Monoclonal antibody neo-201 for treatment of human cancer

Abstract

To provide a method for selectively targeting cancerous cells while sparing normal healthy tissues.SOLUTION: Provided are a method of killing carcinoma cells comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof; a method of treating a carcinoma comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof; and a method of preventing the recurrence of a carcinoma comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 592,778, filed November 30, 2017, and U.S. Provisional Application No. 62 / 581,380, filed November 3, 2017, each of which is incorporated herein by reference in its entirety.

[0002] Sequence listing information This application contains biological sequences listed in a file named "43282o4402.txt" having a size of 32563 bytes and created on November 2, 2018, which is incorporated by reference herein in its entirety as part of this disclosure. [Background technology]

[0003] Cancer is one of the most frequent causes of death worldwide, with an estimated 20 million new cases predicted annually as early as 2025 (Ferlay et al., 2015). Traditional cancer treatments, such as surgery, radiation, and chemotherapy, often induce severe side effects and are incurable for the majority of patients with advanced disease, resulting in recurrence (Bodey et al., 1996). More recent therapies have been developed to selectively target cancerous cells, primarily while sparing normal, healthy tissue. Among these, immunotherapy has revolutionized the field of cancer care and has become an important treatment option for cancer patients.

[0004] The underlying principle of cancer immunotherapy is known as immunoediting (Mittal et al., 2014), an extrinsic mechanism of tumor suppression that initiates only after cellular transformation and intrinsic mechanisms of tumor suppression have failed. The immunoediting process occurs in three phases: elimination, equilibrium, and escape. During the elimination and equilibrium phases, immune rejection of cancer cells dominates or balances with cancer cell proliferation, controlling malignant growth. However, during the escape phase, once suppressed, cancer cells may escape immune recognition due to insensitivity to immune effector mechanisms and / or the induction of immunosuppression in the tumor microenvironment. Cancer cells that escape immune recognition can proliferate and grow more freely, potentially resulting in clinically evident disease (Dunn et al., 2004). The goal of cancer immunotherapy is to generate and / or amplify antitumor immune responses to suppress tumor growth, delay tumor recurrence, and prolong survival, thereby maintaining cancer cells in a state of elimination and / or equilibrium (Carter, 2001; Hodge et al., 2006; Vergati et al., 2010; Gabitzsch et al., 2015). Therapeutic approaches include treating patients with checkpoint inhibitor antibodies, antitumor vaccines, and chimeric antigen receptor (CAR) T cells, all of which exploit T cell-mediated adaptive immunity. 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).

[0005] NEO-201 is a novel humanized IgG1 mAb generated against the Hollinshead allogeneic colon cancer vaccine platform (Hollinshead et al., 1970; Hollinshead et al., 1972). The immunogenic component of this vaccine was tumor-associated antigens (TAA) derived from pooled tumor membrane fractions from surgical resections of 79 patients with colon cancer (Hollinshead et al., 1985). These membrane fractions were semi-purified, screened for delayed-type hypersensitivity (DTH) in colon cancer patients versus healthy volunteers, and evaluated in clinical trials in patients with refractory colon cancer (Hollinshead et al., 1985; Hollinshead, US4810781, 1989; Bristol & Kantor, US7829678, 2010). These trials reported clinical benefit, defined by both antitumor responses and significantly prolonged overall survival, in patients who developed sustained IgG responses in addition to cell-mediated responses to the vaccine, suggesting that the vaccine contained immunogenic components capable of generating antitumor antibodies (Hollinshead, 1991). This first colon cancer vaccine was used to generate monoclonal antibodies in mice, including the previously described ensituximab (NPC-1C / NEO-102) (Luka et al., 2011; Patel et al., 2013; Beg et al., 2016; Kim et al., 2017) and NEO-201. Preliminary studies suggest that NEO-201 may bind to tumor-associated variants of CEACAM family members (Zeligs et al., 2017), and efforts are underway to further characterize the antigen(s) and specific epitope(s) recognized by NEO-201.

[0006] The human carcinoembryonic antigen (CEA) family consists of 29 genes tandemly arranged on chromosome 19q13.2. Based on nucleotide homology, these genes are classified into two major subfamilies: CEACAM and pregnancy-specific glycoprotein subgroups. CEACAM-encoded proteins include CEA (CEACAM5) and CEA-related cell adhesion molecules (CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, and CEACAM8). The CEACAM family belongs to the Ig superfamily. Structurally, each human CEACAM contains a single N-terminal domain of 108–110 amino acids that is homologous to Ig variable domains, followed by a variable number (0–6) of Ig C2-type constant-like domains. CEACAM proteins can interact with each other via homophilic and heterophilic mechanisms. CEACAM1 is unique within this family because it contains a PD1-like ITIM (immunoreceptor tyrosine-based inhibitory motif) within its cytoplasmic domain. This inhibitory effect is caused by the phosphorylation of tyrosine residues by ITIMs, resulting in the recruitment of Src homology 2 domain-containing tyrosine phosphatases-1 and -2. The CEACAM1 protein is expressed on various immune cells, including monocytes, granulocytes, activated T cells, B cells, and NK cells. CEACAM1 occurs in several isoforms, the two major of which are CEACAM1-L and CEACAM1-S, with long (L) and short (S) cytoplasmic domains, respectively. CEACAM1-S expression is completely absent on human leukocytes. CEACAM1-L is expressed on a subpopulation of activated human NK cells that are negative for CD16 but positive for CD56.

[0007] Monoclonal antibodies (mAbs) consist of a unique antigen-binding region (fragment antigen-binding, Fab) that is specific to a given mAb and a constant region (fragment crystallizable, Fc) that is common to all mAbs of the same isotype. The Fc region can regulate immune cell activity by engaging with members of the Fc receptor (FcR) family expressed on the surface of specific immune cell types. In particular, human IgG1 mAbs can interact with Fc gamma receptor IIIa (FcγRIIIa, CD16) expressed on macrophages and NK cells. This interaction stimulates macrophages to phagocytose mAb-opsonized cancer cells and activates NK cells to degranulate and lyse cancer cells through a mechanism known as antibody-dependent cellular cytotoxicity (ADCC). ADCC has been shown to be a primary mediator of antitumor effects in vivo in numerous 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 can mediate ADCC include trastuzumab, which targets the HER2 receptor in breast cancer (Seidel et al., 2013; Petricevic 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 cancers (Seidel et al., 2013; Levy et al., 2009; Kawaguchi et al., 2007; Lopez-Albaitero et al., 2009); and avelumab, which targets the immunosuppressive ligand PD-L1 in Merkel cell carcinoma and bladder cancer (Boyerinas et al., 2015). Additionally, the Fc region can interact with the C1 complex to activate complement-dependent cytotoxicity (CDC), in which a proteolytic cascade leads to the formation of pores in the plasma membrane that cause lysis of the antibody-targeted cell.Even when antitumor CDC is demonstrated in vitro, whether it is important for the clinical efficacy of mAb therapy in cancer remains controversial ( Meyer et al., 2014 ).

[0008] Applicant's previous U.S. Patent Nos. 5,688,657, 7,314,622, 7,491,801, 7,763,720, 7,829,678, 8,470,326, 8,524,456, 8,535,667, 8,802,090, 9,034,588, 9,068,014, 9,371,375, 9,592,290, 9,718,866, and RE39,760 (each of which is incorporated herein by reference in its entirety) disclose various anti-cancer antibodies, cancer antigens, and related technology. Summary of the Invention

[0009] The studies described in the Examples herein evaluate the in vitro binding characteristics and 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 was not observed to bind to the majority of healthy tissues. Furthermore, NEO-201 exhibited both ADCC and CDC activity against human carcinoma cells in vitro, and NEO-201 significantly attenuated the growth of human pancreatic xenograft tumors in vivo, both alone and in combination with human peripheral blood mononuclear cells (PBMCs) as a source of ADCC effector cells. Finally, a single-dose toxicity study in non-human primates demonstrated the safety and tolerability of NEO-201, with the only adverse effect observed being a transient decrease in circulating neutrophils. These studies provide evidence for the potential clinical utility of NEO-201 as a novel therapeutic agent for the treatment of various solid tumors. Furthermore, the observed CDC activity of the subject antibodies opens up the opportunity to treat immunocompromised patients in whom ADCC is not expected to be effective, e.g., patients who are immunocompromised due to disease or as a result of radiation, chemotherapy, and other disease treatments.

[0010] We previously reported the preclinical antitumor activity (Patel et al., 2013) and clinical safety and efficacy (Beg et al., 2016; Kim et al., 2017) of a mAb (termed ensituximab (NPC-1C / NEO-102)) generated against the Hollinshead allogeneic colorectal cancer vaccine platform. In this report, we describe the properties of a second tumor antigen-targeting mAb derived from the same vaccine platform, termed NEO-201. NEO-201 has been shown to positively stain a variety of human carcinoma cell lines in vitro, including cells derived from various tumor types, histological subtypes, and mutational profiles. NEO-201 positivity was observed more frequently in tumor cell lines derived from lung adenocarcinoma versus squamous cell carcinoma, and in HER2-positive breast cancer cell lines versus triple-negative lines. Staining of human tumor samples demonstrated positive staining for NEO-201 in a variety of cancer tissues, including colon, pancreas, stomach, lung, breast, and uterine tumors. Expanded studies using larger sample sizes may reveal that NEO-201 can distinguish between various histological and / or molecular subtypes of carcinomas. Interestingly, a higher proportion of tumor tissues responded to NEO-201, in contrast to cultured cancer cell lines. This observation may indicate that the targets recognized by NEO-201 are more readily expressed in vivo than in vitro. This suggests that target expression is at least partially dependent on tumor cell interactions with factors from within the local microenvironment. Experiments are currently underway to further characterize the antigen(s) and epitope(s) recognized by NEO-201 and to determine the regulatory control mechanism(s) governing their expression in tumor tissues but not in normal tissues.

[0011] This study revealed that the staining profile of NEO-201 is remarkably tumor-specific, as the vast majority of healthy normal tissues and normal tissues adjacent to tumor tissues were negative for NEO-201. NEO-201 positivity was observed in normal tongue and cervical tissue, but the staining intensity was weak and represented only the smallest sample size (n = 2) in the microarray. Further expanded analysis of NEO-201 staining in normal tissue samples will be performed to confirm these observations. Furthermore, NEO-201 administration did not induce any macroscopic toxicity in mice and was well tolerated when administered to non-human primates. The observed depletion of neutrophils in non-human primates suggests that the antigen(s) reactive with NEO-201 are expressed on these immune cells, and evaluation of NEO-201 reactivity with hematopoietic cell types is ongoing. These promising results suggest that 1) NEO-201 may have diagnostic utility in distinguishing benign from cancerous tissue in patient biopsies, and 2) NEO-201 can effectively target tumors without causing significant toxicities or off-target effects other than neutropenia. Efforts to further evaluate the safety and tolerability of NEO-201 are currently underway, and clinical trials for the treatment of cancer using NEO-201 are planned.

