Monoclonal Antibody NEO-201 for the Treatment of Human Cancer

Abstract

NEO-201 is a humanized IgG1 monoclonal antibody (mAb) that is highly reactive against most tumor tissues from many different carcinomas, including colon, pancreatic, gastric, lung, breast, and uterine carcinomas, but is not recognized by this antibody in the vast majority of normal tissues. Functional assays revealed that NEO-201 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against tumor cells. In addition, treatment with NEO-201 alone and in combination with human peripheral blood mononuclear cells (PBMCs), which serve as a source of effector cells for ADCC, greatly attenuated the growth of human pancreatic xenograft tumors in vivo. In vivo biodistribution studies in mice bearing human tumor xenografts revealed that NEO-201 preferentially accumulates in tumors rather than in organ tissues. Single-dose toxicity studies in non-human primates demonstrated the safety and tolerability of NEO-201, as the only relevant side effect observed was a transient decrease in circulating neutrophils.

Description

[0001] Related application publication

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 592,778, filed November 30, 2017, and U.S. Provisional Application Serial No. 62 / 581,380, filed November 3, 2017, each of which is hereby incorporated by reference in its entirety.

[0003] Sequence listing information

[0004] This application includes a biological sequence listing in the form of a file named "43282o4402.txt" as part of its disclosure. The biological sequence listing was created on November 2, 2018, and is 32,563 bytes in size. The biological sequence listing is hereby incorporated by reference in its entirety. Background of the Invention

[0005] Cancer represents one of the most common causes of death worldwide, with an estimated 20 million new cases per year as early as 2025 (Ferlay et al., 2015). Conventional methods of treating cancer such as surgery, radiation, and chemotherapy often cause severe side effects and fail to cure most patients with advanced disease, leading to recurrence (Bodey et al., 1996). Recent treatment modalities have evolved to selectively target cancerous cells while largely sparing normal healthy tissue. Among these, immunotherapy has emerged as an important treatment option for cancer patients because it has revolutionized the field of cancer medicine.

[0006] The underlying principle of cancer immunotherapy is known as immunoediting (Mittal et al., 2014), which is an extrinsic mechanism of cancer containment that is initiated only after cellular transformation has occurred and the intrinsic mechanisms of cancer containment have failed. The immunoediting process occurs in three phases: elimination, equilibrium, and escape. During the elimination and equilibrium phases, immune rejection of cancer cells predominates or balances with cancer cell proliferation, respectively, to control malignant growth. However, during the escape phase, once kept in check, cancer cells can evade immune recognition due to insensitivity to immune effector mechanisms and / or induction of immune containment in the tumor microenvironment. Cancer cells that have evaded immune recognition can then proliferate more freely and grow into clinically apparent disease (Dunn et al., 2004). The goal of tumor immunotherapy is to keep cancer cells in the elimination and / or equilibrium phases by generating and / or augmenting anti-tumor immune responses to counteract tumor growth, delay tumor recurrence, and prolong survival (Carter, 2001; Hodge et al., 2006; Vergati et al., 2010; Gabitzsch et al., 2015). Treatment methods include treating patients with checkpoint inhibitory antibodies, anti-tumor vaccines, and chimeric antigen receptor (CAR)-T cells, all of which utilize the adaptive immunity of T cells. However, innate immunity can also generate and enhance anti-tumor responses, and tumor-targeted monoclonal antibodies (mAbs) can be used to stimulate innate anti-tumor immunity (Topalian et al., 2011).

[0007] NEO-201 is a novel humanized IgG1 mAb generated against the Hollinshead allogeneic colorectal cancer vaccine platform (Hollinshead et al., 1970; Hollinshead et al., 1972). The immunogenic component of this vaccine is tumor-associated antigen (TAA) from a tumor cell membrane fraction pooled from surgical resection specimens of 79 colon cancer patients (Hollinshead et al., 1985). These cell 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 colorectal cancer (Hollinshead et al., 1985; Hollinshead, US4810781, 1989; Bristol and Kantor, US Patent No. 7829678, 2010). These trials reported clinical benefits such as significant prolongation of anti-tumor response and overall survival in patients who developed a sustained IgG response in addition to a cell-mediated response against the vaccine, indicating that the vaccine contains an immunogenic component capable of generating anti-tumor antibodies (Hollinshead, 1991). This original colorectal cancer vaccine was used to generate monoclonal antibodies in mice, resulting in the previously described ensituximab (NPC-1C / NEO-102) (Luka et al., 2011; Patel et al., 2013; Beg et al., 2016; Kim et al., 2017) and NEO-201. Preliminary investigations have shown that NEO-201 can bind to tumor-associated variants of CEACAM family members (Zeligs et al., 2017), and efforts are underway to further characterize one or more antigens and one or more specific epitopes recognized by NEO-201.

[0008] The human carcinoembryonic antigen (CEA) family consists of 29 genes arranged in tandem on chromosome 19q13.2. Based on nucleotide homology, these genes are classified into two major subfamilies, the CEACAM and pregnancy-specific glycoprotein subgroups. The proteins encoded by CEACAM include CEA (CEACAM5), CEA-related cell adhesion molecules (CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, and CEACAM8). The CEACAM family belongs to the Ig superfamily. Structurally, each of the human CEACAMs contains an N-terminal domain that consists of 108-110 amino acids and is homologous to the Ig variable domain, followed by a variable number (0 to 6) of Ig C2-type constant-like domains. The CEACAM proteins can interact homophilically and heterophilically with each other. CEACAM1 is a unique protein in this family because it contains an ITIM (immunoreceptor tyrosine-based inhibitory motif)-like PD1 in its cytoplasmic domain. This inhibition is triggered by phosphorylation of tyrosine residues in the ITIM, which leads to the recruitment of tyrosine phosphatases-1 and -2 containing Src homology 2 domains. The CEACAM1 protein is expressed on a variety of immune cells, including monocytes, granulocytes, activated T cells, B cells, and NK cells. CEACAM1 occurs as several isoforms, and the two major isoforms are CEACAM1-L and CEACAM1-S, which have long (L) or short (S) cytoplasmic domains, respectively. CEACAM1-S expression is completely absent in human leukocytes. CEACAM1-L is expressed on a subset of activated human NK cells that are negative for CD16 but positive for CD56.

[0009] Monoclonal antibodies (mAbs) consist of a unique antigen-binding region (antigen-binding fragment, Fab) that is specific for a given mAb and a constant region (fragment crystallizable, Fc) that is common to all mAbs of the same isotype. The Fc region is capable of regulating 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 the Fc gamma receptor IIIa receptor (FcγRIIIa, CD16) expressed on macrophages and NK cells. This interaction can stimulate macrophages to phagocytose mAb-opsonized cancer cells and can activate NK cells to degranulate and lyse cancer cells by a mechanism known as antibody-dependent cell cytotoxicity (ADCC). ADCC has been shown to be a key mediator of in vivo anti-tumor effects in many preclinical studies and plays an important role in the mechanism of action of several mAbs used in cancer therapy (Seidel et al., 2013). Examples of clinically approved mAbs that mediate ADCC include trastuzumab, which targets the HER2 receptor in breast cancer (Seidel et al., 2013; 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 cancer and head and neck cancer (Seidel et al., 2013; Levy et al., 2009; Kawaguchi et al., 2007; Lopez-Albaitero et al., 2009); and avelumab, which targets the immune checkpoint ligand PD-L1 in Merkel cell carcinoma and bladder cancer (Boyerinas et al., 2015). Additionally, the Fc region can potentially interact with the C1 complex to activate complement-dependent cytotoxicity (CDC), where a proteolytic cascade culminates in the formation of pores in the cytoplasmic membrane (resulting in lysis of the cell targeted by the antibody). Even in cases where anti-tumor CDC has been demonstrated in vitro, it remains controversial whether anti-tumor CDC is critical for the clinical efficacy of mAb therapy for cancer (Meyer et al., 2014).

