC control groups, NEO-201 + PBMC significantly reduced tumor growth induced at both dose levels. Mice without tumors from the control group showed no palpable tumors on day 36. In the NEO-201 100 μg + PBMC and NEO-201 250 μg + PBMC groups, 1 (10%) and 4 (40%) of the remaining 10 mice, respectively, had no palpable tumors. Figure 4B In addition, administration of NEO-201 to another group of mice without the addition of human PBMCs resulted in a significant reduction in tumor growth compared to the control group. Figure 4A Importantly, monitoring the body weight of tumor-bearing mice revealed no weight loss in any treatment group (4C). Figure 4D In summary, these results demonstrate that NEO-201 can significantly reduce tumor growth through both ADCC and non-ADCC mechanisms (such as CDC) without inducing significant toxicity in mice.

[0152] Example 5

[0153] NEO-201 is targeted at xenograft tumor sites.

[0154] Biodistribution of radiolabeled NEO-201 was studied in female and male NU / NU nude mice with established CFPAC-1 xenograft tumors. These mice were intravenously injected with the radiolabeled antibody, and blood, organs, and tumors were harvested at different time points after injection for analysis. Low levels of radioactivity were found in the pancreas, spleen, kidney, liver, stomach, intestine, and lungs of both male and female mice at all time points. Figure 5A (5B). However, the normalized uptake of radioactivity was significantly higher in the tumor compared to all other tissues at all time points, and by day 7, the tumor radioactivity gradually increased to a level 20-30 times higher than that in the blood. Figure 5A (5B). Quantitatively similar results were obtained for both female and male mice. These results indicate that NEO-201 preferentially localizes to malignant tissues expressing the target antigen and does not accumulate in normal tissues.

[0155] Example 6

[0156] Pharmacokinetics and toxicity evaluation of NEO-201 in non-human primates

[0157] Single-dose studies were conducted in purposefully bred cynomolgus monkeys to determine the pharmacokinetics and associated toxicities of NEO-201. Cynomolgus monkeys were chosen because this species is closely related to humans in terms of phylogeny and physiology and is commonly used for non-clinical toxicity evaluation. Female and male animals received a single intravenous infusion of NEO-201 diluted in saline at dose levels of 5 mg / kg, 20 mg / kg, and 49 mg / kg, representing the highest dose achievable per infusion volume. Blood samples were collected from all animals at different time points before and after injection up to 14 days, and serum NEO-201 levels were assessed by ELISA. Quantifiable and dose-dependent serum concentrations of NEO-201 (14 days post-dose) were observed at the last collection time point, as depicted in Table 3. As expected for intravenous administration, Tmax values ​​peaked at 10 minutes (10 / 12, 83%) in most animals from all groups, except for one male and one female from the 5 mg / kg group. Within the evaluated dose range, peak (Cmax) exposure was dose-proportional; total (AUC) exposure was greater than dose-proportional at the lowest dose and approximately proportional from 20 mg / kg to 49 mg / kg. The difference in exposure at the lowest dose was attributed to approximately twice the greater mean clearance (CL) and smaller volume of distribution (Vz). At higher doses, the mean half-life (HL) was 167 (20 mg / kg) hours or 170 (49 mg / kg) hours, approximately 3.7 times greater than at the 5 mg / kg dose (46.2 hours). No sex differences were observed.

[0158] Observations and examinations used to determine toxicity during the 14-day study period include 1) periodic clinical evaluation; 2) measurement of food consumption and body weight; and 3) urinary and blood evaluations, including urinalysis, hematology, coagulation tests, serological chemistry, and toxicokinetics. Figure 6A As shown, no dose level group experienced a change in body weight >3% compared to their pre-injection weight, and no individual monkey experienced a change >7%. Food consumption remained constant for all animals, except for two animals in the 5 mg / kg dose group who had only low consumption on day 11. No significant changes were found in any of the serum chemistry, urine analysis, or coagulation tests from baseline (before NEO-201 injection) to day 15 (see Materials and Methods for details). The main laboratory change in blood cell counts was a decrease in neutrophil count relative to baseline. Figure 6B The decline varied in magnitude, ranging from slight to significant, and a clear dose response was not evident. For most animals, this was a transient finding, as improvement was usually noticed by day 8. Figure 6BBy day 15, almost complete or partial recovery of neutrophil counts was observed in the 5 mg / kg group and the 20 mg / kg and 49 mg / kg groups, respectively. Figure 6B The recovery of neutrophil counts by day 15 was statistically significant in comparison with 0 mg / kg animals on day 2 for all three dose levels (p<0.05), but for two of the three dose groups, there were no significant differences at days 8 and 15 (p>0.05). Figure 6C ).