[0012] Innate immune effector mechanisms have been shown to play a key role in promoting and enhancing host antitumor immunity. It is well known that the Fc portion of human IgG1 mAbs can activate innate immunity against opsonized targets and mediate ADCC and / or CDC (Strome et al., 2007; Hayes J, et al., 2017). In particular, the ability to mediate ADCC has been considered a key factor in 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 within the FCGR3A gene (encoding FcγRIIIa) is associated with differences in the affinity of human IgG1 mAbs (Koene et al., 1997; Wu et al., 1997), and immune cells from donors with the high-affinity V / V genotype exhibit higher trastuzumab-mediated ADCC activity in vitro (Musolino et al., 2008). The V / V genotype has also been shown to significantly correlate with objective response rate and progression-free survival in breast cancer patients treated with trastuzumab (Musolino et al., 2008), providing indirect clinical evidence for the role of ADCC in mAb-based therapy. Treatment of tumor cells with NEO-201 enhanced NK cell cytotoxicity by 2-5-fold, and ADCC activity was maintained even at low antibody concentrations (0.1 μg / mL), demonstrating that NEO-201 can mediate ADCC in vitro. These data raise the possibility that patients with the V / V genotype may derive additional benefit from NEO-201 treatment. An additional perspective is that enhancing NK cell function through cytokine stimulation may improve ADCC activity and possibly the potential clinical benefit of NEO-201.IL-2 is known to be 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 using a novel fusion protein superagonist of IL-15 signaling, termed ALT-803, have demonstrated significant enhancement of the proliferation, activation, and lytic capacity 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 substantially enhance NK cell degranulation, IFN-γ production, and rituximab-mediated ADCC against B-cell lymphoma cell lines and primary follicular lymphoma cells in vitro, and combination treatment with ALT-803 and rituximab in two B-cell lymphoma models in vivo significantly reduced tumor burden and improved survival ( Rosario et al., 2016 ).

[0013] Another innate immune effector mechanism that MAbs can engage is activation of the complement system, which promotes CDC. NEO-201 has been shown to mediate CDC and kill tumor cells. While the contribution of CDC to the therapeutic efficacy of MAbs remains controversial, it has been suggested that, at least in some specific cases, it may be beneficial for cancer treatment (Meyer et al., 2014). Furthermore, several different complement regulatory proteins (CRPs) function to inhibit complement activation, and specific membrane-bound CRPs, such as CD46, CD55, and CD59, have been reported to be aberrantly expressed in various cancers, likely conferring resistance to CDC (Seya et al., 1994; Niehans et al., 1996; Donin et al., 2003). Future investigations will determine whether strategies to block CRPs can enhance NEO-201-mediated CDC in resistant tumor cells.

[0014] In vivo evaluation of NEO-201 revealed significant antitumor effects when administered in combination with activated human immune effector cells. This combination even resulted in complete regression in some mice (5 / 20, 25%) in both combination groups. Furthermore, NEO-201 was found to preferentially localize to xenograft tumor tissue 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, without the addition of human immune cells to immunodeficient mice. In vitro, treatment of CFPAC-1 tumor cells with NEO-201 did not induce substantial toxicity in ADCC assays, so this phenomenon may be specific to conditions occurring in vivo. One hypothesis for NEO-201 activity in the absence of immune effector cells could be the induction of CDC. The CDC activity of NEO-201 was directly demonstrated in further experiments described in Example 3.

[0015] In summary, this study demonstrates that NEO-201 is a highly tumor-specific antibody capable of engaging innate immune effector mechanisms, including both ADCC and CDC, to kill tumor cells. Furthermore, NEO-201 demonstrated safety and antitumor efficacy in an in vivo xenograft model of pancreatic cancer and was well tolerated in nonhuman primates. These findings support the clinical development of NEO-201 as a diagnostic and therapeutic agent for patients with various types of cancer. Because antitumor effects can arise from CDC even in the absence of potent ADCC activity, these results also support the use of NEO-201 in immunocompromised patients (i.e., low NK cell levels). [Brief explanation of the drawings]

[0016] [Figure 1] Figures 1A-1D show flow cytometry results of NEO-201 binding to human carcinoma cell lines. Representative human carcinoma cell lines with varying levels of NEO-201 antigen expression are shown: (Figure 1A) pancreatic CFPAC-1 (high), (Figure 1B) NSCLC H441 (medium), (Figure 1C) breast HCC1937 (low), and (Figure 1D) colon SW1116 (negative). Results are expressed as the % NEO-201 positivity and mean fluorescence intensity (MFI) for each cell line. Red, NEO-201-stained cells; black, unstained cells. NEO-201 positivity was defined as a % positivity of ≥ 10%. [Figure 2] Figures 2A-2C show IHC staining of human tumor samples with NEO-201. Figure 2A shows representative NEO-201 staining from adjacent normal and malignant tissue from colon, pancreas, stomach, and lung samples. All images were acquired at 100X. Figure 2B shows quantification of NEO-201 positive staining from human tumor microarray samples from various cancerous tissues. Figure 2C shows quantification of NEO-201 positive staining from human tumor microarray samples of normal tissue adjacent to tumor tissue. n = number of samples. [Figure 3]Figures 3A-3C show that NEO-201 mediates ADCC and CDC against human tumor cell lines. Figure 3A shows ADCC activity using CFPAC-1 or ASPC-1 cells as target cells. Cells were treated with 10 μg / mL of 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 shows an ADCC assay using CFPAC-1 cells treated with increasing doses of NEO-201. NK cells isolated from healthy donors were used as effector cells at an E:T ratio of 12.5:1. The graph shows the fold change in the % specific lysis of NEO-201-treated tumor cells relative to control cells treated with 10 μg / mL of human IgG1. *Statistically significant by T-test (p<0.05). Figure 3C shows a CDC assay using ASPC-1 cells treated with the indicated doses of NEO-201 for the indicated time periods. *Statistically significant by T-test (p<0.05). [Figure 4] Figures 4A-4D show the antitumor effects of NEO-201 in CFPAC-1 tumor xenografts. Figure 4A shows tumor volume measurements of CFPAC-1 xenografts in each treatment group at various time points. Mice (n = 10 animals per group) were intraperitoneally administered saline, human IgG1 (250 μg), or NEO-201 (100 μg and 250 μg) on ​​days 13, 17, and 20 after tumor cell implantation. Mice were also intraperitoneally administered approximately 1.0 x 10 IL-2-activated human PBMCs on days 14, 18, and 21 as a source of immune effector cells. Figure 4B shows the quantification of the number of mice still bearing palpable tumors on day 36. Figure 4C shows representative images of NEO-201-treated versus saline-treated tumor-bearing mice. FIG. 4D shows the weight measurements of tumor-bearing mice at various time points during the study. [Figure 5]Figures 5A-5B show the biodistribution of NEO-201 in CFPAC-1 xenograft-bearing mice. Normalized radioactivity measurements from indicated tissues of female (Figure 5A) and male (Figure 5B) mice bearing CFPAC-1 tumors were administered intravenously with radiolabeled NEO-201. n=4 animals / time point. Days 1, 2, 4, and 7 represent the amount of time from injection of the radiolabeled antibody to necropsy. [Figure 6] Figures 6A-6C show body weights and neutrophil counts in cynomolgus monkeys treated with NEO-201. Figure 6A shows the percent change in body weight relative to baseline (day -1) measured for monkeys 7 and 14 days after receiving a single dose of NEO-201 at the indicated dose levels. n=4 animals / group (2 females, 2 males). Figure 6B shows the percent change in neutrophil levels relative to baseline (day -7) from the blood of monkeys treated with a single dose of NEO-201 at the indicated dose levels. n=4 animals / group (2 females, 2 males). Figure 6C shows the p-value for neutrophil levels versus the 0 mg / kg control at each dose and time point. *Statistically significant by T-test (p<0.05). [Figure 7]Figures 7A-7C show haNK ADCC assays (4 hours) using NEO-201. Target cells = 3,000 cells / well. Figures 6A-6B show the specific lysis rate of H520 lung cancer (Figure 6A) or OV90 ovarian cancer (Figure 6B) cells treated with NEO-201 (overlined, square symbols) or IgG1 negative control (underlined, circle symbols) at 4 hours as a function of effector:target (E:T) ratio. The E:T ratio was 6.25:1, 12.5:1, or 25:1. The mAb concentration was 10 μg / mL. Values ​​shown are the mean + / - SD of triplicates. Asterisks (*) indicate statistical significance relative to the IgG negative control (p<0.01, two-tailed t-test). Figure 6C shows the percentage of specific lysis of lung (H520, HCC827), breast (ZR-75-1), and ovarian (OV90) cancer cells treated with NEO-201 (right, light gray bars) or negative control IgG (left, black bars) at an E:T ratio of 25:1 for 4 hours. The mAb concentration was 10 μg / mL. Values ​​shown are the mean + / - SD of triplicates. Asterisks (*) indicate statistical significance relative to the IgG negative control (p<0.01, two-tailed t-test). [Figure 8] Figure 1 shows that treatment with ALT-803 enhances ADCC activity mediated by NEO-201. NK cells isolated from two normal donors were treated with ALT-803 (25 ng / ml) or medium control for 48 hours and used as effector cells in a 4-hour non-radioactive ADCC assay using a Celigo Imaging cytometer. 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). [Figure 9] (A) ALT-803 treatment enhanced the expression of TIM-3 and NKG2D on human NK cells. Purified human NK cells from normal donors were cultured with or without ALT-803 (25 ng / ml) for 48 hours. Results are expressed as % positive cells (MFI). [Figure 10](Figure 1) 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 with or without ALT-803 (25 ng / ml) for 48 hours. Results are expressed as % positive cells (MFI). [Figure 11] This figure shows that treatment with ALT-803 enhanced ADCC activity mediated by low concentrations of NEO-201. NK cells (ND#6) isolated from a normal donor were treated with ALT-803 (25 ng / ml) or medium control for 48 hours and used as effector cells in a 4-hour, non-radioactive ADCC assay using a Celigo Imaging cytometer. 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). [Figure 12]This figure shows that treatment with ALT-803 enhanced ADCC activity in a normal donor (ND#8), while NEO-201-mediated ADCC activity was minimal. This activity could be blocked by anti-CD16 and anti-TIM-3 antibodies. NK cells isolated from a normal donor with minimal ADCC activity were treated with ALT-803 (25 ng / ml) or medium control for 48 hours and used as effector cells in a 4-hour non-radioactive ADCC assay using a Celigo Imaging cytometer. 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 hours 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 lysis (SE). *Statistically significant compared to the absence of ALT-803 treatment (p<0.01). #Statistically significant compared to the absence of anti-CD16 and anti-TIM-3 treatment (p<0.01). [Figure 13] Figure 1 shows an NK-92 killing assay (16 hours) using NEO-201. Target tumor cells (ASPC-1, BxPC-3, CFPAC-1, or LS174T) were seeded at 3000 cells / well. Tumor cells were then treated with 10 μg / mL of human IgG1 isotype control antibody or NEO-201, and the natural killer (NK) cell line NK-92 was added 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 Cytometer and GraphPad Prism7 software. Live target cells (calcein AM+ / PI-) were counted in each well, and specific lysis was calculated. Results are presented graphically and tabulated below for each tumor cell type. *Statistically significant (p<0.05). DETAILED DESCRIPTION OF THE INVENTION

[0017] In one aspect, the disclosure provides a method of killing carcinoma cells, comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof.