[0010] Applicant's prior 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 hereby incorporated by reference in its entirety) disclose various anti-cancer antibodies, cancer antigens, and related technologies. Summary of the Invention

[0011] The studies described in the examples herein evaluate the in vitro binding characteristics, as well as the in vivo activity and localization, of NEO-201 in preclinical models. NEO-201 exhibits broad reactivity against a range of human carcinoma cell lines and tumor tissues, but no binding to most healthy tissues was observed. In addition, NEO-201 exhibits ADCC and CDC activity against human carcinoma cells in vitro and, to a large extent, attenuates 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 effector cells for ADCC. Finally, single-dose toxicity studies in non-human primates demonstrated the safety and tolerability of NEO-201, as the only side effect observed was a transient decrease in circulating neutrophils. These studies provide a rationale for the potential clinical utility of NEO-201 as a novel therapeutic agent for the treatment of a variety of solid tumors. In addition, the observed CDC activity of the subject antibody opens the opportunity to treat patients with impaired immune function, in whom ADCC is not expected to be effective, such as patients who are immunocompromised because of their disease or as a result of radiotherapy, chemotherapy, and other disease treatments.

[0012] Preclinical anti-tumor activity of mAbs (termed ensituximab (NPC-1C / NEO-102)) generated against the Hollinshead allogeneic colorectal cancer vaccine platform has been previously reported (Patel et al., 2013) as well as clinical safety and efficacy (Beg et al., 2016; Kim et al., 2017). This report describes the characterization of a second tumor antigen-targeting mAb (termed NEO-201) derived from the same vaccine platform. NEO-201 was shown to positively stain a variety of human cancer cell lines in vitro, including cells derived from multiple tumor types, histological subtypes, and mutational profiles. NEO-201 positivity was more frequently observed in tumor cell lines derived from lung adenocarcinoma versus squamous cell carcinoma tumors and HER2-positive breast cancer cell lines versus triple-negative cancer cell lines. Staining of human tumor samples demonstrated that a wide variety of cancer tissues were positive for NEO-201, including colon, pancreas, stomach, lung, breast, and uterine tumors. An extended investigative study with a larger sample size could reveal that NEO-201 can distinguish histological subtypes from molecular subtypes in various cancers. Intriguingly, a higher proportion of tumor tissues reacted with NEO-201 compared to cultured cancer cell lines. This observation could indicate that the targets recognized by NEO-201 are more readily expressed in vivo compared to in vitro, which would suggest that target expression depends at least in part on tumor cell interactions with factors from the local microenvironment. Experiments are currently underway to further characterize one or more antigens and one or more epitopes recognized by NEO-201 and to determine the known or multiple regulatory control mechanisms governing its expression in tumor tissues rather than normal tissues.

[0013] This investigation also revealed that NEO-201 is significantly tumor-specific in its staining profile, as the vast majority of healthy normal tissues and normal tissues of adjacent tumor tissues were found to be negative for NEO-201. Although NEO-201 positivity was observed in normal tongue and extra-cervical tissues, the staining intensity was weak and the microarray representation was only for a minimal sample size (n = 2). Further extended analysis of NEO-201 staining in normal tissue samples will be conducted to confirm these observations. Additionally, NEO-201 administration did not induce any observable severe toxicity in mice and was well tolerated when administered to non-human primates. The observed depletion of neutrophils in non-human primates suggests that one or more antigens reactive with NEO-201 are expressed on these immune cells, and an assessment of the reactivity of NEO-201 with hematopoietic cell types was conducted. These encouraging results suggest that 1) NEO-201 may have diagnostic utility in differentiating cancerous tissues from benign tissues in patient biopsies; and 2) NEO-201 can effectively target tumors without causing significant toxicity or off-target effects other than neutropenia. Efforts are currently underway to further evaluate the safety and tolerability of NEO-201, and clinical trials using NEO-201 to treat cancerous tumors are being planned.

[0014] Innate immune effector mechanisms have been shown to play a major role in promoting and enhancing host anti-tumor immunity. It is well known that the Fc portion of human IgG1 mAb activates innate immunity against opsonized targets, thereby potentially mediating ADCC and / or CDC (Strome et al., 2007; Hayes J, et al., 2017). In particular, the ability to mediate ADCC is considered a key component of the therapeutic efficacy of various human IgG1 mAbs approved for the treatment of cancer (Boyerinas et al., 2015; Seidel et al., 2013; Petricevic et al., 2013; Dall’Ozzo et al., 2004; Levy et al., 2009; Kawaguchi et al., 2007; Lopez-Albaitero et al., 2009). Importantly, the V158F polymorphism of the FCGR3A gene (encoding FcγRIIIa) is associated with differential affinity for human IgG1 mAbs (Koene et al., 1997; Wu et al., 1997), where immune cells from donors with the high-affinity V / V genotype exhibit greater trastuzumab-mediated ADCC activity in vitro (Musolino et al., 2008). The V / V genotype has also been shown to be significantly associated with objective response rate and progression-free survival in breast cancer patients treated with trastuzumab (Musolino et al., 2008), thus providing indirect clinical evidence for the role of ADCC in mAb-based therapies. NEO-201 can mediate ADCC in vitro, as treatment of tumor cells with NEO-201 enhances the cytotoxic activity of NK cells by 2- to 5-fold, and ADCC activity is retained even at low concentrations of antibody (0.1 μg / mL). These data raise the possibility that patients with the V / V genotype may derive increased benefit from NEO-201 treatment. Another prospect is the potential to enhance ADCC activity and possibly the potential clinical benefit of NEO-201 by enhancing NK cell function and cytokine stimulation. It is well known that IL-2 is a potent activator of NK cells (Hank et al., 1990), and IL-21 has been shown to enhance ADCC activity mediated by trastuzumab and cetuximab (Watanabe et al., 2010). Recent preclinical studies of a novel fusion protein superagonist of IL-15 signaling (termed ALT-803) have demonstrated greatly enhanced proliferation, activation, and lytic capabilities of NK cells (and CD8+ T cells), resulting in significant anti-tumor activity in various animal models of cancer (Han et al., 2011; Gomes-Giacoia et al., 2014; Mathios et al., 2016; Rhode et al., 2016; Kim et al., 2016; Felices et al., 2017).Interestingly, ALT-803 was found to significantly enhance in vitro NK cell degranulation, IFN-γ production, and rituximab-mediated ADCC against B cell lymphoma cell lines and primary follicular lymphoma cells, and treatment with the combination of ALT-803 and rituximab in two B cell lymphoma models resulted in significantly reduced tumor cell burden and improved survival in vivo (Rosario et al., 2016).