[0159] Example 7

[0160] Materials and Methods

[0161] Cell lines and cultures

[0162] The following human cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA): colon (COLO205, HT-29, LS174T, SW1116, SW1463, SW480, SW620), pancreas (ASPC-1, CFPAC-1, PANC-1), breast (AU-565, BT-474, BT-549, HCC1500, HCC1937, HCC38, MDA-MB-231, MDA-MB-468, SK-BR-3, T-47D, ZR-75-1), and lung (CALU-1, H1703, H226, H441, H520, H522, H596, HCC4006, HCC827, SK-LU-1). All cell cultures were propagated and maintained in RPMI 1640, DMEM, or IMDM media as specified by the supplier (Corning, Corning, NY). The medium was supplemented with 10% heat-inactivated HyClone fetal bovine serum of USA origin (GE Healthcare Life Sciences, Issaquah, WA, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin (Corning Life Science, Manassas, VA, USA). PBMCs from healthy volunteer donors were obtained from the National Institutes of Health Clinical Center Blood Bank (NCT00001846) with appropriate institutional review board approval and informed consent.

[0163] Production of humanized NEO-201 monoclonal antibody

[0164] Hollinshead's colon cancer-specific vaccine was used as an immunogenic material to generate monoclonal antibodies in mice. Methods for preparing tumor-associated proteins and peptides have been previously described (Hollinshead, US4810781, 1989). In short, 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, and subsequent low-frequency sonication. The resulting membrane-extracted proteins were fractionated on Sephadex G-200 resin or by electrophoresis, then concentrated and quantified (Hollinshead et al., 1970; Hollinshead et al., 1972; Hollinshead et al., 1985). The TAA formulation was mixed with complete Freund's adjuvant and injected subcutaneously into BALB / c mice. This was followed by three booster injections in incomplete Freund's adjuvant at 2–3 week intervals. Antibody responses to immunogenic antigens in mouse serum were tested by ELISA, and mice with effective responses were used to generate immortalized hybridoma cells by fusing mouse B cells derived from the spleen with the SP2 / 0-Ag14 myeloma cell line and selecting cells that grew and produced mouse immunoglobulins (IgG). Mouse 16C3 clones (m16C3) were selected from these mouse IgGs based on their reactivity with colon tumor cell membrane extracts derived from LS174T or HT-29 cells, as determined by ELISA. cDNAs encoding heavy and light chain IgG1 were identified from RNA isolated from hybridoma clone 16C3E12 and showed to be unique (Bristol & Kantor, US7829678, 2010). The m16C3 protein sequence was humanized to h16C3 and designated NEO-201. Humanization was performed in a computer by replacing the mouse sequences outside the complementarity-determining regions (CDRs) of the Fab regions of the heavy and light chain proteins with human Fab sequences and 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 mAb products. The amino acid sequences were back-translated into DNA, which was then optimized for protein expression in CHO cells. DNA of the heavy and light chains of 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 obtained and stored in a library. The purified recombinant h16C3 was retested in studies, confirming that the humanized 16C3 antibody possessed similar characteristics to the original m16C3 antibody (Bristol and Kantor, US7829678, 2010).

[0165] The NEO-201 antibody sequence used in these examples is shown in the following figures:

[0166] H16C3-Abb* heavy chain

[0167]

[0168] H16C3-Abb* Light Chain

[0169]

[0170] In each sequence, the boundaries between the leader sequence, variable region, and constant region are defined by a forward slash (" / "), and the CDR sequences are displayed in bold, underlined text. The antibody sequences used contain the variable and constant regions shown. These include 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.