[0018] In one aspect, the present disclosure provides a method of treating carcinoma comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof.

[0019] In one aspect, the disclosure provides a method of preventing recurrence of a carcinoma, comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof.

[0020] In one aspect, the present disclosure provides a method of reducing tumor burden in a patient having a carcinoma, comprising administering to the patient in need thereof an effective amount of a NEO-201 antibody.

[0021] This antibody is capable of mediating complement-mediated cytotoxicity (CDC), thereby killing carcinoma cells in patients.

[0022] The patient may be natural killer ("NK") depleted before or at the time of administration. The patient may be severely NK depleted before or at the time of administration. The patient may have an NK cell deficiency (NKD), such as CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD1). The patient may be NK depleted or severely NK depleted as a result of another treatment, for example, cancer therapy such as chemotherapy or radiation therapy. Patients may be treated with one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, suberoylanilide-hydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, aphidicolin, 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).

[0023] The method can include determining whether the patient is NK depleted prior to or at the time of administration.

[0024] The method can include determining whether the patient is severely NK depleted prior to or at the time of administration.

[0025] In this method, prior to or at the time of administration, NK cells may comprise less than 5% of the peripheral blood mononuclear cells (PBMCs) in the individual.

[0026] In this method, prior to or at the time of administration, NK cells may comprise less than 3% of the peripheral blood mononuclear cells (PBMCs) in the individual.

[0027] In this method, prior to or at the time of administration, less than 70% of the patient's PBMC NK cells can be CD56dimCD16+ NK cells.

[0028] In this method, prior to or at the time of administration, less than 50% of the patient's PBMC NK cells can be CD56dimCD16+ NK cells.

[0029] The NEO-201 antibody can 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.

[0030] The NEO-201 antibody may comprise a variable heavy chain sequence having at least 90% identity to SEQ ID NO:38.

[0031] The NEO-201 antibody may comprise a variable light chain sequence having at least 90% identity to SEQ ID NO:39.

[0032] The NEO-201 antibody may comprise a variable heavy chain sequence having at least 90% identity to SEQ ID NO:38 and a variable light chain sequence having at least 90% identity to SEQ ID NO:39.

[0033] The NEO-201 antibody can comprise a heavy chain sequence having at least 90% identity to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence having at least 90% identity to amino acids 20-233 of SEQ ID NO:29.

[0034] The NEO-201 antibody may comprise all six CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0035] The NEO-201 antibody may comprise a human IgG1 constant domain.

[0036] The NEO-201 antibody may be humanized.

[0037] The NEO-201 antibody may be conjugated to another moiety.

[0038] The NEO-201 antibody may be conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

[0039] The method can further include administering to the patient an effective amount of a cytokine agonist to enhance or stimulate cell death of the carcinoma. The cytokine agonist can be interleukin-2 (IL-2), interleukin-21 (IL-21), ALT-803, an IL-15 inhibitor, a checkpoint inhibitor, anti-PD1, anti-PDL1, anti-CTLA-4, anti-41BB, anti-OX40, anti-Tim-3, or a combination thereof.

[0040] The method may further comprise administering to the patient an effective amount of a complement regulatory protein (CRP) antagonist to enhance or stimulate the death of carcinoma 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.

[0041] The cytokine agonist may comprise an IL-15 agonist or an IL-15 superagonist.

[0042] The cytokine agonist may comprise a complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor alpha / IgG1 Fc fusion protein, such as ALT-803.

[0043] The effective dosage of NEO-201 antibody may be reduced compared to treatment with NEO-201 antibody alone without a cytokine agonist.

[0044] The carcinoma can be colon cancer. The carcinoma can be pancreatic cancer. The carcinoma can be ovarian cancer. The carcinoma can be gastric cancer. The carcinoma can be lung cancer. The carcinoma can be breast cancer. The carcinoma can be uterine cancer.

[0045] In another embodiment, the present disclosure provides a method of killing carcinoma cells, comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof, wherein the patient is natural killer ("NK") depleted prior to or at the time of administration. NK depletion can be in a patient having less than 5% or less than 3% peripheral blood mononuclear cells (PBMCs) in a sample, e.g., a blood sample, from the patient that are NK cells. Alternatively or additionally, in this method, less than 70% (optionally less than 50%) of the patient's PBMC NK cells can be CD56dimCD16+ NK cells prior to or at the time of administration.

[0046] In another embodiment, the present disclosure provides a method of treating carcinoma comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof, wherein the patient is natural killer ("NK") depleted prior to or at the time of administration.

[0047] In another embodiment, the present disclosure provides a method for preventing recurrence of carcinoma, comprising administering an effective amount of a NEO-201 antibody to a patient in need thereof, wherein the patient is natural killer ("NK") depleted prior to or at the time of administration.

[0048] In another embodiment, the present disclosure provides a method for reducing tumor burden in a patient having a carcinoma, comprising administering to the patient in need thereof an effective amount of a NEO-201 antibody, wherein the patient is natural killer ("NK") depleted prior to or at the time of administration.

[0049] In the above-mentioned method, the antibody mediates CDC, thereby killing carcinoma cells in the patient, for example, even though there is no effective ADCC because the patient is NK depleted.The patient can be severely NK depleted at the time of administration.Optionally, the method further comprises determining whether the patient is NK depleted or severely NK depleted, for example, at the time of administration or within a period before administration, for example, 1 week or 2 weeks before.NK depletion or severely NK depleted state can be inferred from the patient's medical history, such as the prior use or simultaneous use of another treatment that depletes NK cells.For example, the patient is undergoing or simultaneously undergoing cancer therapy, such as radiotherapy or chemotherapy. Cancer therapy may include administering one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, suberoylanilide-hydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, aphidicolin, 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).

[0050] The patient may have an NK cell deficiency (NKD), such as a CNKD (e.g., CNKD1, CNKD2), or an FNKD (e.g., FNKD1).

[0051] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding or 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.

[0052] In preferred embodiments of the invention, which may be used in conjunction with any of the preceding 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 to SEQ ID NO: 38. A variable heavy chain having the above percent sequence identity may comprise all three CDR sequences contained in SEQ ID NO: 38.

[0053] In preferred embodiments of the invention, which may be used in conjunction with any of the preceding 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 to SEQ ID NO: 39. The variable light chain may comprise all three CDR sequences contained in SEQ ID NO: 39.

[0054] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding 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 to 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 to SEQ ID NO: 39. The variable light chain may comprise all three CDR sequences contained in SEQ ID NO: 39, and the variable heavy chain with percent sequence identity may comprise all three CDR sequences contained in SEQ ID NO: 38.

[0055] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding 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 to 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 to amino acids 20-233 of SEQ ID NO: 29. The light chain may comprise all three CDR sequences contained in SEQ ID NO: 29, and the variable heavy chain having percent sequence identity may comprise all three CDR sequences contained in SEQ ID NO: 28.

[0056] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding or following embodiments, the NEO-201 antibody may comprise a heavy chain variable region sequence contained in SEQ ID NO:28 and a light chain variable region sequence contained in SEQ ID NO:29.

[0057] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding or following embodiments, the NEO-201 antibody may comprise a heavy chain sequence comprising amino acids 20-470 of SEQ ID NO:28 and a light chain sequence comprising amino acids 20-233 of SEQ ID NO:29.

[0058] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding or following embodiments, the NEO-201 antibody comprises a human IgG1 constant domain.

[0059] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding or following embodiments, the NEO-201 antibody may be humanized.

[0060] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding or following embodiments, the NEO-201 antibody may be conjugated to another moiety, such as another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

[0061] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding or following embodiments, the method may further comprise administering to the patient an effective amount of a cytokine agonist to enhance or stimulate carcinoma cell death. The cytokine agonist may be interleukin-2 (IL-2), interleukin-21 (IL-21), ALT-803, an IL-15 inhibitor, a checkpoint inhibitor, anti-PD1, anti-PDL1, anti-CTLA-4, anti-41BB, anti-OX40, anti-Tim-3, or a combination thereof.

[0062] In a preferred embodiment of the present invention, which may be used in conjunction with any of the preceding or following embodiments, the method may further comprise administering to the patient an effective amount of a complement regulatory protein (CRP) antagonist to enhance or stimulate carcinoma cell death. 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 IL-15 superagonist. The cytokine agonist may comprise a complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor α / IgG1 Fc fusion protein. The cytokine agonist may comprise ALT-803.

[0063] In a preferred embodiment of the invention, which may be used in conjunction with any of the preceding or following embodiments, the effective dosage of NEO-201 antibody is reduced compared to treatment with NEO-201 antibody alone without a cytokine agonist.

[0064] In a preferred embodiment of the present invention, which may be used in conjunction with any of the preceding or following embodiments, the cancer may express the NEO-201 antigen. Expression of the NEO-201 antigen may be determined by detecting the NEO-201 antigen in a cancer sample. Detection may be performed by techniques including histological staining, flow cytometry, RT-PCR, dot blot, Western blot, Northern blot, and other well-known techniques. In the case of recurrent or metastatic cancer, expression of the NEO-201 antigen may be predicted from the expression of NEO-201 in the primary cancer or the response of the primary cancer to NEO-201 antibody therapy.

[0065] In a preferred embodiment of the present invention, which may be used in conjunction with any of the preceding or following embodiments, the cancer may be colon cancer.

[0066] In a preferred embodiment of the present invention, which may be used in conjunction with any of the preceding or following embodiments, the cancer may be pancreatic cancer.

[0067] In a preferred embodiment of the present invention, which may be used in conjunction with any of the preceding or following embodiments, the cancer may be ovarian cancer.

[0068] In a preferred embodiment of the present invention, which may be used in conjunction with any of the preceding or following embodiments, the cancer may be gastric cancer.

[0069] In a preferred embodiment of the present invention, which may be used in conjunction with any of the preceding or following embodiments, the cancer may be lung cancer.

[0070] In a preferred embodiment of the present invention, which may be used in conjunction with any of the preceding or following embodiments, the cancer may be breast cancer.

[0071] In a preferred embodiment of the present invention, which may be used in conjunction with any of the preceding or following embodiments, the cancer may be uterine cancer.

[0072] definition

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the present invention or testing of the present invention, suitable methods and materials are described herein. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0074] As used in this description and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural referents unless the context clearly dictates otherwise.

[0075] As used herein, "amino acid" broadly refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons attached to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid but function in a manner similar to a naturally occurring amino acid.