[0015] Another innate immune effector mechanism potentially engaged by mAbs is the activation of the complement system to promote CDC, and NEO-201 has been found to have the ability to mediate CDC to kill tumor cells. The contribution of CDC to the therapeutic efficacy of mAbs is controversial, but has been proposed to be beneficial for cancer therapy, at least in some specific situations (Meyer et al., 2014). Additionally, several different complement regulatory proteins (CRPs) function to inhibit complement activation, and certain membrane-bound CRPs such as CD46, CD55, CD59 have been reported to be aberrantly expressed in various cancers (Seya et al., 1994; Niehans et al., 1996; Donin et al., 2003), which may confer resistance to CDC. Future investigations will determine whether strategies to block CRPs can enhance the CDC of NEO-201-mediated resistant tumor cells.

[0016] Evaluation of NEO-201 in vivo revealed a substantial antitumor effect when administered in combination with activated human immune effector cells. This combination even led to complete regression in some mice (5 / 20, 25%) from both combination groups. In addition, NEO-201 was found to preferentially localize to xenograft tumor tissues, but not to various healthy tissues. These data confirm that the mechanism of action of NEO-201 against tumors is ADCC-dependent lysis of tumor cells by innate immune cells. However, it should be noted that antitumor activity was also observed in the case of NEO-201 alone without adding human immune cells to immunodeficient mice. This phenomenon may be specific to the conditions encountered in vivo, as treatment of CFPAC-1 tumor cells with NEO-201 did not induce significant toxicity in an in vitro ADCC assay. One hypothesis for the activity of NEO-201 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.

[0017] In summary, the present investigation demonstrates that NEO-201 is a remarkable tumor-specific antibody capable of engaging innate immune effector mechanisms, including both ADCC and CDC, to kill tumor cells. In addition, NEO-201 has demonstrated safety and anti-tumor efficacy in in vivo xenograft models of pancreatic cancer, as well as tolerance in non-human primates. These findings provide a supportive rationale for the clinical development of NEO-201 as a diagnostic and therapeutic agent for patients with a variety of carcinomas. The results also support the use of NEO-201 in patients with impaired immune function (with low NK cell levels), as anti-tumor effects can be generated by CDC even in the absence of robust ADCC activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figures 1A - 1D : Flow cytometry of NEO-201 bound to human carcinoma cell lines. Representative human carcinoma cell lines with various levels of NEO-201 antigen expression, ( Figure 1A ) pancreatic CFPAC-1 (high), ( Figure 1B ) NSCLC H441 (medium), ( Figure 1C ) breast HCC1937 (low), and ( Figure 1D ) colon SW1116 (negative). For each cell line, the results are presented as % NEO-201 positive and mean fluorescence intensity (MFI). Red, NEO-201-stained cells; black, unstained cells. NEO-201 positivity was defined as % positive ≥ 10%.

[0019] Figures 2A - 2C : IHC staining of NEO-201 on human tumor samples. ( Figure 2A ) Representative NEO-201 staining of adjacent normal and malignant tissues from colon, pancreas, stomach, and lung samples. All images were obtained at 100X. ( Figure 2B ) Quantification of NEO-201 positive staining on human tumor microarray samples from various carcinoma tissues. ( Figure 2C ) Quantification of NEO-201 positive staining on human tumor microarray samples from normal tissues adjacent to tumor tissues. n = number of samples.

[0020] Figures 3A - 3C : NEO-201 mediates ADCC and CDC against human tumor cell lines. ( Figure 3A ) 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 ratios. *, statistically significant by t-test (p < 0.05). ( Figure 3B) ADCC assay of 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 figure depicts the fold increase (%) in specific lysis of NEO-201-treated tumor cells compared to control cells treated with 10 μg / mL human IgG1. *, Statistically significant by t-test (p < 0.05).( Figure 3C ) CDC assay using ASPC-1 cells treated with the indicated doses of NEO-201 for the indicated durations. *, Determined to be statistically significant by t-test (p < 0.05).

[0021] Figures 4A - 4D : Antitumor efficacy of NEO-201 in CFPAC-1 tumor xenografts.( Figure 4A ) Tumor volume measurements of CFPAC-1 xenografts from each treatment group at different time points. Saline solution, human IgG1 (250 μg), or NEO-201 (100 and 250 μg) were administered intraperitoneally to mice (n = 10 animals / group) on days 13, 17, and 20 after tumor cell implantation. Approximately 1.0 x 10 7 IL-2-activated PBMCs were also administered intraperitoneally to mice on days 14, 18, and 21.( Figure 4B ) Quantification of the number of mice still bearing palpable tumors on day 36.( Figure 4C ) Representative images of NEO-201-treated versus saline-treated tumor-bearing mice.( Figure 4D ) Body weight measurements of tumor-bearing mice at different time points during the study.

[0022] Figures 5A - 5B : Biodistribution of NEO-201 in mice bearing CFPAC-1 xenografts. Measurement of the normalized radioactivity of the indicated tissues from female( Figure 5A ) and male( Figure 5B ) mice bearing CFPAC-1 tumors that received intravenously administered radiolabeled NEO-201. n = 4 animals / time point. Days 1, 2, 4, and 7 represent the amount of time between radiolabeled antibody injection and necropsy.

[0023] Figures 6A - 6C : Body weight and neutrophil count from cynomolgus monkeys treated with NEO-201.( Figure 6A ) The percentage change in monkey body weight relative to baseline (day -1) was measured at 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)Percentage change in neutrophil levels in blood from monkeys treated with a single dose of NEO-201 at the indicated dose levels relative to baseline (Day -7). n = 4 animals / group (2 females, 2 males).( Figure 6C )For each dose and time point, p-value of neutrophil levels compared to 0 mg / kg control. * Statistically significant by t-test (p < 0.05).

[0024] Figures 7A - 7C : haNK ADCC assay (4 hours) using NEO-201. Target cells = 3000 cells / well.( Figures 6A - 6B )Percentage specific lysis of H520 lung cancer tumors( Figure 6A )or OV90 ovarian cancer tumors( Figure 6B )cells treated with NEO-201 (upper line, square symbols) or IgG1 negative control (lower line, circular symbols) at 4 hours, varying with effector:target (E:T) ratio. E:T ratios were 6.25:1, 12.5:1, or 25:1. mAb concentration was 10 μg / mL. Values shown are mean ± SD of 3 replicates. Asterisk (*) indicates statistical significance compared to IgG negative control (p < 0.01, two-tailed t-test).( Figure 6C )Percentage specific lysis of lung (H520, HCC827), breast (ZR-75-1), and ovarian (OV90) cancer tumor cells treated with NEO-201 (right, light gray bars) or negative control IgG (left, solid black bars) at a constant E:T ratio of 25:1 for four hours. mAb concentration was 10 μg / mL. Values shown are mean ± SD of 3 replicates. Asterisk (*) indicates statistical significance compared to IgG negative control (p < 0.01, two-tailed t-test).

[0025] Figure 8: 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 media 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 percentage specific lysis (SE).

[0026] Figure 9: Treatment with ALT-803 enhanced the expression of TIM-3 and NKG2D on human NK cells. Purified human NK cells from normal donors were cultured for 48 hours with or without ALT-803 (25 ng / ml). Results are expressed as % positive cells (MFI).

[0027] Figure 10: Treatment with ALT - 803 Enhances the Expression of TIM - 3 and NKG2D on Human NK Cells Purified human NK cells from another normal donor were cultured for 48 hours with or without ALT-803 (25 ng / ml). Results are expressed as % of positive cells (MFI).

[0028] Figure 11: Treatment with ALT-803 enhances ADCC activity mediated by low concentrations of NEO-201. NK cells isolated from a normal donor (ND#6) were treated with ALT-803 (25 ng / ml) or media 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).