[0171] Flow cytometry

[0172] The binding of NEO-201 to human cancer cell lines was analyzed by flow cytometry. Cells (1.0 × 10⁻⁶ cells) were cultured at 4°C. 6 Cells were incubated with 1 μL of 1X phosphate-buffered saline (PBS) containing LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes per test to distinguish between live and dead cells. Cells were then centrifuged, washed twice with cold PBS, and stained for 30 minutes at 4°C with Pacific Blue-conjugated NEO-201 antibody (BioLegend, San Diego, CA) in 1X PBS + 1% BSA (Teknova, Hollister, CA, USA). After staining, cells were washed twice with cold PBS and examined using a FACSQuite flow cytometer (BD Biosciences, San Jose, CA, USA). Cell fluorescence analysis was performed using BD FACSQuite software (BD Biosciences, San Jose, CA, USA). A staining value >10% positive was considered positive for NEO-201 expression. Positive cell lines were sorted according to their quantified expression levels (positive% × MFI) and then classified into low (<200), intermediate (200-1000), and high (<1000) expression groups.

[0173] Immunohistochemistry (IHC)

[0174] Tissue microarrays of colon samples (CO808, CO951) were obtained from USBiomax (Rockville, MD), and AccuMax tissue microarrays of colon (A303(I)), pancreas (A207(II), A307), stomach (A209), lung (A206(V), A306), breast (A202(VI), A712), uterus (A212), ovary (A212, A213(II)), prostate (A302(IV)), and various normal (A103(VII)) samples were obtained from Accurate Chemical and Scientific Corporation (Westbury, NY). NEO-201 was biotinylated using a biotinylated protein labeling kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Slides were baked at 60°C for 20 minutes, dewaxed with xylene, and rehydrated with a gradient of ethanol. Then, the slides were blocked with peroxidized I solution (Biocare Medical, Concord, CA) for 2 minutes, with avidin solution (Biocare Medical, Concord, CA) for 10 minutes, with biotin solution (Biocare Medica, Concord, CA) for 10 minutes, and with CAS-blocking histochemistry reagent (Thermo Fisher Scientific, Waltham, MA) for 10 minutes. The slides were then incubated at room temperature for 2 hours with 10 μg / mL biotinylated human IgG1κ (Ancell, Bayport, MN) or biotinylated NEO-201 diluted in 1X PBS as a negative control. Detection was performed for 30 minutes with 1:300 Dako streptoavidin-HRP conjugate (Agilent Technologies, Santa Clara, CA), followed by incubation with DAB peroxidase substrate (Thermo Fisher Scientific, Waltham, MA) for 1–3 minutes, and counterstaining with hematoxylin. The cell staining intensity of each microarray tissue spot was evaluated using an optical microscope using the following scale: 0 (negative), ± (ambiguous), 1+ (weak), 2+ (moderate), 3+ (strong). A tissue spot containing cells stained with an intensity ≥ +1 was recorded as positive.

[0175] Antibody-dependent cytotoxicity (ADCC) assay

[0176] ADCC assays were performed using a modified procedure as previously described (Boyerinas et al., 2015). NK cells were selected from normal human donor PBMCs negatively using the EasySep Human NK Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's protocol. The purified NK cells were incubated overnight in RPMI-1640 medium supplemented with L-glutamine, 10% FBS, and antibiotics. On the day of assay, target cells (CFPAC-1, ASPC-1) were labeled with 10 μM calcein AM cell-permeable dye (Termo Fisher Scientific, Waltham, MA, USA) for 30 min, and then injected at 3.0 × 10⁻⁶ cells / mL. 3 Three copies of each cell / well were seeded into a black-walled, flat-bottomed 96-well culture plate (#655090 Greiner bio-one, Germany). Tumor cells were then treated with 10 μg / mL of human IgG1 isotype control antibody (Thermo Fisher Scientific, Waltham, MA, USA) or NEO-201 (unless otherwise specified), and then NK cells were added at effector to target (E:T) ratios of 12.5:1 and 25:1. After incubation at 37°C for 4 hours, 10 μg / mL propidium iodide (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well, and the plate was imaged and analyzed using a Celigo imaging system (Nexcelom Bioscence LLC, Lawrence, MA, USA). The number of viable target cells (calcein AM+ / PI-) in each well was counted, and specific ADCC lysis was calculated as follows: Specific lysis % = 100 – [(mean viable target count)] 实验值 Average live target count 对照 (x 100).