[0076] As used herein, the term "NK depletion" or "natural killer depletion" refers to patients with low natural killer (NK) cell levels compared to the normal range. NK cells are cytotoxic innate immune lymphocytes. Typically, NK cells comprise 5-20% of peripheral blood mononuclear cells (PBMCs) in healthy individuals. Patients with NK cells comprising less than 5% of PMBCs are referred to as NK depleted. Furthermore, if NK cells comprise less than 3% of PMBCs, patients are referred to as severely NK cell depleted. Furthermore, in normal individuals, up to 90% of PBMC NK cells express CD56 dim CD16 + NK cells, which are considered the most cytotoxic subset. Less than 70% of PBMC NK cells express CD56 dim CD16 + If the NK cells are CD56+, the patient is said to be NK-depleted. dim CD16 + If it is NK cells, the patient is referred to as severely NK depleted. A given patient may be referred to as NK depleted or severely NK depleted based on meeting one or both of these individual criteria. Generally speaking, a patient's status as NK depleted or severely NK depleted is determined by examining a sample taken from the patient, for example, a blood sample, for example, a sample obtained and examined within one or two weeks. A patient's status as NK depleted or severely NK depleted can also be inferred from the diagnosis and / or course of treatment of a disease associated with such depletion of NK cells.

[0077] NK 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 among peripheral blood lymphocytes, and functional NKD (FNKD), characterized by the presence of NK cells within peripheral blood lymphocytes but defective NK cell activity. In both CNKD and FNKD, NK cell abnormalities are the primary immunological defect, resulting in an inadequate ADCC response. CNKD and FNKD can be further classified based on patient characteristics, such as the identity of the causative gene(s) or 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.

[0078] "Antibody," as used herein, broadly refers to any polypeptide chain-containing molecular structure with a specific shape that fits and recognizes an epitope, with one or more noncovalent interactions stabilizing the complex between the molecular structure and the epitope. The prototypical antibody molecule is an immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, and IgD, from all sources, e.g., human, rodent, rabbit, bovine, ovine, porcine, canine, and chicken, are considered "antibodies." Antibodies include, but are not limited to, chimeric antibodies, human antibodies and other nonhuman mammalian antibodies, humanized antibodies, single-chain antibodies (scFv), camelidbodies, nanobodies, IgNARs (single-chain antibodies derived from sharks), small modular immunopharmaceuticals (SMIPs), and antibody fragments (e.g., Fab, Fab', F(ab')2). Numerous antibody coding sequences have been described; others may be generated by methods well known in the art. See Streltsov, et al. (2005) Protein Sci. 14(11):2901-9; Greenberg, et al. (1995) Nature 374(6518):168-173; Nuttall, et al. (2001) Mol Immunol. 38(4):313-26; Hamers-Casterman, et al. (1993) Nature 363(6428):446-8; Gill, et al. (2006) Curr Opin Biotechnol. 17(6):653-8.

[0079] "NEO-201 antibody" refers to an antibody comprising the heavy and light chains of SEQ ID NOs: 28 and 29, or the variable regions, optionally together with the constant regions contained therein, and fragments and variants thereof. Such variants include sequences comprising one, two, three, four, five, or preferably all six of the CDR sequences contained in SEQ ID NOs: 28 and 29, i.e., heavy chain CDR1 of SEQ ID NO: 32, heavy chain CDR2 of SEQ ID NO: 33, heavy chain CDR3 of SEQ ID NO: 34, light chain CDR1 of SEQ ID NO: 35, light chain CDR2 of SEQ ID NO: 36, and light chain CDR3 of SEQ ID NO: 37. This antibody may be humanized. This antibody may be expressed containing one or more leader sequences that can be removed during expression and / or processing and secretion of the antibody. This antibody may be presented in monovalent, bivalent, or higher polyvalent forms, including, but not limited to, bispecific or multispecific antibodies comprising NEO-201 antibody sequences and binding fragments of different antibodies. Typically, this antibody specifically binds to carcinoma cells and competes for binding to carcinoma cells with an antibody comprising the variable heavy chain of SEQ ID NO: 38 and the variable light chain of SEQ ID NO: 39, or the heavy chain of SEQ ID NO: 28 and the light chain of SEQ ID NO: 29. One or more of the CDR sequences contained in SEQ ID NO: 28 and / or SEQ ID NO: 29 may be replaced with a variant sequence, such as a light chain CDR1 of SEQ ID NO: 1 or 4; a light chain CDR2 of SEQ ID NO: 2 or 5; a light chain CDR3 of SEQ ID NO: 3 or 6; a heavy chain CDR1 of SEQ ID NO: 7; a heavy chain CDR2 of SEQ ID NO: 8, 10, 30, or 31; or a heavy chain CDR3 of SEQ ID NO: 9 or 11 or SEQ ID NO: 30-31. The light chain may comprise a CDR contained in the light chain sequence of SEQ ID NO: 14, 16, 17, 18, 19, 20, 21, or 29. The heavy chain can comprise the CDRs contained in the heavy chain sequence of SEQ ID NO: 15, 22, 23, 24, 25, 26, 27, or 29.The antibody may comprise a variable heavy chain sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 38 and / or a variable light chain sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 39, and optionally the heavy and / or light chain sequences comprise one, two, three, four, five, or preferably all six of the CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO: 29, i.e., heavy chain CDR1 of SEQ ID NO: 32, heavy chain CDR2 of SEQ ID NO: 33, heavy chain CDR3 of SEQ ID NO: 34, light chain CDR1 of SEQ ID NO: 35, light chain CDR2 of SEQ ID NO: 36, and light chain CDR3 of SEQ ID NO: 37. The antibody may be conjugated to another moiety, such as a cytotoxic moiety, a radioactive moiety, a label, or a purification tag.

[0080] As used herein, "antigen" broadly refers to a molecule or a portion of a molecule that can be bound by an antibody that can further induce an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen may have one epitope or more than one epitope. The specific reaction referred to herein indicates that the antigen reacts with the corresponding antibody in a highly selective manner and does not react with the numerous other antibodies that may be elicited by other antigens. The antigen may be tumor-specific (e.g., expressed by neoplastic cells of pancreatic and colon cancers).

[0081] As used herein, "cancer" refers broadly to any neoplastic disease (invasive or metastatic) characterized by uncontrolled, abnormal cell division resulting in malignant growth or tumor.

[0082] As used herein, "chimeric antibody" refers broadly to an antibody molecule in which the antigen-binding site (variable region) has been modified, substituted, or exchanged with a constant region of a different or altered class, effector function, and / or species, or with an entirely different molecule, e.g., an enzyme, toxin, hormone, growth factor, drug, that confers new properties to the chimeric antibody; or the variable region or a portion thereof has been modified, substituted, or exchanged with a variable region having a different or altered antigen specificity.

[0083] As used herein, the term "conservatively modified variants" applies to both amino acid and nucleic acid sequences, and with respect to a particular nucleic acid sequence, refers broadly to conservatively modified variants, referring to those nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. Such nucleic acid variations are "silent variations," a type of conservatively modified variation. Any nucleic acid sequence herein that encodes a polypeptide describes all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be altered to produce a functionally identical molecule.

[0084] As used herein, "complementarity-determining region," "hypervariable region," or "CDR" broadly refers to one or more hypervariable or complementarity-determining regions (CDRs) found in the variable region of an antibody's light or heavy chain. See Kabat, et al. (1987) "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, MD. These expressions include the hypervariable regions defined by Kabat et al. ((1983) "Sequences of Proteins of Immunological Interest," US Department of Health and Human Services) or the hypervariable loops in the three-dimensional structure of an antibody. Chothia and Lesk (1987) J Mol. Biol. 196:901-917. The CDRs within each chain are held in close proximity by framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site. Within the CDRs are selected amino acids that have been described as selectivity determining regions (SDRs), which represent important contact residues used by the CDRs in the interaction of the antibody with the antigen. Kashmiri (2005) Methods 36:25-34.

[0085] " Control amount " as used herein refers broadly to marker, and can be any amount or amount range that is compared with the test amount of marker.For example, the control amount of marker can be the amount of marker in patients with specific disease or condition or in people who do not have such disease or condition.The control amount can be either absolute amount (for example, microgram / ml) or relative amount (for example, relative intensity of signal).

[0086] " Differentially present " as used herein broadly refers to the difference in the quantity or quality of markers present in the sample collected from patients with disease or condition compared with the equivalent sample collected from patients without one of the diseases or conditions.For example, when measured by hybridization / or NAT-based assay, for example, if the amount of nucleic acid fragment in one sample is significantly different from the amount of nucleic acid fragment in another sample, nucleic acid fragments can optionally be present differently between two samples.If the amount of polypeptide in one sample is significantly different from the amount of polypeptide in another sample, polypeptides will be present differently between two samples.It should be noted that if a marker can be detected in one sample and not detected in another sample, such marker can be considered to be present differently.Optionally, a relatively small amount of upregulation can serve as a marker.

[0087] "Diagnostic," as used herein, broadly refers to identifying the presence or nature of a disease state. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and test negative in the assay are referred to as "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it is sufficient if the method provides a positive indication that aids in diagnosis.

[0088] "Diagnosing," as used herein, broadly refers to classifying a disease or condition, determining the severity of a disease, monitoring the progression of a disease, and predicting the outcome and / or prognosis for recovery from a disease. The term "detecting" may also optionally encompass any of the foregoing. Diagnosis of a disease according to the present invention may, in some embodiments, be affected by determining the level of a polynucleotide or polypeptide of the present invention in a biological sample obtained from a subject, the determined level being correlated with a predisposition to the disease, or the presence or absence of the disease. It should be noted that a "biological sample obtained from a subject" may optionally include a sample that has not been physically removed from the subject.

[0089] As used herein, "effective amount" broadly refers to the amount of a compound, antibody, antigen, or cell that, when administered to a patient to treat a disease, is sufficient to effect such treatment of the disease. An effective amount can be a prophylactically effective amount and / or a preventatively effective amount. An effective amount can be an amount effective to reduce the onset of signs / symptoms, an amount effective to prevent, reduce the severity of the onset of signs / symptoms, eliminate the onset of signs / symptoms, delay the development of the onset of signs / symptoms, prevent the development of the onset of signs / symptoms, and / or effect prevention of the onset of signs / symptoms. An "effective amount" can vary depending on the disease and its severity, as well as the age, weight, medical history, susceptibility, and pre-existing conditions of the patient being treated. The term "effective amount" is synonymous with "therapeutically effective amount" for purposes of the present invention.

[0090] As used herein, "expression vector" refers broadly to any recombinant expression system intended to express a nucleic acid sequence of the present invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect, or mammalian cells. The term includes linear or cyclic expression systems. The term includes expression systems that remain episomal or that integrate into the host cell genome. Expression systems can be autonomously replicating or non-autonomously replicating, i.e., capable of driving only transient expression in a cell. The term includes recombinant expression cassettes that contain only the minimal elements required for transcription of the recombinant nucleic acid.

[0091] As used herein, "framework region" or "FR" refers broadly to one or more of the framework regions within the light and heavy chain variable regions of an antibody. See Kabat, et al. (1987) "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, MD. These expressions include the amino acid sequence regions interposed between the CDRs within the light and heavy chain variable regions of an antibody.