[0029] Figure 12: Treatment with ALT-803 enhances ADCC activity of a normal donor (ND#8) with the lowest ADCC activity mediated by NEO-201, and the activity can be blocked by anti-CD16 and anti-TIM-3 antibodies. NK cells isolated from a normal donor with the lowest ADCC activity were treated with ALT-803 (25 ng / ml) or media 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. 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 no ALT-803 treatment (p < 0.01). # Statistically significant compared to no anti-CD16 and anti-TIM-3 treatment (p < 0.01).

[0030] Figure 13: 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. The cells were then treated with 10 μg / mL of human IgG1 isotype control antibody or NEO-201 and then 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 incubation at 37 °C for 16 hours, cell viability was quantified using a Celigo imaging cytometer and GraphPad Prism 7 software. Live target cells (calcein AM+ / PI-) in each well were counted and specific lysis was calculated. Results are tabulated and graphed below for each tumor cell type. *Statistically significant (p<0.05). Detailed Description

[0031] In one aspect, the present disclosure provides a method of killing cancerous tumor cells, the method comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody.

[0032] In one aspect, the present disclosure provides a method of treating a cancerous tumor, the method comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody.

[0033] In one aspect, the present disclosure provides a method of preventing recurrence of a cancerous tumor, the method comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody.

[0034] In one aspect, the present disclosure provides a method of reducing the tumor burden of a patient having a cancerous tumor, the method comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody.

[0035] The antibody may mediate complement-mediated cytotoxicity (CDC), thereby killing cancerous tumor cells in the patient's body.

[0036] Before or at the time of said administration, the patient may be natural killer cell ("NK") depleted. Before or at the time of said administration, the patient may be severely NK depleted. The patient may have natural killer 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 due to another therapy, such as a cancer therapy (e.g., chemotherapy or radiotherapy). The patient 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., radicicol, 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel) and / or immunomodulatory drugs (e.g., lenalidomide).

[0037] The method may include determining, before or at the time of said administration, whether the patient is NK depleted.

[0038] The method may include determining, before or at the time of said administration, whether the patient is severely NK depleted.

[0039] In the method, before or at the time of said administration, NK cells may account for less than 5% of peripheral blood mononuclear cells (PBMCs) in the individual.

[0040] In the method, before or at the time of said administration, NK cells may account for less than 3% of peripheral blood mononuclear cells (PBMCs) in the individual.

[0041] In the method, before or at the time of said administration, less than 70% of PBMC NK cells in the patient may be CD56dimCD16+ NK cells.

[0042] In the method, before or at the time of said administration, less than 50% of PBMC NK cells in the patient may be CD56dimCD16+ NK cells.

[0043] 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.

[0044] The NEO-201 antibody may comprise a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38.

[0045] The NEO-201 antibody may comprise a variable light chain sequence having at least 90% identity with SEQ ID NO: 39.

[0046] The NEO-201 antibody may comprise a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38 and a variable light chain sequence having at least 90% identity with SEQ ID NO: 39.

[0047] The NEO-201 antibody may comprise a heavy chain sequence having at least 90% identity with amino acids 20-470 of SEQ ID NO: 28 and a light chain sequence having at least 90% identity with amino acids 20-233 of SEQ ID NO: 29.

[0048] The NEO-201 antibody may comprise all six of the CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO: 29.

[0049] The NEO-201 antibody may comprise a human IgG1 constant domain.

[0050] The NEO-201 antibody may be a humanized antibody.

[0051] The NEO-201 antibody may be conjugated to another moiety.

[0052] The NEO-201 antibody may be conjugated to another cytotoxic moiety, label, radioactive moiety, or affinity tag.

[0053] The method may further comprise administering to the patient an effective amount of a cytokine agonist to enhance or stimulate killing of the cells of the cancerous tumor. The cytokine agonist may include 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.

[0054] The method may further comprise administering to the patient an effective amount of a complement regulatory protein (CRP) antagonist to enhance or stimulate killing of the cells of the cancerous tumor. The CRP antagonist may antagonize one or more of CD46, CD55, or CD59. The CRP antagonist may comprise an antibody or an antigen-binding fragment thereof.

[0055] The cytokine agonist may include an IL-15 agonist or an IL-15 superagonist.

[0056] The cytokine agonist may include a complex consisting of an IL-15 mutant (IL-15N72D) that binds to an IL-15 receptor α / IgG1 Fc fusion protein, such as ALT-803.

[0057] The effective dose of the NEO-201 antibody may be reduced compared to treatment with the NEO-201 antibody alone in the absence of the cytokine agonist.

[0058] The cancer tumor may include colon cancer. The cancer tumor may include pancreatic cancer. The cancer tumor may include ovarian cancer. The cancer tumor may include gastric cancer. The cancer tumor may include lung cancer. The cancer tumor may include breast cancer. The cancer tumor may include uterine cancer.

[0059] In another embodiment, the present disclosure provides a method of killing cancer tumor cells, the method comprising administering to a patient in need thereof an effective amount of the NEO-201 antibody, wherein prior to or at the time of the administration, the patient is natural killer cell ("NK") depleted. The NK depletion may include that less than 5% or less than 3% of the patient's peripheral blood mononuclear cells (PBMCs) in a sample derived from the patient, such as a blood sample, are NK cells. Alternatively or in addition, prior to or at the time of the administration, less than 70% (or optionally less than 50%) of the PBMC NK cells in the patient may be CD56dimCD16+ NK cells.

[0060] In another embodiment, the present disclosure provides a method of treating a cancer tumor, the method comprising administering to a patient in need thereof an effective amount of the NEO-201 antibody, wherein prior to or at the time of the administration, the patient is natural killer cell ("NK") depleted.

[0061] In another embodiment, the present disclosure provides a method of preventing recurrence of a cancer tumor, the method comprising administering to a patient in need thereof an effective amount of the NEO-201 antibody, wherein prior to or at the time of the administration, the patient is natural killer cell ("NK") depleted.

[0062] In another embodiment, the present disclosure provides a method of reducing the tumor burden of a patient suffering from a cancer tumor, the method comprising administering to a patient in need thereof an effective amount of the NEO-201 antibody, wherein prior to or at the time of the administration, the patient is natural killer cell ("NK") depleted.

[0063] In the foregoing method, the antibody can mediate CDC to kill cancer cells in the patient. For example, although there is no effective ADCC because the patient is NK-depleted. At the time of the administration, the patient can be severely NK-depleted. Optionally, the method further includes determining whether the patient is NK-depleted or severely NK-depleted, for example, at the time of the administration or within a certain period before the administration, such as within the previous 1 or 2 weeks. The NK-depleted or severely NK-depleted status can also be inferred from the patient's medical history, such as the previous or concurrent use of another therapy that depletes NK cells. For example, the patient has undergone or is concurrently undergoing cancer therapy, such as radiotherapy or chemotherapy. The cancer therapy can include the administration of 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 drugs (e.g., lenalidomide).

[0064] The patient can have NK cell deficiency (NKD), such as CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD1).

[0065] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, 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.

[0066] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody can 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. The variable heavy chain having the percentage of sequence identity can comprise all 3 of the CDR sequences contained in SEQ ID NO:38.

[0067] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody may comprise a variable light chain sequence having at least 80%, at least 85%, at least 90% or most preferably at least 95% identity to SEQ ID NO:39. The variable light chain may comprise all 3 of the CDR sequences contained in SEQ ID NO:39.