[0177] Complement-dependent cytotoxicity (CDC) assay

[0178] CDC assays were performed using a modified version of the previously described procedure (Konishi et al., 2008). ASPC-1 target cells were labeled with calcein AM as described above, and at a concentration of 5.0 × 10⁻⁶. 3Cells were seeded per well into black 96-well plates. Cells were then conditioned at 37°C with 0.5 or 5.0 μg / mL NEO-201 for 15 minutes, and purified rabbit complement (MPBiomedicals, Santa Ana, CA) was added to each well at a 1:8 dilution. After incubation at 37°C for 30, 60, or 120 minutes, propidium iodide was added, the plates were imaged, and analysis was performed using a Celigo imaging hemocytometer. Specific lysis was calculated as described above for ADCC activity.

[0179] Xenograft Antitumor Assay

[0180] Tumors were established in 6-week-old female athymic NU / NU nude mice (Charles River Laboratories International, Wilmington, MA) by subcutaneous implantation of cultured tumor cells in a suspension of 1XPBS into the right ventral region of the mice. Once the tumor reached approximately 100 mm... 3 Mice were sorted by tumor size and randomly divided into 5 groups (n = 10 animals). On days 13, 17, and 20 post-implantation, mice were intraperitoneally injected with either a single carrier (saline solution), human IgG1 (250 μg), or NEO-201 (100 and 250 μg). Mice also received approximately 1.0 x 10⁻⁶ mmol / L on days 14, 18, and 21. 7 Intraperitoneal injection of IL-2-activated human PBMCs (treated overnight in culture at 200 U / mL) as a source of immune effector cells. A group of mice were treated similarly with NEO-201 but did not receive human PBMCs. Tumors were measured every 2–3 days using digital calipers, and tumor volume (width) was calculated according to the following formula. 2 (×length) / 2=mm 3 The width is the shorter of the two measurements. Mice were also weighed weekly as a measure of overall health. Tumors >2000 mm² were considered a subset of overall health. 3 The mice were euthanized according to the IACUC guidelines.

[0181] Biological distribution analysis

[0182] Biodistribution studies were evaluated in tumor-bearing mice using radiolabeled NEO-201 (ComparativeBiosciences, Sunnyvale, CA) following a previously described procedure (Patel et al., 2013). Briefly, male and female athymic NU / NU nude mice (Charles River Laboratories International, Wilmington, MA) were subcutaneously injected with 4.0 × 10⁻⁶ NEO-201 in the lateral abdomen. 6200 μL of CFPAC-1 cells were suspended in 1X PBS. On day 14 post-transplantation, mice were intravenously injected with 20 μCi of [the solution / resource]. 125 I-labeled NEO-201, and then autopsies were performed at 1, 2, 4, or 7 days later. Blood, tumor tissue, and internal organs (lung, kidney, liver, spleen, pancreas, intestine, and stomach) (n = 4 animals) were harvested at each time point. All tissues were weighed, and the radioactivity in the tissues was measured using a gamma counter. Data for each mouse were first calculated as cpm / mg tissue, and then the tissue cpm value was normalized relative to the blood cpm value.

[0183] Single-dose toxicity study in cynomolgus monkeys

[0184] Single-dose toxicity studies were conducted in purposefully bred cynomolgus monkeys to test the pharmacokinetics and toxicity of NEO-201 following a single dose. The study duration was 15 days from dose administration, with an additional 14 days of isolation prior to dose administration to allow the monkeys to acclimatize to the laboratory. NEO-201 diluted in saline solution was administered to eight male and eight female animals (2 animals / sex / group) via slow intravenous infusion (approximately 30 min ± 5 min) at dose levels of 0 mg / kg, 5 mg / kg, 20 mg / kg, and 49 mg / kg, representing the highest achievable concentration of the antibody. Blood samples were drawn from all animals receiving NEO-201 at the following time points: before dose, 10 min, and 1, 2, 4, 6, 24, 48, 72, 96, 168, and 336 hours. Serum was prepared from the blood samples used for pharmacokinetic and toxicological analyses. Whole blood was used for cytological analysis. The level of NEO-201 in serum was measured by ELISA using a human therapeutic IgG1 ELISA kit (Cayman Chemical, Ann Arbor, MI) in accordance with the manufacturer’s instructions.