[0092] As used herein, "heterologous," when referring broadly to a portion of a nucleic acid, indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, the nucleic acid is typically recombinantly produced, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0093] As used herein, "high affinity" refers to an affinity of at least 10 for a target antigen. -8 M, more preferably at least 10 -9 M, and even more preferably at least 10 -10The term "high affinity" broadly refers to an antibody having a KD of at least 10 M. However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is defined as binding of at least 10 M. -7 M, more preferably at least 10 -8 It refers to an antibody with a KD of M.

[0094] As used herein, "homology" broadly refers to the degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence, or between a polypeptide sequence and a reference polypeptide sequence. Homology can be partial or total. Complete homology indicates that the nucleic acid or amino acid sequences are identical. A partially homologous nucleic acid or amino acid sequence is one that is not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The term "sequence identity" can be used synonymously with "homology."

[0095] As used herein, "host cell" refers broadly to a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect (e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa, HEK-293, including cultured cells, explants, and in vivo cells.

[0096] As used herein, "hybridization" refers broadly to the physical interaction of complementary (including partially complementary) polynucleotide strands due to the formation of hydrogen bonds between complementary nucleotides when the strands are positioned antiparallel to one another.

[0097] As used herein, "K-assoc" or "Ka" broadly refers to the association rate of a particular antibody-antigen interaction, while the term "Kdiss" or "Kd" as used herein refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well established in the art.

[0098] "Immunoassay," as used herein, refers broadly to an assay that uses an antibody to specifically bind an antigen. Immunoassays can be characterized by using the specific binding properties of a particular antibody to isolate, target, and / or quantify the antigen.

[0099] "Isolated," as used herein, refers broadly to a material that has been removed from its original environment in which it naturally occurs and has therefore been modified by the hand of man from that natural environment. An isolated material can be, for example, an exogenous nucleic acid contained in a vector system, an exogenous nucleic acid contained within a host cell, or any material that has been removed from its original environment and has therefore been modified by the hand of man (e.g., an "isolated antibody").

[0100] A "label" or a "detectable moiety," as used herein, refers broadly to a composition detectable by microscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[0101] As used herein, "low stringency," "moderate stringency," "high stringency," or "very stringent conditions" refer broadly to nucleic acid hybridization and washing conditions. Guidance for performing hybridization reactions is provided in Ausubel, et al. (2002) Short Protocols in Molecular Biology (5th ed.), John Wiley & Sons, NY. Exemplary specific hybridization conditions include, but are not limited to, the following: (1) low stringency hybridization conditions at about 45°C in 6X sodium chloride / sodium citrate (SSC), followed by at least two washes at 50°C in 0.2X SSC, 0.1% SDS (for low stringency conditions, the wash temperature can be increased to 55°C); (2) medium stringency hybridization conditions at about 45°C in 6X SSC, followed by one or more washes at 60°C in 0.2X SSC, 0.1% SDS; (3) high stringency hybridization conditions at about 45°C in 6X SSC, followed by one or more washes at 65°C in 0.2X SSC, 0.1% SDS; and (4) very high stringency hybridization conditions at 65°C in 0.5M sodium phosphate, 7% SDS, followed by one or more washes at 65°C in 0.2X SSC, 1% SDS.

[0102] "Mammal," as used herein, broadly refers to any and all warm-blooded vertebrates of the mammalian class, including humans, characterized by a hairy skin and, in females, milk-producing mammary glands for rearing their young. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cows, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammals include, but are not limited to, bovines, canines, equines, felines, murines, ovines, porcines, primates, and rodents. Mammals also include any and all of the mammals of the world listed by the National Museum of Natural History, Smithsonian Institution (Washington, DC).

[0103] As used herein, "nucleic acid" or "nucleic acid sequence" broadly refers to deoxyribonucleotide or ribonucleotide oligonucleotides in either single- or double-stranded form. The term encompasses nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones. Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0104] As used herein, "operably linked" refers broadly to when two DNA segments are joined so that the amino acid sequences encoded by the two DNA segments remain in-frame.

[0105] As used herein, "paratope" broadly refers to the portion of an antibody that recognizes an antigen (e.g., the antigen-binding site of an antibody). The paratope is a small region (e.g., 15-22 amino acids) of the Fv region of an antibody and may include portions of the heavy and light chains of the antibody. See Goldsby, et al., Antigens (Chapter 3), Immunology (5th Ed.), New York: W.H. Freeman and Company, pp. 57-75.

[0106] "Patient," as used herein, broadly refers to any animal in need of treatment to either alleviate a disease state or prevent the occurrence or recurrence of a disease state. "Patient," as used herein, also broadly refers to any animal that has risk factors, a medical history, susceptibility, symptoms, or signs, has previously been diagnosed with a disease, is at risk for a disease, or is a member of a patient population for a disease. A patient can be a clinical patient, such as a human, or a companion animal, livestock animal, farm animal, exotic animal, or zoo animal. The term "subject" can be used interchangeably with the term "patient."

[0107] The terms "polypeptide," "peptide," and "protein" are used interchangeably and refer broadly to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. Polypeptides can be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide," and "protein" include glycoproteins as well as non-glycoproteins.

[0108] As used herein, "promoter" broadly refers to an array of nucleic acid sequences that directs transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, for example, a TATA element in the case of a polymerase II type promoter. A promoter also optionally includes distal enhancer or repressor elements, which may be located as far away as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.

[0109] As used herein, a "prophylactically effective amount" refers broadly to the amount of a compound that, when administered to a patient for the prevention of a disease or the prevention of recurrence of a disease, is sufficient to effect such prevention of the disease or recurrence. A prophylactically effective amount can be an amount effective to prevent the occurrence of signs and / or symptoms. A "prophylactically effective amount" can vary depending on the disease and its severity, as well as the age, weight, medical history, predisposition to the condition, and pre-existing conditions of the patient being treated.

[0110] As used herein, "prevention" broadly refers to a course of treatment in which signs and / or symptoms are absent, in remission, or previously present in a patient. Prevention includes preventing disease from occurring after a patient has been treated for the disease. Furthermore, prevention includes treating patients who may potentially develop the disease, particularly those who are susceptible to the disease (e.g., members of a patent population, patients with risk factors, or patients at risk of developing the disease).

[0111] As used herein, "recombinant" refers broadly to a product, e.g., a cell, or a nucleic acid, protein, or vector, and indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the modification of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell may express genes that are not found within the native (non-recombinant) form of the cell, or may express aberrantly expressed naturally occurring genes that would otherwise be expressed poorly or not at all.

[0112] As used herein, the terms "specifically (or selectively) bind" or "specifically (or selectively) immunoreact" or "specifically interact or bind" to an antibody refer broadly to a protein or peptide (or other epitope) and, in some embodiments, to a binding reaction that is determinative of the protein's presence in a heterogeneous population of proteins and other biologics. For example, under specified immunoassay conditions, a particular antibody binds to a particular protein with at least two-fold greater than background (nonspecific signal) and does not substantially bind in significant amounts to other proteins present in the sample. Typically, a specific or selective reaction is at least two-fold greater than background signal or noise, and more typically about 10-100-fold greater than background.

[0113] As used herein, "specifically hybridizable" and "complementary" broadly refer to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional type. The binding free energy of a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid, such as RNAi activity, to proceed. The determination of the binding free energy of a nucleic acid molecule is well known in the art. For example, see Turner, et al. (1987) CSH Symp. Quant. Biol. LII: 123-33; Frier, et al. (1986) PNAS 83: 9373-77; Turner, et al. (1987) J. Am. Chem. Soc. 109: 3783-85. Percent complementarity refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., at least about 5, 6, 7, 8, 9, 10 out of 10 are about 50%, 60%, 70%, 80%, 90%, and 100% complementary, inclusive). "Fully complementary" or 100% complementarity broadly refers to all of the contiguous residues of a nucleic acid sequence that hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" refers to polynucleotide strands that exhibit at least about 90% complementarity, excluding regions of the polynucleotide strand, such as overhangs, that are selected to be non-complementary. Specific binding requires a sufficient degree of complementarity to avoid nonspecific binding of the oligomeric compound to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions under which the assay is performed in the case of in vitro assays. The non-target sequences may typically differ by at least five nucleotides.

[0114] As used herein, a "sign" of disease refers broadly to any abnormality detectable on examination of a patient that indicates an objective sign of disease, as opposed to a symptom, which is a subjective sign of disease.

[0115] As used herein, "solid support," "support," and "substrate" refer broadly to any material that provides a solid or semi-solid structure to which another material can be attached, including, but not limited to, smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces), as well as textured and porous materials.

[0116] As used herein, "subject" refers broadly to any human suitable for treatment according to the present invention, including, but not limited to, avian and mammalian subjects, preferably mammals. Mammals of the present invention include, but are not limited to, dogs, cats, cows, goats, horses, sheep, pigs, rodents (e.g., rats and mice), rabbits, primates, and humans. Any mammalian subject in need of treatment according to the present invention is suitable. Human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult) can be treated according to the present invention. The present invention can also be practiced on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, cows, goats, sheep, and horses, for veterinary purposes and for drug screening and drug development purposes. "Subject" is used synonymously with "patient."

[0117] As used herein, a "symptom" of a disease refers broadly to any pathological phenomenon or deviation from normal structure, function, or sensation experienced by a patient that is indicative of the disease.

[0118] "Therapy," "therapeutic," "treating," or "treatment," as used herein, broadly refer to treating a disease, halting or reducing the development of a disease or its clinical symptoms, and / or alleviating a disease or causing regression of a disease or its clinical symptoms. Therapy includes preventing, treating, repairing, reducing, alleviating, and / or causing relief of a disease, disease signs, and / or symptoms. Therapy includes alleviating signs and / or symptoms in patients with ongoing disease signs and / or symptoms (e.g., tumor growth, metastasis). Therapy also encompasses "prevention." The term "reduced," for purposes of therapy, broadly refers to a clinically significant reduction in signs and / or symptoms. Therapy includes treating relapsing or recurrent signs and / or symptoms (e.g., tumor growth, metastasis). Therapy includes, but is not limited to, eliminating the appearance of signs and / or symptoms at any time, reducing existing signs and / or symptoms, and eliminating existing signs and / or symptoms. Therapy includes treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing the recurrence or recurrence of signs and / or symptoms (eg, tumor growth, metastasis).

[0119] As used herein, "variable region" or "VR" refers broadly to the domains within each pair of light and heavy chains of an antibody that are directly involved in binding the antibody to an antigen. Each heavy chain contains a variable domain (V H ), followed by several constant domains. Each light chain has at one end a variable domain (V L ) and at its other end a constant domain, the constant domain of the light chain aligned with the first constant domain of the heavy chain, and the variable domain of the light chain aligned with the variable domain of the heavy chain.

[0120] As used herein, "vector" refers broadly to a plasmid, cosmid, phagemid, phage DNA, or other DNA molecule capable of autonomous replication in a host cell and characterized by one or a few restriction endonuclease recognition sites, at which such DNA sequences can be cleaved in a determinable manner without abolishing the essential biological function of the vector, and into which DNA can be inserted to effect its replication and cloning. Vectors may further contain markers suitable for use in identifying cells transformed with the vector.