[0068] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody may comprise a variable heavy chain sequence having at least 80%, at least 85%, at least 90% or most preferably at least 95% identity 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 3 of the CDR sequences contained in SEQ ID NO:39, and the variable heavy chain having the percentage of sequence identity may comprise all 3 of the CDR sequences contained in SEQ ID NO:38.

[0069] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody may comprise a heavy chain sequence having at least 80%, at least 85%, at least 90% or most preferably at least 95% identity 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 3 of the CDR sequences contained in SEQ ID NO:29, and the heavy chain having the percentage of sequence identity may comprise all 3 of the CDR sequences contained in SEQ ID NO:28.

[0070] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody may comprise the heavy chain variable region sequence contained in SEQ ID NO:28 and the light chain variable region sequence contained in SEQ ID NO:29.

[0071] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody may comprise a heavy chain sequence containing amino acids 20-470 of SEQ ID NO:28 and a light chain sequence containing amino acids 20-233 of SEQ ID NO:29.

[0072] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody comprises a human IgG1 constant domain.

[0073] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody can be a humanized antibody.

[0074] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the NEO-201 antibody can be conjugated to another moiety, such as another cytotoxic moiety, label, radioactive moiety, or affinity tag.

[0075] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the method can further comprise administering to the patient an effective amount of a cytokine agonist to enhance or stimulate the killing of the cells of the cancerous tumor. The cytokine agonist can include 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.

[0076] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the method can further comprise administering to the patient an effective amount of a complement regulatory protein (CRP) antagonist to enhance or stimulate the killing of the cells of the cancerous tumor. The CRP antagonist can antagonize one or more of CD46, CD55, or CD59. The CRP antagonist can comprise an antibody or an antigen-binding fragment thereof. The cytokine agonist can include an IL-15 agonist or an IL-15 superagonist. The cytokine agonist can include a complex consisting of an IL-15 mutant (IL-15N72D) that binds to an IL-15 receptor α / IgG1 Fc fusion protein. The cytokine agonist can include ALT-803.

[0077] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the effective dose of the NEO-201 antibody is reduced compared to treatment with the NEO-201 antibody alone in the absence of the cytokine agonist.

[0078] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the cancer may express the NEO-201 antigen. The expression of the NEO-201 antigen can be determined by detecting the NEO-201 antigen in a sample of the cancer. The detection can be carried out by techniques including but not limited to the following: histological staining, flow cytometry, RT-PCR, dot blotting, Western blotting, Northern blotting, and other techniques known in the art. In the case of recurrent or metastatic cancer, the expression of the NEO-201 antigen can also be inferred by the expression of NEO-201 in the primary cancer or by the responsiveness of the primary cancer to NEO-201 antibody therapy.

[0079] In a preferred embodiment of the invention that can be used in conjunction with any of the foregoing or following embodiments, the cancer may include colon cancer.

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

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

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

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

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

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

[0086] Definitions

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those 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 practice or testing of the present invention, suitable methods and materials are described herein. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0088] Unless the context clearly indicates otherwise, as used in the description herein and throughout the following claims, the meanings of "a / an" and "the" include plural referents.

[0089] As used herein, "amino acid" broadly refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids subsequently modified, such as, for example, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid, the basic chemical structure being an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetic" refers to a chemical compound that has a structure different from the general chemical structure of an amino acid, but acts in a manner similar to a naturally occurring amino acid.

[0090] As used herein, the term "NK-depleted" or "natural killer cell-depleted" refers to a patient having low natural killer (NK) cell levels relative to the normal range. NK cells are cytotoxic innate immune lymphocytes. Typically, NK cells comprise 5%-20% of peripheral blood mononuclear cells (PBMCs) in healthy individuals. A patient having less than 5% of NK cells of PMBCs is referred to as NK-depleted. Additionally, if the NK cells comprise less than 3% of PMBCs, the patient is referred to as severely NK cell-depleted. Additionally, in normal individuals, up to 90% of PBMC NK cells are CD56 dim CD16 + NK cells, and these are considered the most cytotoxic subset. If less than 70% of PBMC NK cells are CD56 dim CD16 + NK cells, then the patient is referred to as NK-depleted. Additionally, if less than 50% of PBMC NK cells are CD56 dim CD16 +NK cells, and the patient is then 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, the status of a patient as NK-depleted or severely NK-depleted is determined by testing a sample obtained from the patient, such as a blood sample, e.g., a sample obtained and tested within one or two weeks prior. The status of a patient as NK-depleted or severely NK-depleted may also be inferred from a disease diagnosis and / or a treatment course associated with such depletion of NK cells.

[0091] NK-depleted also includes subjects with NK cell deficiency (NKD). Exemplary NKD conditions include classical NKD (CNKD), which is characterized by the absence of NK cells and their function in peripheral blood lymphocytes; functional NKD (FNKD), which is characterized by the presence of NK cells within peripheral blood lymphocytes with defective NK cell activity. In both CNKD and FNKD, the NK cell abnormality is a primary immunodeficiency that results in an insufficient ADCC response. CNKD and FNKD can be further subdivided based on patient characteristics, such as the identity of one or more disease-causing genes and other patient characteristics. CNKD includes CNKD subtype 1 (CNKD1), which is autosomal dominant and associated with a defect in the GATA2 gene; and CNKD subtype 2 (CNKD2), which is autosomal recessive and associated with a defect in the MCM4 gene. FNKD includes FNKD1, which is autosomal recessive and associated with a defect in the FCCR3A gene.

[0092] As used herein, the term "antibody" broadly refers to any polypeptide chain-containing molecular structure having a specific shape that mates with and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. Typical antibody molecules are immunoglobulins, and all types of immunoglobulins IgG, IgM, IgA, IgE, IgD from all sources (e.g., human, rodent, rabbit, cow, sheep, pig, dog, chicken) are considered "antibodies". Antibodies include, but are not limited to, chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single-chain antibodies (scFv), camel antibodies, nanobodies, IgNAR (single-chain antibodies obtained from sharks), small molecule immune pharmaceuticals (SMIP), and antibody fragments (e.g., Fab, Fab’, F(ab’)2). Many antibody-encoding sequences have been described; and other antibody-encoding sequences can 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.