[0185] Laboratory tests included hematology and coagulation (baseline (BL), day 2, day 8, day 15): CBC and differential, activated partial thromboplastin kinase time, fibrinogen and prothrombin time; serum chemistry (BL, day 2, day 8, day 15): albumin, alkaline phosphatase, ALT, AST, total bilirubin, calcium, total cholesterol, creatine kinase, creatinine, glucose, inorganic phosphorus, total protein, triglycerides, sodium, potassium, chloride, globulin, albumin / globulin ratio, BUN; urinalysis (BL, day 15): color, clarity, glucose, ketones, occult blood, protein, bilirubin, nitrite, pH, urobilinogen, white blood cells, volume, specific gravity; bioassay (using ELISA) – (BL, 10 min, 1, 2, 4, 6 h, 24, 48, 72, 96, 168, and 336 h) from groups 2 to 4 using Phoenix WinNonlin. Version 6.1 software (Certara USA, Princeton, NJ). Animal body weight measurements were recorded (BL, 7, and 14), and neutrophil counts were assessed (BL, day 2, day 8, and day 15).

[0186] Statistical analysis

[0187] Data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA). Comparisons between two groups were performed using t-tests, and p < 0.05 was considered statistically significant. The figure depicts the mean ± SD from representative experiments conducted in triplicate.

[0188] Example 8

[0189] ALT-803 enhances ADCC mediated by NEO-201.

[0190] ALT-803 is a novel IL-15 superagonist complex composed of an IL-15 mutant (IL-15N72D) that binds to the IL-15 receptor α / IgG1 Fc fusion protein. This example tests the ability of ALT-803 to modulate ADCC mediated by NEO-201.

[0191] method

[0192] NK cells were isolated from normal donors and treated with different concentrations of ALT-803 for 48 hours before being used as effector cells. Human cancer cell lines expressing the NEO-201 antigen were used as targets in an in vitro non-radioactive ADCC assay. The ability of ALT-803 to affect the phenotype of NK cells and regulate NK cell gene expression was evaluated by flow cytometry and by using Nanostring analysis.

[0193] result

[0194] Treatment with ALT-803 significantly enhanced NEO-201-mediated ADCC activity against NEO-201-positive tumor cells. Figure 8 , Figure 11 The effect of ALT-803 was dose-dependent and achieved statistical significance at all tested doses compared to the control treatment. Treatment of NK cells with ALT-803 enhanced ADCC activity from donors also with minimal ADCC activity and reduced the effective dose of NEO-201 required to initiate an ADCC response compared to untreated NK cells. Figure 12 Furthermore, ADCC activity can be blocked by using anti-CD16 and anti-TIM3 blocking antibodies. Figure 12 ).

[0195] Phenotypic analysis of NK cells treated with 25 ng / ml ALT-803 for 48 hours showed that ALT-803 enhanced the expression of TIM3 and NKG2D in CD16 / CD56 positive NK cells, as well as the mean fluorescence intensity (MFI) of granzyme B and CD107a. Figure 9 ).

[0196] Nanostring analysis of human NK cells treated with different concentrations of ALT-803 for 48 hours showed that ALT-803 could regulate the mRNA expression of 62 genes (a fold change of 1.6 log2 compared with the control was considered significant).

[0197] ALT-803 treatment upregulated the mRNA expression of 43 genes (including NK activation receptors, factors involved in NK cell activity, cytokines, and their receptors) and downregulated the mRNA expression of 19 genes (including NK inhibitory receptors and factors involved in apoptosis activation).

[0198] Therefore, ALT-803 enhanced ADCC activity against human cancer cells mediated by NEO-201. The enhancement of ADCC activity may be attributed in part to increased expression of TIM3, NKG2D, granzyme B, and CD107a-positive NK cells, as well as to the regulation of transcripts involved in NK activation and cytotoxicity.