[0121] Techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook, et al. (2001) Molec. Cloning: Lab. Manual [3rd Ed] Cold Spring Harbor Laboratory Press. Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The nomenclatures utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as these laboratory procedures and techniques, are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0122] Example

[0123] The invention generally described herein will be more readily understood by reference to the following examples, which are included merely to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0124] Example 1

[0125] NEO-201 binds to a variety of human carcinoma cell lines.

[0126] Using flow cytometry analysis, we profiled a panel of human carcinoma cell lines for NEO-201 binding. The staining profiles are summarized in Table 1, and representative histograms from cell lines with high, moderate, low, and negative staining are shown in Figure 1A–C. Evaluation of NEO-201 binding activity revealed high positivity in 3 / 6 (50%) colon cancer cell lines and 4 / 5 (80%) pancreatic cancer cell lines. When non-small cell lung cancer (NSCLC) cell lines of various histological subtypes were profiled, we found that 3 / 5 (60%) adenocarcinoma cell lines reacted with NEO-201, whereas only 1 / 4 (25%) squamous cell carcinoma cell lines were positive. We also screened breast cancer cell lines. Among cell lines that expressed either the estrogen receptor (ER) or progesterone receptor (PR), either alone or in combination with HER2, 2 / 4 (50%) stained positive for NEO-201. Among HER2+ cell lines, three-quarters (75%) were recognized by NEO-201, either alone or in combination with ER or PR. However, NEO-201 staining was found at low levels in only one-quarter (25%) of triple-negative breast cancer cell lines. Overall, 15 / 30 (50%) of tumor cell lines tested were recognized by NEO-201. These data demonstrate that NEO-201 is reactive against a wide range of in vitro cultured tumor cell lines and indicate that distinct differences in antibody reactivity can occur based on tumor subtype.

[0127] Example 2

[0128] NEO-201 tissue staining is highly tumor specific

[0129] We investigated NEO-201 reactivity in human tumor samples using immunohistochemistry and tissue microarrays representing several dozen samples for each cancer type. As shown in Figure 2A, NEO-201 immunoreactivity (7,829,678) was completely absent in normal colon, pancreas, and lung tissue, but was highly positive in tumor tissues from these organs. Surprisingly, staining was only observed on tumor cells, as surrounding stromal cells were not stained (Figure 2A). In IHC staining and microarray samples, NEO-201 was determined to be highly reactive in colon cancer (72%), pancreatic cancer (80%), gastric cancer (71%), lung cancer (61%), breast cancer (55%), and uterine cancer (54%). Furthermore, a significant minority of ovarian cancer (26%) samples also exhibited positive staining, while no staining was observed in prostate cancer tissue (Figure 2B). Overall, 258 / 345 (74.7%) of the tumor tissues sampled stained positive for NEO-201. Importantly, NEO-201 reactivity was almost completely absent in normal healthy tissues (Table 2) and normal tumor-adjacent tissues, except for some uterine and ovarian samples (Figure 2C). However, the number of tissues in this series was limited (5 and 9 samples, respectively). Collectively, these data demonstrate that NEO-201 recognizes tumor tissues from a variety of carcinomas and is highly tumor-specific.

[0130] Experimental Example 3

[0131] NEO-201 mediates ADCC and CDC to kill tumor cells

[0132] As a humanized IgG1 antibody, NEO-201 is theorized to be capable of mediating ADCC to kill tumor cells expressing the NEO-201 antigen. To investigate this potential mechanism of action, ADCC assays using human natural killer (NK) cells isolated from PBMCs from two different healthy donors were performed on cell lines (CFPAC-1 and ASPC-1) highly positive for NEO-201 staining. Treatment with NEO-201 was observed to enhance cell killing of both CFPAC-1 and ASPC-1 cells to levels 2-6-fold higher than that of control IgG1-treated tumor cells (Figure 3A). Titration assays were also performed, revealing that NEO-201 retains the ability to significantly induce ADCC at doses as low as 0.1 μg / mL (Figure 3B).

[0133] CDC is a complex cascade of proteolytic cleavages that leads to the activation of membrane attack complexes, which lyse antibody-bound target cells. Certain human IgG1 antibodies can mediate CDC, but CDC depends on the antigen specificity of the antibody. CDC assays revealed that NEO-201 induced complement-mediated lysis of ASPC-1 cells in a manner that depended on both the mAb dose and incubation time (Figure 3C). Collectively, these data demonstrate that NEO-201 effectively engages innate immune effector mechanisms to specifically lyse antibody-bound tumor cells in vitro.

[0134] Example 4

[0135] NEO-201 alone and in combination with human PBMC effector cells suppresses the growth of tumor xenografts.

[0136] To determine the potential antitumor effects of NEO-201, CFPAC-1 cells were grown as tumor xenografts in immunodeficient NU / NU nude mice. These cells were selected based on their high expression levels of the NEO-201 antigen and their susceptibility to NEO-201-mediated ADCC. CFPAC-1 tumors were approximately 100 mm in size. 3Once tumor-bearing mice had grown to 100 μg, they were injected three times with saline, 250 μg human IgG1, 100 μg NEO-201, or 250 μg NEO-201, followed by 1.0 × 107 7 IL-2-activated (200 U / mL) human PBMCs were injected three times to serve as ADCC-mediating effector cells. As shown in Figure 4A, NEO-201 + PBMCs induced a substantial reduction in tumor growth at both dose levels compared to either the saline + PBMC or human IgG + PBMC control groups. While none of the mice in the control group were tumor-free by day 36, 1 out of 10 (10%) and 4 out of 10 (40%) mice in the NEO-201 100 μg + PBMC and NEO-201 + 250 μg PBMC groups, respectively, had no palpable tumors remaining (Figure 4B). Furthermore, when a separate group of mice was administered NEO-201 without the addition of human PBMCs, a significant reduction in tumor growth was observed compared to the control group (Figure 4A, C). Importantly, monitoring the body weight of tumor-bearing mice revealed no weight loss in either treatment group (Figure 4D). Taken together, these results demonstrate that NEO-201 can significantly reduce tumor growth in mice through both ADCC and non-ADCC mechanisms (such as CDC) without inducing significant toxicity.

[0137] Example 5

[0138] NEO-201 localizes to xenograft tumor sites

[0139] Biodistribution studies were performed using radiolabeled NEO-201 in female and male NU / NU nude mice bearing established CFPAC-1 xenograft tumors. These mice were intravenously injected with the radiolabeled antibody, and blood, organs, and tumors were collected for analysis at various time points post-injection. 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, B). However, normalized radioactivity uptake was significantly higher in tumors compared to all other tissues at all time points, with tumor radioactivity gradually increasing to levels 20–30 times higher than blood radioactivity by day 7 (Figure 5A, B). Quantitatively similar results were obtained in 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.

[0140] Example 6

[0141] Pharmacokinetic and Toxicity Evaluation of NEO-201 in Non-Human Primates

[0142] To determine the pharmacokinetics and associated toxicity of NEO-201, a single-dose study was conducted in captive cynomolgus monkeys. Cynomolgus monkeys were chosen because they are closely related to humans both phylogenetically and physiologically and are a commonly used species for nonclinical toxicity evaluation. Male and female animals received a single intravenous infusion of NEO-201 diluted in saline at doses of 5 mg / kg, 20 mg / kg, and 49 mg / kg, which was the maximum achievable dose per injection. Blood samples were collected from all animals before and at various time points up to 14 days after injection, and serum preparations were evaluated for NEO-201 levels by ELISA. As shown in Table 3, quantifiable, dose-dependent serum concentrations of NEO-201 were observed at the last collection time point (14 days post-dose). As expected for intravenous administration, Tmax values ​​peaked at 10 minutes for most animals (10 / 12, 83%) in all groups, except for one male and one female in the 5 mg / kg group. Peak (Cmax) exposure was dose-proportional across the dose range evaluated. Total (AUC) exposure was greater than dose-proportional at the lowest dose and nearly proportional from approximately 20 mg / kg to 49 mg / kg. The difference in exposure at the lowest dose was attributable to a roughly two-fold greater mean clearance (CL) and smaller volume of distribution (Vz). The mean half-life (HL) was 167 (20 mg / kg) or 170 (49 mg / kg) hours at the higher doses, approximately 3.7-fold longer than the 5 mg / kg dose (46.2 hours). No gender differences were observed.

[0143] Toxicity was determined over the course of the 14-day study by the following observations and tests: 1) periodic clinical evaluations; 2) measurements of food intake and body weight; and 3) urinalysis and blood tests (e.g., urinalysis, hematology, coagulation, serum chemistry, and toxicokinetics). As shown in Figure 6A, none of the dose groups experienced a change in body weight greater than 3% from pre-injection, and no individual monkey experienced a change greater than 7%. Food intake remained unchanged for all animals except for two in the 5 mg / kg dose group, which consumed less food only on Day 11. There were no significant changes from baseline (before NEO-201 injection) to Day 15 in any of the serum chemistry, urinalysis, or coagulation tests (see Materials and Methods for details). The primary change in laboratory blood counts was a decrease in neutrophil counts compared to baseline (Figure 6B). The decrease varied in magnitude from mild to significant, and no clear dose-response pattern was evident. For the majority of animals, this was a transient finding, as improvement was usually observed by Day 8 (Figure 6B). By day 15, neutrophil counts were observed to recover almost completely or partially in the 5 mg / kg or 20 mg / kg and 49 mg / kg groups, respectively (Figure 6B). The recovery of neutrophil counts by day 15 is reflected in statistical comparisons with 0 mg / kg animals, which were significantly different at day 2 (p<0.05) at all three dose levels, but not significantly different at days 8 and 15 (p>0.05) for two of the three treatment groups (Figure 6C).

[0144] Example 7

[0145] Materials and Methods

[0146] Cell lines and culture

[0147] The following human carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA): colon (COLO 205, HT-29, LS174T, SW1116, SW1463, SW480, SW620), pancreatic (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 maintained in RPMI 1640, DMEM, or IMDM culture medium (Corning, Corning, NY) as specified by the supplier for propagation and maintenance. Culture medium was supplemented with 10% heat-inactivated HyClone fetal bovine serum (GE Healthcare Life Sciences, Issaquah, WA, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin (Corning Life Sciences, Manassas, VA, USA). PBMCs from healthy volunteer donors were obtained from the National Institutes of Health Clinical Center Blood Bank (NCT00001846) under appropriate institutional review board approval and informed consent.