[0093] "NEO-201 antibody" refers to an antibody containing the heavy and light chains of SEQ ID NO: 28 and 29, or the variable regions contained therein, optionally together with the constant regions, as well as fragments and variants thereof. Such variants include sequences containing one, two, three, four, five, or preferably all six of the CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO: 29, which CDR sequences are the heavy chain CDR1 of SEQ ID NO: 32, the heavy chain CDR2 of SEQ ID NO: 33, the heavy chain CDR3 of SEQ ID NO: 34, the light chain CDR1 of SEQ ID NO: 35, the light chain CDR2 of SEQ ID NO: 36, and the light chain CDR3 of SEQ ID NO: 37. The antibody can be a humanized antibody. The antibody can be expressed as containing one or more leader sequences, which can be removed during the expression and / or processing and secretion of the antibody. The antibody can be presented in monovalent, divalent, or higher multivalent forms, including but not limited to bispecific or multispecific antibodies containing the NEO-201 antibody sequence and binding fragments of different antibodies. Generally, the antibody specifically binds to cancerous cells and competes with an antibody containing the variable heavy chain of SEQ ID NO: 38 and the variable light chain of SEQ ID NO: 39 or an antibody containing the heavy chain of SEQ ID NO: 28 and the light chain of SEQ ID NO: 29 for binding to cancerous cells. One or more of those CDR sequences contained in SEQ ID NO: 28 and / or SEQ ID NO: 29 can be replaced by variant sequences such as the light chain CDR1 of SEQ ID NO: 1 or 4; the light chain CDR2 of SEQ ID NO: 2 or 5; the light chain CDR3 of SEQ ID NO: 3 or 6; the heavy chain CDR1 of SEQ ID NO: 7; the heavy chain CDR2 of SEQ ID NO: 8, 10, 30, or 31; the heavy chain CDR3 of SEQ ID NO: 9 or 11 or SEQ ID NO: 30 - 31. The light chain can contain the CDRs contained in the light chain sequences of SEQ ID NO: 14, 16, 17, 18, 19, 20, 21, or 29. The heavy chain can contain the CDRs contained in the heavy chain sequences of SEQ ID NO: 15, 22, 23, 24, 25, 26, 27, or 29.The antibody may comprise a variable heavy chain sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity 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, optionally wherein the heavy chain and / or light chain sequence contains one, two, three, four, five or preferably all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29, the CDR sequences being the heavy chain CDR1 of SEQ ID NO:32, the heavy chain CDR2 of SEQ ID NO:33, the heavy chain CDR3 of SEQ ID NO:34, the light chain CDR1 of SEQ ID NO:35, the light chain CDR2 of SEQ ID NO:36 and the light chain CDR3 of SEQ ID NO:37. The antibody may be conjugated to another moiety, such as a cytotoxic moiety, a radioactive moiety, a label or a purification tag.

[0094] As used herein, "antigen" broadly refers to a molecule or a portion of a molecule capable of being bound by an antibody, and which is additionally capable of inducing an animal to produce an antibody capable of binding to an epitope of the antigen. An antigen may have one epitope or may have more than one epitope. The specific reaction referred to herein indicates that the antigen will react with its corresponding antibody in a highly selective manner and will not react with a variety of other antibodies that may be induced by other antigens. An antigen may be tumor-specific (e.g., expressed by neoplastic cells of pancreatic cancer tumors and colon cancer tumors).

[0095] As used herein, "cancer" broadly refers to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division, resulting in malignant growth or a tumor.

[0096] As used herein, "chimeric antibody" broadly refers to an antibody molecule in which the constant region or a portion thereof is altered, replaced or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or to a completely different molecule (e.g., an enzyme, a toxin, a hormone, a growth factor, a drug) that confers new properties to the chimeric antibody; or the variable region or a portion thereof is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0097] As used herein, "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, a conservatively modified variant refers broadly to those nucleic acids that encode the same or substantially the same amino acid sequence, or, when the nucleic acid does not encode an amino acid sequence, to sequences that are substantially the same. Because of 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" and represent one species of conservatively modified variation. Each nucleic acid sequence encoding a polypeptide herein also describes every possible nucleic acid silent variation. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except for the AUG which is normally the only codon for methionine and the TGG which is normally the only codon for tryptophan) can be modified to yield a functionally identical molecule.

[0098] As used herein, "complementary determining region", "hypervariable region", or "CDR" refers broadly to one or more hypervariable or complementary determining regions (CDRs) found in the variable region of a light or heavy chain of an antibody. See Kabat, et al. (1987) " Sequences of Proteins of Immunological Interest "National Institutes of Health, Bethesda, MD. These terms include hypervariable regions as defined by Kabat, et al. (1983) " Sequences of Proteins of Immunological Interest "U.S. Dept. of Health and Human Services or hypervariable loops in the three-dimensional structure of an antibody. Chothia and Lesk (1987) J Mol.Biol. 196:901–917. The CDRs in each chain are held in close proximity by framework regions and together with the CDRs from the other chain contribute to the formation of the antigen-binding site. Within the CDRs, there are selected amino acids that have been described as selectivity determining regions (SDRs) which represent key contact residues used by the CDRs in antibody-antigen interactions. Kashmiri (2005) Methods 36:25–34.

[0099] As used herein, "control amount" refers broadly to any amount or series of amounts of a marker that can be used for comparison with a test amount of the marker. For example, a control amount of a marker can be the amount of the marker in a patient with a particular disease or disorder or in a person without such disease or disorder. A control amount can be an absolute amount (e.g., micrograms / ml) or a relative amount (e.g., relative intensity of a signal).

[0100] As used herein, "differentially present" broadly refers to a difference in the amount or quality of a marker present in a sample taken from a patient having a disease or disorder as compared to a comparable sample taken from a patient not having one of the diseases or disorders. For example, if the amount of a nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in another sample, the nucleic acid fragment may optionally be differentially present between the two samples, such as as measured by hybridization and / or NAT-based assays. If the amount of a polypeptide in one sample is significantly different from the amount of the polypeptide in another sample, the polypeptide may optionally be differentially present between the two samples. It should be noted that if the marker is detectable in one sample and undetectable in another sample, the marker may be considered to be differentially present. Optionally, a relatively low level of upregulation may serve as a marker.

[0101] As used herein, "diagnosis" broadly refers to the identification of the presence or nature of a pathological disorder. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (the percentage 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 called "true negatives". The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those who test positive but do not have the disease. Although a particular diagnostic method may not provide a definitive diagnosis of a disorder, it is qualified if the method provides a positive indication that aids in the diagnosis.

[0102] As used herein, "diagnosis" broadly refers to grading a disease or symptom, determining the severity of a disease, monitoring disease progression, predicting the consequences of a disease and / or the prospects for recovery. The term "detection" may also optionally encompass any of the foregoing. In some embodiments, the diagnosis of a disease according to the present invention may be affected by determining the level of a polynucleotide or polypeptide of the present invention in a biological sample obtained from a subject, wherein the determined level may be related to the susceptibility to the disease, or the presence or absence of the disease. It should be noted that a "biological sample obtained from a subject" may also optionally include a sample that has not been removed from the subject's body.

[0103] As used herein, an "effective amount" broadly refers to the amount of a compound, antibody, antigen, or cell that is sufficient to effect such treatment of a disease when administered to a patient for treating the disease. An effective amount can be an amount effective for prophylaxis, and / or an amount effective for prevention. An effective amount can be an amount effective to reduce, prevent the occurrence of signs / symptoms, reduce the severity of the occurrence of signs / symptoms, eliminate the occurrence of signs / symptoms, slow the progression of the occurrence of signs / symptoms, prevent the progression of the occurrence of signs / symptoms, and / or effectively prevent the occurrence of signs / symptoms. An "effective amount" can vary depending on the disease and its severity of the patient to be treated, as well as the age, weight, medical history, susceptibility, and pre-existing conditions. For the purposes of the present invention, the term "effective amount" is synonymous with "therapeutically effective amount".

[0104] As used herein, an "expression vector" broadly refers to any recombinant expression system for the purpose of constitutively or inducibly expressing the nucleic acid sequences of the present invention in vitro or in vivo in any cell, including prokaryotic cells, yeast cells, fungal cells, plant cells, insect cells, or mammalian cells. The term includes linear or circular expression systems. The term includes expression systems that remain free or integrate into the host cell genome. The expression system can have the ability to self-replicate or not self-replicate (i.e., drive only transient expression in the cell). The term includes recombinant expression cassettes that contain only the minimal elements required for transcription of the recombinant nucleic acid.