[0199] In summary, treatment of NK cells isolated from normal donors with ALT-803 enhanced NEO-201-mediated ADCC activity. Phenotypic analysis of ALT-803-treated NK cells isolated from normal donors demonstrated that ALT-803 enhanced the expression of TIM-3 and NKG2D on CD16 / CD56-positive NK cells. Treatment of normal NK cells with ALT-803 also increased the MFI of granzyme B in CD16 / CD56-positive NK cells. Treatment of normal NK cells with ALT-803 also increased the MFI of CD107a in CD16 / CD56-positive NK cells from one of the two donors tested. TIM-3 is an inducible human NK cell receptor that enhances interferon-γ production. It is also a marker of maturation. The enhancement of NEO-201-mediated ADCC activity after ALT-803 treatment may be partly due to the increased expression of TIM-3-positive, NKG2D-positive, granzyme B-positive, and CD107a-positive NK cells, although this theory is not intended to be limiting. Treatment of NK cells with ALT-803 enhanced ADCC activity mediated by lower concentrations of NEO-201. When NK cells were treated with ALT-803, lower concentrations of Mab were used to mediate ADCC activity and produced equivalent levels of cytotoxicity compared to NK cells treated with higher concentrations of NEO-201 without ALT-803 treatment. This result suggests that lower doses of Mab can be used in combination with ALT-803 in clinical trials for cancer treatment.

[0200] Example 9

[0201] NEO-201 enhances NK cell-dependent killing of tumor cells by blocking the inhibitory CEACAM5 / CEACAM1 immune checkpoint pathway.

[0202] Immunotherapy using checkpoint blocking antibodies targeting effector cell inhibitory receptors such as PD-1 and CTLA-4 has elicited some significant and durable responses in several tumor types. Carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1) is a cell surface protein expressed by both immune cells and tumor cells, and it can suppress T cell function similarly to PD-1 and CTLA-4. CEACAM1 is also a potent inhibitor of natural killer (NK) cell function; the binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling mediated by NKG2D, preventing NK cell lysis and allowing tumor cells to evade NK killing.

[0203] NEO-201 binds to members of the CEACAM family and can activate innate immune mechanisms, such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), to kill tumor cells. This study was designed to determine whether NEO-201 blocks the CEACAM1 inhibitory pathway to restore the antitumor function of NK cells.

[0204] method

[0205] In vitro assays using human tumor cell lines were performed to identify CEACAM family members bound by NEO-201. Functional assays were performed to evaluate the ability of NEO-201 to enhance the in vitro killing of tumor cells and mediate ADCC by the NK cell line NK-92 (which expresses CEACAM1 and lacks CD16).

[0206] Kill assays were performed using a modified version of the previously described procedure (David et al., 2017). In short, target cells derived from pancreatic (ASPC-1, BxPC-3, CFPAC-1) and colonic (LS174T) tumors were labeled with 10 μM calcein AM cell-permeable dye (Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes, and then analyzed at 3.0 × 10⁻⁶. 3 Three copies of each cell were seeded into 96-well black-walled, flat-bottomed culture plates. Tumor cells were then treated with 10 μg / mL of human IgG1 isotype control antibody (Thermo Fisher Scientific, Waltham, MA, USA) or NEO-201, followed by the addition of the natural killer (NK) cell line NK-92 at effector-to-target (E:T) ratios of 1.5625:1, 3.125:1, 6.25:1, and 12.5:1. After incubation at 37°C for 16 hours, propidium iodide (PI; Thermo Fisher Scientific, Waltham, MA, USA) was added to each well at a final concentration of 1.67 μg / mL, and the plates were centrifuged. Imaging was performed using a Celigo imaging hematology counter (Nexcelom Bioscence LLC, Lawrence, MA, USA), and analysis was performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). Count the number of live target cells (calcein AM+ / PI-) in each well and calculate specific lysis as follows: Specific lysis % = 100 – [(mean live target count)] 实验值 Average live target count 对照 (x 100).