[0148] Generation of humanized NEO-201 monoclonal antibody

[0149] The Hollinshead colon cancer-specific vaccine was used as an immunogen to generate monoclonal antibodies in mice. The method for preparing tumor-associated proteins and peptides has been previously described (Hollinshead, US 4,810,781, 1989). Briefly, cancer tissue was minced and used to generate a single-cell suspension, followed by hypotonic saline membrane extraction, a series of centrifugation steps, and low-frequency sonication. The resulting membrane-extracted proteins were fractionated on Sephadex G-200 resin or electrophoretically, then concentrated and quantified (Hollinshead et al., 1970; Hollinshead et al., 1972; Hollinshead et al., 1985). The TAA preparation was mixed with complete Freund's adjuvant and injected subcutaneously into BALB / c mice. This was followed by three booster injections with incomplete Freund's adjuvant at 2-3 week intervals. Mouse sera were tested for antibody responses to the immunizing antigen by ELISA. Mice with strong responses were used to generate immortalized hybridoma cells by fusing mouse B cells from the spleen with the SP2 / 0-Ag14 myeloma cell line, growing them, and selecting for cells that produced mouse immunoglobulin (IgG). From these mouse IgGs, the mouse 16C3 clone (m16C3) was selected based on its reactivity with colon tumor cell membrane extracts derived from LS174T or HT-29 cells as determined by ELISA. The cDNA encoding the heavy and light IgG1 chains was determined from RNA isolated from the hybridoma clone 16C3 E12 and shown to be unique (Bristol & Kantor, US7829678, 2010). The m16C3 protein sequence was humanized as h16C3 and designated NEO-201. Humanization was performed in silico by replacing murine sequences outside the complementarity-determining regions (CDRs) of the Fab regions of both the heavy and light chain proteins with human Fab sequences, while retaining the three murine CDR sequences from each chain. The Fc regions of the heavy and light chains were selected from the human IgG1 isotype, which has been used in other humanized, approved mAb products. The amino acid sequences were reverse-translated into DNA optimized for protein expression in CHO cells.Next, the heavy and light chain h16C3 DNA was chemically synthesized, cloned into mammalian expression plasmids, and transfected into mammalian cell lines (HEK293T and CHO). Several stable CHO cell lines expressing recombinant h16C3 were derived and maintained. Purified recombinant h16C3 was retested in a study to confirm that the humanized 16C3 antibody had properties similar to those of the original m16C3 antibody (Bristol & Kantor, US7829678, 2010).

[0150] The NEO-201 antibody sequence used in these examples is included in the following figure.

number

[0151] The boundaries between the expression leader sequence, variable region, and constant region are separated by a slash (" / ") in each sequence, and the CDR sequences are indicated by bold, underlined text. The antibody sequences used contained the variable and constant regions shown. These regions 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.

[0152] Flow cytometry

[0153] Binding of NEO-201 to human carcinoma cell lines was analyzed by flow cytometry. Cells (1.0x10 6) were incubated in 1X phosphate-buffered saline (PBS) containing 1 μL LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) per test for 30 minutes at 4°C to differentiate live versus dead cells. Cells were then centrifuged, washed twice with cold PBS, and then stained with Pacific Blue-conjugated NEO-201 antibody (BioLegend, San Diego, CA) in 1X PBS + 1% BSA (Teknova, Hollister, CA, USA) for 30 minutes at 4°C. After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis of cell fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). A staining value of >10% positive was considered positive for NEO-201 expression. Positive cell lines were ranked according to quantified expression levels (% positive x MFI) and divided into low (<200), medium (200-1000), and high (<1000) expression groups.

[0154] Immunohistochemistry (IHC)

[0155] Tissue microarrays of colon samples (CO808, CO951) were obtained from US Biomax (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 biotin protein labeling kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Slides were baked at 60°C for 20 minutes, deparaffinized in xylene, and rehydrated in a graded ethanol series. Slides were subjected to peroxide blocking using peroxidase I solution (Biocare Medical, Concord, CA) for 2 minutes, avidin blocking using avidin solution (Biocare Medical, Concord, CA) for 10 minutes, biotin blocking using biotin solution (Biocare Medical, Concord, CA) for 10 minutes, and protein blocking using CAS-Block histochemistry reagent (Thermo Fisher Scientific, Waltham, MA) for 10 minutes. Slides were then incubated for 2 hours at room temperature with either negative control biotinylated human IgG1 kappa (Ancell, Bayport, MN) or biotinylated NEO-201 at 10 μg / mL diluted in 1X PBS. Detection was enabled by incubation with Dako streptavidin-HRP conjugate (Agilent Technologies, Santa Clara, CA) at 1:300 for 30 minutes, DAB peroxidase substrate (Thermo Fisher Scientific, Waltham, MA) for 1–3 minutes, and counterstaining with hematoxylin. Each microarray tissue spot was assessed by light microscopy for cell staining intensity using the following scale: 0 (negative), ± (borderline), 1+ (weak), 2+ (moderate), 3+ (strong).Tissue spots containing cells stained with an intensity of +1 or greater were scored as positive.

[0156] Antibody-dependent cellular cytotoxicity (ADCC) assay

[0157] ADCC assays were performed using a modification of a previously described procedure (Boyerinas et al., 2015). Negative selection of NK cells from normal human donor PBMCs was performed using the EasySep Human NK Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's protocol. Purified NK cells were incubated overnight in RPMI-1640 medium supplemented with L-glutamine, 10% FBS, and antibiotics. On the day of the assay, target cells (CFPAC-1, ASPC-1) were labeled with 10 μM calcein AM cell-permeant dye (Termo Fisher Scientific, Waltham, MA, USA) for 30 minutes and then incubated at 3.0 × 10 3 Cells were seeded in triplicate at 1000 cells / well into black-walled, flat-bottom 96-well culture plates (#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, and NK cells were added at effector-to-target (E:T) ratios of 12.5:1 and 25:1, respectively, unless otherwise specified. After 4 hours of incubation at 37°C, 10 μg / mL of propidium iodide (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well, and the plates were imaged and analyzed using a Celigo Imaging Cytometer (Nexcelom Bioscience LLC, Lawrence, MA, USA). Viable target cells (calcein AM / PI-) were counted in each well, and specific ADCC lysis was calculated as follows: % specific lysis = 100 - [(mean viable target counts experimental / Average number of live targets control )x100].

[0158] Complement-dependent cytotoxicity (CDC) assay

[0159] CDC assays were performed using a modification of a previously described procedure (Konishi et al., 2008). ASPC-1 target cells were labeled with Calcein AM as described above and 5.0 × 10 3 Cells were seeded at 1000 cells / well into black-walled 96-well plates. Cells were then opsonized by treatment with 0.5 or 5.0 μg / mL NEO-201 for 15 minutes at 37°C, and purified rabbit complement (MP Biomedicals, Santa Ana, CA) was added to each well at a 1:8 dilution. After incubation for 30, 60, or 120 minutes at 37°C, propidium iodide was added, and plates were imaged and analyzed using a Celigo Imaging Cytometer. Specific lysis was calculated for ADCC activity as described above.

[0160] Xenograft antitumor assay

[0161] Tumors were established in 6-week-old female athymic NU / NU nude mice (Charles River Laboratories International, Wilmington, MA) by subcutaneously implanting a suspension of cultured tumor cells in 1X PBS into the right flank of the mice. Tumors were approximately 100 mm in size. 3 Upon reaching tumor volume, mice were sorted by tumor volume and randomly divided into five groups (n=10 animals). Mice were then intraperitoneally injected with vehicle alone (saline), human IgG1 (250 μg), or NEO-201 (100 μg and 250 μg) on ​​days 13, 17, and 20 post-implantation. Mice were also injected with approximately 1.0x10 IL-2-activated human IgG1 (IgG1) on days 14, 18, and 21 as a source of immune effector cells. 7 One group of mice was treated similarly with NEO-201 but did not receive human PBMCs. Tumors were measured every 2–3 days with digital calipers, and tumor volume was calculated using the formula (width x width). 2x length) / 2 = mm 3 The width was calculated as follows: where the width was the shorter of the two measurements. Mice were also weighed weekly as a measure of general health. Tumor volumes of 2000 mm were excluded according to IACUC guidelines. 3 Mice exceeding this number were sacrificed.

[0162] Biodistribution analysis

[0163] Biodistribution studies were evaluated in tumor-bearing mice using radiolabeled NEO-201 (Comparative Biosciences, Sunnyvale, CA) using a previously described procedure (Patel et al., 2013). Briefly, male and female athymic NU / NU nude mice (Charles River Laboratories International, Wilmington, MA) were injected with 4.0x10 6 Mice were subcutaneously injected with 200 μL of a 1X PBS suspension containing CFPAC-1 cells. On day 14 after engraftment, 20 μCi of CFPAC-1 cells were administered. 125 I-labeled NEO-201 was intravenously injected, and animals were autopsied 1, 2, 4, or 7 days later. Blood, tumor tissue, and internal organs (lungs, kidneys, liver, spleen, pancreas, intestines, and stomach) were collected at each time point (n = 4 animals). All tissues were weighed and radioactivity in the tissues was measured using a gamma counter. Data for each mouse were first calculated as cpm / mg tissue, and then tissue cpm values ​​were normalized to blood cpm values.

[0164] Single-dose toxicity study in cynomolgus monkeys

[0165] A single-dose toxicity study was conducted in captive cynomolgus monkeys to examine the pharmacokinetics and toxicity of NEO-201 after a single dose. The study period was 15 days from dose administration, with an additional 14 days of quarantine before dose administration to allow the monkeys to acclimate to the laboratory. Eight male and female animals (two per sex per group) were administered NEO-201 diluted in saline by slow intravenous infusion (approximately 30 minutes ± 5 minutes infusion) at dose levels of 0 mg / kg, 5 mg / kg, 20 mg / kg, and 49 mg / kg (which was the highest achievable concentration of antibody) using an infusion pump and a plastic disposable syringe with catheter extension tubing. Blood samples were collected from all animals receiving NEO-201 at the following time points: pre-dose, 10 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 168 hours, and 336 hours. Serum was prepared from blood samples for pharmacokinetic and toxicological analyses. Whole blood was used for cellular analysis. Serum NEO-201 levels were measured by ELISA using a Human Therapeutic IgG1 ELISA kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions.

[0166] Laboratory tests included hematology and coagulation (baseline (BL), days 2, 8, and 15); CBC and differential, activated partial thromboplastin time, fibrinogen, and prothrombin time; serum chemistry (BL, days 2, 8, and 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, and BUN; urinalysis (BL, day 15): color, clarity, glucose, ketones, occult blood, protein, bilirubin, nitrite, pH, urobilinogen, white blood cells, volume, and specific gravity; bioanalysis (using ELISA)—(BL, 10 min, 1 h, 2 h, 4 h, 6 h, 24 h, 48 h, 72 h, 96 h, 168 h, and 336 h), Phoenix Using WinNonlin version 6.1 software (Certara USA, Princeton, NJ), groups 2 to 4 were included. Animal body weight measurements were recorded (BL, days 7 and 14), and neutrophil counts were assessed (BL, days 2, 8, and 15).

[0167] statistical analysis

[0168] Data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA). Comparisons between two groups were performed by T-test, and p<0.05 was considered statistically significant. Graphs show the mean ± SD from one representative experiment performed in triplicate.

[0169] Example 8

[0170] ALT-803 enhances NEO-201-mediated ADCC

[0171] ALT-803 is a novel IL-15 superagonist complex consisting of an IL-15 mutant (IL-15N72D) linked to an IL-15 receptor α / IgG1 Fc fusion protein. In this example, the ability of ALT-803 to modulate ADCC by NEO-201 is tested.