[0105] As used herein, a "framework region" or "FR" broadly refers to one or more framework regions within the variable region of the light or heavy chain of an antibody. See Kabat, et al. (1987) " Sequences of Proteins of Immunological Interest " National Institutes of Health, Bethesda, MD. These expressions include those amino acid sequence regions inserted between the CDRs within the variable regions of the light and heavy chains of an antibody.

[0106] As used herein, "heterologous" broadly refers to a portion of a nucleic acid, indicating that the nucleic acid contains two or more subsequences that do not find the same relationship in nature to each other. For example, a nucleic acid is typically recombinantly produced such that two or more sequences from unrelated genes are arranged to prepare a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates a protein that contains two or more subsequences that do not find the same relationship in nature to each other (e.g., a fusion protein).

[0107] As used herein, "high affinity" broadly refers to an antibody having at least 10 –8 M, more preferably at least 10–9 M and even more preferably at least 10 –10 KD of M. However, for other antibody isotypes, "high affinity" binding may vary. For example, for the IgM isotype, "high affinity" binding refers to an antibody having at least 10 –7 M, more preferably at least 10 –8 M KD.

[0108] 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 complete. Complete homology indicates that the nucleic acid or amino acid sequence is identical. Partially homologous nucleic acid or amino acid sequences are nucleic acid or amino acid sequences that are not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The term "sequence identity" may be used interchangeably with "homology".

[0109] As used herein, "host cell" broadly refers to a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells can be prokaryotic cells such as Escherichia coli, or eukaryotic cells such as yeast, insect (e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa, HEK-293, e.g., cultured cells, explants, and in vivo cells.

[0110] As used herein, "hybridization" broadly refers to the physical interaction of complementary (including partially complementary) polynucleotide strands by forming hydrogen bonds between complementary nucleotides when the strands are arranged antiparallel to each other.

[0111] As used herein, the term "K-assoc" or "Ka" broadly refers to the association rate of a specific antibody-antigen interaction; and as used herein, the term "Kdiss" or "Kd" refers to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as molar concentration (M). The KD value of an antibody can be determined using well-established methods in the art.

[0112] As used herein, "immunoassay" broadly refers to an assay that uses an antibody that specifically binds to an antigen. The characteristics of an immunoassay can be characterized by using the specific binding properties of a specific antibody to isolate, target, and / or quantify the antigen.

[0113] As used herein, "isolated" broadly refers to a material that has been removed from its original environment in which it naturally occurs and thus altered artificially from its natural environment. An isolated material can be, for example, an exogenous nucleic acid contained in a vector system, an exogenous nucleic acid contained within a host cell, or any material that has been removed from its original environment and thus artificially altered (e.g., "isolated antibody").

[0114] As used herein, "label" or "detectable moiety" broadly refers to a composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[0115] As used herein, "low stringency", "medium stringency", "high stringency", or "very high stringency conditions" broadly refer to conditions for nucleic acid hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel, et al. (2002) Short Protocols in Molecular Biology (5 th (Ed.) John Wiley & Sons, NY. Exemplary specific hybridization conditions include, but are not limited to: (1) low stringency hybridization conditions of hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing at least twice in 0.2X SSC, 0.1% SDS at 50°C (for low stringency conditions, the washing temperature can be increased to 55°C); (2) medium stringency hybridization conditions of hybridization in 6X SSC at about 45°C, followed by washing one or more times in 0.2X SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions of hybridization in 6X SSC at about 45°C, followed by washing one or more times in 0.2X SSC, 0.1% SDS at 65°C; and (4) very high stringency hybridization conditions are hybridization in 0.5M sodium phosphate, 7% SDS at 65°C, followed by washing one or more times in 0.2X SSC, 1% SDS at 65°C.

[0116] As used herein, "mammal" broadly refers to any and all warm-blooded vertebrates of the class Mammalia, including humans, characterized by the presence of hair on the skin and, in the female, mammary glands for nourishing the young. Examples of mammals include, but are not limited to, alpaca, armadillo, capybara, cat, camel, chimpanzee, chinchilla, cow, dog, goat, gorilla, hamster, horse, human, lemur, llama, mouse, non-human primate, pig, rat, sheep, shrew, squirrel, and tapir. Mammals include, but are not limited to, bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammals also include any and all mammals listed in the World Mammal Species maintained by the National Museum of Natural History, Smithsonian Institution, Washington, D.C.

[0117] As used herein, "nucleic acid" or "nucleic acid sequence" refers to deoxyribonucleotide or ribonucleotide oligonucleotides in single-stranded or double-stranded form. The term encompasses nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0118] As used herein, the term "operably linked" broadly refers to the joining of two DNA fragments such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0119] As used herein, "complementary site" broadly refers to a portion of an antibody that recognizes an antigen (e.g., the antigen-binding site of an antibody). A complementary site can be a small region of the Fv region of an antibody (e.g., 15-22 amino acids) and can contain portions of both 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.

[0120] As used herein, "patient" broadly refers to any animal in need of treatment to alleviate a disease state or to prevent the occurrence or recurrence of a disease state. In addition, as used herein, "patient" broadly refers to any animal having a previously diagnosed risk factor, medical history, susceptibility, symptom, sign, or being at risk of or a member of a patient population of a disease. A patient can be a clinical patient, such as a human, or a veterinary patient, such as a companion animal, domestic animal, livestock, exotic animal, or zoo animal. The term "subject" can be used interchangeably with the term "patient".

[0121] "Polypeptide", "peptide", and "protein" are used interchangeably herein and broadly refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the 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 the 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.

[0122] As used herein, "promoter" broadly refers to a nucleic acid sequence that directs the transcription of a nucleic acid. As used herein, "promoter" includes the essential nucleic acid sequences near the transcription start site, such as the TATA element in the case of type II polymerase promoters. A promoter also optionally includes distal enhancer or repressor elements that can be located up to several thousand base pairs away from the transcription start site. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulatory conditions.

[0123] As used herein, "therapeutically effective amount" broadly refers to the amount of a compound that is sufficient to effect such treatment of a disease or recurrence of a disease when administered to a patient for treating the disease or preventing recurrence of the disease. A therapeutically effective amount can be an amount effective to prevent the occurrence of signs and / or symptoms. A "therapeutically effective amount" can vary depending on the disease and its severity in the patient to be treated as well as the age, weight, medical history, susceptibility to the disorder, and pre-existing disorders of the patient.

[0124] As used herein, "treatment" broadly refers to a process in which signs and / or symptoms are absent in a patient, in remission, or were previously present in the patient. Treatment includes preventing a disease from occurring subsequent to treatment of a patient's disease. In addition, prevention includes treating patients who may potentially develop a disease, particularly patients who are predisposed to the disease (e.g., members of a patient population, patients having risk factors, or patients at risk of developing a disease).

[0125] As used herein, with respect to a product, "recombinant" broadly refers to, for example, a cell or nucleic acid, protein, or vector, indicating that the cell or nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by alteration of a native nucleic acid or protein, or indicating that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene that is not found in the native (non-recombinant) form of the cell or expresses a native gene that is abnormally expressed, under-expressed, or not expressed at all in other forms.

[0126] As used herein, "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreacts with" or "specifically interacts or binds to" broadly refers to a binding reaction of a protein or peptide (or other epitope) that, in some embodiments, is used to determine the presence of a protein in a heterogeneous population of proteins and other biological agents. For example, under specified immunoassay conditions, a specific antibody binds to a particular protein at least two-fold greater than background (non-specific signal) and does not substantially bind to other proteins present in the sample in significant amounts. Typically, a specific or selective reaction will be at least two-fold background signal or background noise and more typically greater than about 10-fold to 100-fold background.