[0207] result

[0208] NEO-201 was found to respond to different variants of CEACAM5 and CEACAM6, but not to CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell lines expressed different levels of the native forms of CEACAM5 / 6 compared to NEO-201-responsive variants of these molecules. Functionally, NEO-201 treatment enhanced the cytolytic activity of NK-92 cells against NEO-201+ tumor cells expressing CEACAM5, but not against NEO-201+ cells expressing only CEACAM6. Figure 13 ).

[0209] in conclusion

[0210] NEO-201 responds to tumor-associated variants of CEACAM5 / 6 and can block the interaction between CEACAM5 on tumor cells and CEACAM1 on NK cells to reverse CEACAM1-dependent inhibition of NK cell activity.

[0211] abbreviation

[0212] 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 rate (CL), observed maximum plasma concentration (Cmax), dose-normalized measured maximum plasma concentration (Cmax / D), estrogen receptor (ER), half-life (HL), immunohistochemistry (IHC), natural killer cells (NK), non-small cell lung cancer (NSCLC), peripheral blood mononuclear cells (PBMC), progesterone receptor (PR), tumor-associated antigen (TAA), time to observed maximum plasma concentration (Tmax), volume of distribution (Vz).

[0213] References

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[0264] Table 1: Flow cytometry analysis of NEO-201 binding with cultured tumor cell lines derived from various types of solid tumors. Percentage of positive cells and mean fluorescence intensity (MFI) values ​​are detailed for each cell line. NEO-201 positive cell lines are shown in bold. NEO-201 positivity is defined as >10% positivity.

[0265]

[0266]

[0267] Table 2: IHC spectra of NEO-201 staining in normal human microarray tissues.

[0268]

[0269]

[0270] Table 3: Pharmacokinetic results of a single dose of NEO-201 in cynomolgus monkeys. Eight male and eight female animals (2 animals / sex / group) were intravenously injected with 0 mg / kg (saline solution) or 5 mg / kg, 20 mg / kg, or 49 mg / kg of NEO-201. Blood samples were collected from all animals receiving NEO-201 at different time points (before dose, 10 minutes after dose, and 1, 2, 4, 6, 24, 48, 72, 96, 168, and 336 hours after dose), and pharmacokinetic measurements from the serum preparation were obtained by ELISA. The values ​​in the table represent the mean from 2 animals / sex / group (M, F) or from all 4 animals (all).

[0271] Abbreviations: 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); Clearance (CL); Observed maximum plasma concentration (Cmax); Dose-normalized maximum measured plasma concentration (Cmax / D); Half-life (HL); Time to the observed maximum plasma concentration (Tmax); Volume of distribution (Vz).

[0272]

Claims

1. A method for killing cancer cells, the method comprising administering an effective amount of NEO-201 antibody to a patient in need.

2. A method for treating cancer, the method comprising administering an effective amount of NEO-201 antibody to a patient in need.

3. A method for preventing cancer recurrence, the method comprising administering an effective amount of NEO-201 antibody to a patient in need.

4. A method for reducing the tumor burden in a patient with cancer, the method comprising administering an effective amount of NEO-201 antibody to the patient in need.

5. The method of any of the preceding claims, wherein the antibody mediates complement-mediated cytotoxicity (CDC) to kill cancer cells in the patient.

6. The method as claimed in any of the preceding claims, wherein the patient is of natural killer ("NK") depletion type before or during the administration.

7. The method as claimed in any of the preceding claims, wherein the patient is a severe NK cell depletion type before or during the administration.

8. The method of claim 6, further comprising determining whether the patient is NK-reduced type before or during the administration.

9. The method of claim 6, further comprising determining, before or during the administration, whether the patient is a severe NK cell depletion type.

10. The method of any of the preceding claims, wherein the patient suffers from NK cell deficiency (NKD), the NK cell deficiency optionally including CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD1).

11. The method of any of the preceding claims, wherein the patient is NK-reduced or severely NK-reduced due to another therapy.

12. The method as described in any of the preceding claims, wherein the patient is undergoing cancer therapy.

13. The method as described in any of the preceding claims, wherein the patient is undergoing chemotherapy or radiation therapy.