[0172] method

[0173] NK cells were isolated from normal donors and treated with various concentrations of ALT-803 for 48 hours before being used as effector cells. Human carcinoma 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 NK cell phenotype and modulate NK cell gene expression was assessed using flow cytometry and Nanostring analysis, respectively.

[0174] result

[0175] Treatment with ALT-803 significantly enhanced NEO-201-mediated ADCC activity against NEO-201-positive carcinoma cells (Figures 8 and 11). The effect of ALT-803 was dose-dependent, achieving statistical significance at all doses tested compared with vehicle control treatment. Treatment of NK cells with ALT-803 also enhanced ADCC activity from donors with minimal ADCC activity and reduced the effective dose of NEO-201 required to initiate an ADCC response compared with untreated NK cells (Figure 12). Furthermore, ADCC activity can be blocked using anti-CD16 and anti-TIM3 blocking antibodies (Figure 12).

[0176] Phenotypic analysis of NK cells treated with 25 ng / ml ALT-803 for 48 h showed that ALT-803 enhanced the expression of TIM3 and NKG2D, as well as the mean fluorescence intensity (MFI) of granzyme B and CD107a in CD16 / CD56-positive NK cells (Figure 9).

[0177] Nanostring analysis of human NK cells treated with ALT-803 at various concentrations for 48 hours showed that ALT-803 could regulate the mRNA expression of 62 genes (a fold change of 1.6 log2 compared to vehicle control was considered significant).

[0178] ALT-803 treatment upregulated the mRNA expression of 43 genes, including NK activating receptors, factors involved in NK cytotoxicity, cytokines and their receptors, and downregulated the mRNA expression of 19 genes, including NK inhibitory receptors and factors involved in the activation of apoptosis.

[0179] Thus, ALT-803 enhances NEO-201-mediated ADCC activity against human carcinoma cells, which may be due in part to increased expression of TIM3, NKG2D, granzyme B, and CD107a-positive NK cells, as well as modulation of transcripts involved in NK activation and cytotoxicity.

[0180] In summary, treatment of NK cells isolated from normal donors with ALT-803 can enhance NEO-201-mediated ADCC activity. Phenotypic analysis of ALT-803-treated NK cells isolated from normal donors demonstrated that ALT-803 can enhance 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 on CD16 / CD56-positive NK cells. Treatment of normal NK cells with ALT-803 also increased the MFI of CD107a on CD16 / CD56-positive NK cells in one of two donors tested. TIM-3 is an inducible human NK cell receptor that enhances interferon-gamma production and is also a maturation marker. The enhanced ADCC activity mediated by NEO-201 after treatment with ALT-803 may be due in part to 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 can enhance ADCC activity mediated by low concentrations of NEO-201. Low concentrations of MAbs can be used to mediate ADCC activity when NK cells are treated with ALT-803, and can result in comparable levels of cytotoxicity compared to NK cells treated with high concentrations of NEO-201 without ALT-803 treatment. These results suggest that lower doses of MAbs can be used in combination with ALT-803 in clinical trials for cancer treatment.

[0181] Example 9

[0182] NEO-201 enhances NK cell-dependent killing of tumor cells through blocking the inhibitory CEACAM5 / CEACAM1 immune checkpoint pathway.

[0183] Immunotherapy using checkpoint-blocking antibodies targeting effector cell inhibitory receptors such as PD-1 and CTLA-4 has elicited some dramatic and durable responses in several tumor types. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell surface protein expressed by immune cells and tumor cells that, like PD-1 and CTLA-4, can inhibit T cell function. CEACAM1 is also a potent inhibitor of natural killer (NK) cell function. Binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activating signaling by NKG2D, thereby preventing NK cell lysis and allowing tumor cells to evade NK killing.

[0184] NEO-201 binds to members of the CEACAM family and can activate innate immune mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), to kill tumor cells. This study was designed to determine whether NEO-201 could block the CEACAM1 inhibitory pathway and restore antitumor function to NK cells.

[0185] method

[0186] In vitro assays using human tumor cell lines were performed to identify the CEACAM family members bound by NEO-201. Functional assays were performed to evaluate the ability of NEO-201 to enhance in vitro killing of tumor cells by the NK-92 NK cell line, which expresses CEACAM1, lacks CD16, and is incapable of mediating ADCC.

[0187] Killing assays were performed using a modification of a previously described procedure (David et al., 2017). Briefly, target cells derived from pancreatic cancer (ASPC-1, BxPC-3, CFPAC-1) and colon cancer (LS174T) were labeled with 10 μM calcein AM cell-permeant dye (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min, then transfected with 3.0 × 10 cells in triplicate.3 Tumor cells were seeded at 1000 cells / well into black-walled, flat-bottom 96-well culture plates. Subsequently, tumor cells were treated with 10 μg / mL of a human IgG1 isotype control antibody (Thermo Fisher Scientific, Waltham, MA, USA) or NEO-201, and the natural killer (NK) cell line NK-92 was added 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, propidium iodide (PI; Thermo Fisher Scientific, Waltham, MA, USA) was added to each well at a final concentration of 1.67 μg / mL. The plates were centrifuged and imaged using a Celigo Imaging Cytometer (Nexcelom Bioscience LLC, Lawrence, MA, USA). Images were analyzed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). Live target cells (calcein AM / PI-) were counted in each well, and specific lysis was calculated as follows: % specific lysis = 100 - [(mean live target count experimental / Average number of live targets control )x100].

[0188] result

[0189] NEO-201 was found to react with different variants of CEACAM5 and CEACAM6, but not CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell lines express different levels of negative forms of CEACAM5 / 6 and NEO-201-responsive variant forms of these molecules. Functionally, NEO-201 treatment enhanced the cytolytic activity of NK-92 cells against NEO-201+ tumor cells that expressed CEACAM5, but not against NEO-201+ cells that expressed only CEACAM6 (Figure 13).

[0190] conclusion

[0191] NEO-201 reacts with tumor-associated variants of CEACAM5 / 6 and can block the interaction between tumor cell CEACAM5 and NK cell CEACAM1, reversing the CEACAM1-dependent inhibition of NK cytotoxicity.

[0192] Abbreviation Antibody-dependent cellular cytotoxicity (ADCC), area under the plasma concentration-time curve from time 0 to infinity (AUCinf), dose-normalized area under the plasma concentration-time curve from time 0 to infinity (AUCinf / D), baseline (BL), complement-dependent cytotoxicity (CDC), clearance (CL), maximum observed plasma concentration (Cmax), dose-normalized maximum observed plasma concentration (Cmax / D), estrogen receptor (ER), half-life (HL), immunohistochemistry (IHC), natural killer (NK), non-small cell lung cancer (NSCLC), peripheral blood mononuclear cells (PBMC), progesterone receptor (PR), tumor-associated antigen (TAA), time to maximum observed plasma concentration (Tmax), volume of distribution (Vz).

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Table 1

Table 2

Table 3

Claims

1. A method of killing carcinoma cells comprising administering to a patient in need thereof an effective amount of NEO-201 antibody.

2. A method of treating carcinoma comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody.

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

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

5. 10. The method of any one of the preceding claims, wherein the antibody mediates complement-mediated cytotoxicity (CDC), thereby killing carcinoma cells in the patient.

6. 10. The method of any one of the preceding claims, wherein the patient is natural killer ("NK") depleted prior to or at the time of said administering.

7. 10. The method of any one of the preceding claims, wherein said patient is severely NK depleted prior to or at the time of said administering.

8. 7. The method of claim 6, further comprising determining whether the patient is NK depleted prior to or at the time of said administering.

9. 7. The method of claim 6, further comprising determining whether the patient is severely NK depleted prior to or at the time of said administering.

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

11. 10. The method of any one of the preceding claims, wherein the patient is NK-depleted or severely NK-depleted as a result of another therapy.

12. 10. The method of any one of the preceding claims, wherein the patient is undergoing treatment for cancer.

13. 10. The method of any one of the preceding claims, wherein the patient is undergoing chemotherapy or radiation therapy.

14. 14. The method of claim 13, wherein the chemotherapy comprises administering one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, suberoylanilide-hydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, aphidicolin, 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).

15. 10. The method of any one of the preceding claims, wherein prior to or at the time of said administering, NK cells comprise less than 5% of peripheral blood mononuclear cells (PBMCs) in said individual.

16. 10. The method of any one of the preceding claims, wherein prior to or at the time of said administering, NK cells comprise less than 3% of peripheral blood mononuclear cells (PBMCs) in said individual.

17. prior to or at the time of said administration, less than 70% of said patient's PBMC NK cells are CD56 ｄｉｍ CD16 ＋ 10. The method of any one of the preceding claims, wherein the cells are NK cells.

18. prior to or at the time of said administration, less than 50% of said patient's PBMC NK cells are CD56 ｄｉｍ CD16 ＋ 10. The method of any one of the preceding claims, wherein the cells are NK cells.

19. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises at least one, two, three, four, five, or all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:

29.

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

38.

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

39.

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

39.

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

39.

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

29.

24. The method of claim 22 or 23, wherein the NEO-201 antibody comprises all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:

29.

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

29.

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

29.

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

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

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

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

31. 10. The method of any one of the preceding claims, further comprising administering to the patient an effective amount of a cytokine agonist to enhance or stimulate death of cells of the carcinoma.

32. 32. The method of claim 31, wherein the cytokine agonist is interleukin-2 (IL-2), interleukin-21 (IL-21), ALT-803, an IL-15 inhibitor, a checkpoint inhibitor, anti-PD1, anti-PDL1, anti-CTLA-4, anti-41BB, anti-OX40, anti-Tim-3, or a combination thereof.

33. 10. The method of any one of the preceding claims, further comprising administering to the patient an effective amount of a complement regulatory protein (CRP) antagonist to enhance or stimulate cell death of the carcinoma.

34. 34. The method of claim 33, wherein the CRP antagonist antagonizes one or more of CD46, CD55, or CD59.

35. 35. The method of claim 33 or 34, wherein the CRP antagonist comprises an antibody or an antigen-binding fragment thereof.

36. 32. The method of claim 31, wherein the cytokine agonist comprises an IL-15 agonist or an IL-15 superagonist.

37. 32. The method of claim 31, wherein the cytokine agonist comprises a complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor alpha / IgG1 Fc fusion protein.

38. 38. The method of claim 37, wherein the cytokine agonist comprises ALT-803.

39. The method of any one of claims 31 to 38, wherein the effective dosage of the NEO-201 antibody is reduced compared to treatment with the NEO-201 antibody alone without the cytokine agonist.

40. 10. The method of any one of the preceding claims, wherein the cancer is colon cancer.

41. 10. The method of any one of the preceding claims, wherein the cancer is pancreatic cancer.

42. 10. The method of any one of the preceding claims, wherein the cancer is ovarian cancer.

43. 10. The method of any one of the preceding claims, wherein the cancer is gastric cancer.

44. 10. The method of any one of the preceding claims, wherein the cancer is lung cancer.

45. 10. The method of any one of the preceding claims, wherein the cancer is breast cancer.

46. 10. The method of any one of the preceding claims, wherein the cancer is uterine cancer.

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