14. The method of claim 13, wherein the chemotherapy comprises administration of one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, succinylaniline-hydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, afedipine, mitomycin, methotrexate, etoposide), GSK inhibitors (e.g., LiCl, BIO, SB21), BET inhibitors (e.g., JQ1), HSP90 inhibitors (e.g., rhizobacterin, 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel), and / or immunomodulatory agents (e.g., lenalidomide).

15. The method of any of the preceding claims, wherein NK cells constitute less than 5% of peripheral blood mononuclear cells (PBMCs) in the individual before or during the administration.

16. The method as described in any of the preceding claims, wherein NK cells constitute less than 3% of peripheral blood mononuclear cells (PBMCs) in the individual before or during the administration.

17. The method as claimed in any of the preceding claims, wherein less than 70% of the PBMC NK cells in the patient are CD56 prior to or at the time of administration. dim CD16 + NK cells.

18. The method as described in any of the preceding claims, wherein less than 50% of the PBMC NK cells in the patient are CD56 prior to or at the time of administration. dim CD16 + NK cells.

19. The method of any 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.

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

38.

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

39.

22. The method of any of the preceding claims, 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.

23. The method of any of the preceding claims, wherein the NEO-201 antibody comprises the variable heavy chain sequence of SEQ ID NO: 38 and the variable light chain of SEQ ID NO:

39.

24. The method of any of the preceding claims, wherein the NEO-201 antibody comprises 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.

25. The method of claim 23 or 24, wherein the NEO-201 antibody comprises all six of the CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO:

29.

26. The method of any of the preceding claims, wherein the NEO-201 antibody comprises the heavy chain variable region sequence contained in SEQ ID NO: 28 and the light chain variable region sequence contained in SEQ ID NO:

29.

27. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a heavy chain sequence containing amino acids 20-470 of SEQ ID NO:28 and a light chain sequence containing amino acids 20-233 of SEQ ID NO:

29.

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

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

30. The method of any of the preceding claims, wherein the NEO-201 antibody is conjugated to another portion.

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

32. The method as described in any of the preceding claims, further comprising administering an effective amount of a cytokine agonist to the patient to enhance or stimulate the killing of cells of the tumor.

33. The method of claim 32, wherein the cytokine agonist comprises interleukin-2 (IL-2), interleukin-21 (IL-21), ALT-803, IL-15 inhibitor, checkpoint inhibitor, anti-PD1, anti-PDL1, anti-CTLA-4, anti-41BB, anti-OX40, anti-Tim-3, or combinations thereof.

34. The method of any of the preceding claims, further comprising administering to the patient an effective amount of a complement regulatory protein (CRP) antagonist to enhance or stimulate the killing of cells of the tumor.

35. The method of claim 34, wherein the CRP antagonist antagonizes one or more of CD46, CD55, or CD59.

36. The method of claim 34 or 35, wherein the CRP antagonist comprises an antibody or an antigen-binding fragment thereof.

37. The method of claim 32, wherein the cytokine agonist comprises an IL-15 agonist or an IL-15 superagonist.

38. The method of claim 32, wherein the cytokine agonist comprises a complex consisting of an IL-15 mutant (IL-15N72D) that binds to the IL-15 receptor α / IgG1 Fc fusion protein.

39. The method of claim 38, wherein the cytokine agonist comprises ALT-803.

40. The method of any one of claims 32-39, wherein the effective dose of the NEO-201 antibody is reduced compared to treatment with the NEO-201 antibody alone in the absence of the cytokine agonist.

41. The method as claimed in any of the preceding claims, wherein the cancer includes colon cancer.

42. The method as claimed in any of the preceding claims, wherein the cancer includes pancreatic cancer.

43. The method as claimed in any of the preceding claims, wherein the cancer includes ovarian cancer.

44. The method as described in any of the preceding claims, wherein the cancer includes gastric cancer.

45. The method as claimed in any of the preceding claims, wherein the cancer includes lung cancer.

46. ​​The method as claimed in any of the preceding claims, wherein the cancer includes breast cancer.

47. The method as claimed in any of the preceding claims, wherein the cancer includes uterine cancer.

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