[0127] As used herein, "specifically hybridizable" and "complementary" generally refer to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by traditional Watson-Crick type or other non-traditional types. The free energy of binding of a nucleic acid molecule to its complementary sequence is sufficient to permit the relevant functions of the nucleic acid, such as RNAi activity. Determination of the free energy of binding of nucleic acid molecules is well known in the art. See, e.g., Turner, et al. (1987) CSH Symp.Quant.Biol. LII:123–33; Frier, et al. (1986) PNAS 83:9373–77; Turner, et al. (1987) J.Am.Chem.Soc. 109:3783–85. The percentage of complementarity indicates the percentage of 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 at least about 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" or 100% complementarity generally refers to all contiguous residues of a nucleic acid sequence hydrogen bonding with the same number of contiguous residues in a second nucleic acid sequence. "Substantial complementarity" means that, excluding regions of the polynucleotide chain that are chosen to be non-complementary, such as overhangs, the polynucleotide chain exhibits at least about 90% complementarity. Specific binding requires a degree of complementarity sufficient to avoid non-specific binding of an oligomeric compound to non-target sequences under conditions where specific binding is required (i.e., under physiological conditions in an in vivo assay or therapeutic treatment, or under the conditions under which an in vitro assay is performed). Non-target sequences typically differ by at least 5 nucleotides.

[0128] As used herein, a "sign" of a disease generally refers to any abnormality that indicates a disease and can be detected upon examination of a patient; it is an objective indication of a disease as compared to the subjective indication of symptoms of the disease.

[0129] As used herein, "solid support", "support", and "substrate" generally refer to any material that provides a solid or semi-solid structure to which another material can be attached, including but not limited to smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) and textured and porous materials.

[0130] As used herein, "subject" broadly refers to a subject suitable for treatment according to the present invention, including but not limited to avian and mammalian subjects, and preferably mammalian subjects. Mammals of the present invention include but are not limited to dogs, cats, cows, goats, horses, sheep, pigs, rodents (e.g., rats and mice), lagomorphs, 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, young adult, adult) can be treated according to the present invention. The present invention can also be practiced in 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" can be used interchangeably with "patient".

[0131] As used herein, "symptom" of a disease broadly refers to any pathological phenomenon or deviation from normal structure, function, or sensation that a patient experiences and that indicates the disease.

[0132] As used herein, "therapy", "therapeutic", "treating", or "treatment" broadly refers to treating a disease, arresting or reducing the development of the disease or its clinical symptoms, and / or alleviating the disease such that the disease or its clinical symptoms regress. Therapy encompasses prophylaxis, treatment, remediation, reduction, mitigation, and / or alleviation of a disease, the signs and / or symptoms of a disease. Therapy encompasses alleviation of the signs and / or symptoms of a patient having signs and / or symptoms of a developing disease (e.g., tumor growth, metastasis). Therapy also encompasses "prophylaxis". For purposes of therapy, the term "reduction" broadly refers to a clinically significant reduction of signs and / or symptoms. Therapy includes treating recurrent or relapsing signs and / or symptoms (e.g., tumor growth, metastasis). Therapy encompasses but is not limited to precluding the appearance of signs and / or symptoms at any time and reducing and eliminating existing signs and / or symptoms. Therapy includes treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing recurrence or relapse of signs and / or symptoms (e.g., tumor growth, metastasis).

[0133] As used herein, "variable region" or "VR" broadly refers to the domains within each pair of light and heavy chains in an antibody that are directly involved in the binding of the antibody to an antigen. Each heavy chain has a variable domain (V H ) at one end, followed by a plurality of constant domains. Each light chain has a variable domain (V L ) at one end and a constant domain at its other end; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the light chain variable domain aligns with the variable domain of the heavy chain.

[0134] As used herein, a "vector" broadly refers to a plasmid, cosmid, phagemid, phage DNA or other DNA molecule that is capable of autonomous replication in a host cell and is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences can be cut in a determinable manner without loss of the basic biological functions of the vector, and into which DNA can be inserted for its replication and cloning. The vector may also contain a marker suitable for use in the identification of cells transformed with the vector.

[0135] The techniques and procedures are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook, et al. (2001) Molec.Cloning: Lab.Manual [3rd ed.] Cold Spring Harbor Laboratory Press. Standard techniques are available for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly accomplished in the art or as described herein. The nomenclature used in the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein and the laboratory procedures and techniques of the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are available for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0136] Examples

[0137] The invention has now been generally described, and it will be more readily understood by reference to the following examples, which are included only for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0138] Example 1

[0139] NEO-201 Binding to Various Human Cancer Cell Lines

[0140] Flow cytometry analysis was used to profile NEO-201 binding in a panel of human cancer cell lines. The staining profiles are summarized in Table 1, and representative histograms from cell lines with high, medium, low, and negative staining are shown in Figures 1A - 1CShown in. Evaluation of the binding activity of NEO-201 revealed that 3 / 6 (50%) of the colon cancer cell lines and 4 / 5 (80%) of the pancreatic cancer cell lines were highly positive. When profiling non-small cell lung cancer (NSCLC) cell lines of various histological subtypes, it was determined that 3 / 5 (60%) of the adenocarcinoma cell lines reacted with NEO-201, while only 1 / 4 (25%) of the squamous cell carcinoma cell lines were found to be positive. Screening of breast cancer cell lines was also performed. Among cell lines expressing estrogen receptor (ER) or progesterone receptor (PR), 2 / 4 (50%) were positive for NEO-201 staining, whether used alone or in combination with HER2. Among HER2+ cell lines, 3 / 4 (75%) were recognized by NEO-201, whether used alone or in combination with ER or PR. However, NEO-201 staining was found at low levels on only 1 / 4 (25%) of the triple-negative breast cancer cell lines. In total, 15 / 30 (50%) of the tested tumor cell lines were recognized by NEO-201. These data indicate that NEO-201 is reactive against a wide range of in vitro cultured tumor cell lines and suggest that significant differences in antibody reactivity can occur based on tumor subtype.

[0141] Example 2

[0142] NEO-201 tissue staining is highly tumor-specific

[0143] Using tissue microarrays representing many samples of each cancer type, immunohistochemical studies were used to investigate NEO-201 reactivity from human tumor samples. As Figure 2A shown, immunoreactivity with NEO-201 7,829,678 was completely absent in normal colon, pancreas, and lung tissues but was highly positive in tumor tissues from these organs. Notably, staining was found only on tumor cells as the surrounding stromal cells were not stained ( Figure 2A ). IHC staining of the microarray samples determined that NEO-201 had high reactivity against colon cancer (72%), pancreatic cancer (80%), gastric cancer (71%), lung cancer (61%), breast cancer (55%), and uterine cancer (54%). Additionally, a significant minority of ovarian cancer (26%) samples also showed positive staining, but no staining was observed in prostate cancer tissues ( Figure 2B ). Overall, 258 / 345 (74.7%) of the sampled tumor tissues were positive for NEO-201 staining. Importantly, NEO-201 reactivity was almost completely absent in normal healthy tissues (Table 2) and in normal adjacent tissues to the tumors (except for some uterine and ovarian samples) ( Figure 2C)。However, the number of tissues in this set of uterine and ovarian tissues was limited (5 and 9 samples, respectively). In summary, these data indicate that NEO-201 recognizes tumor tissues from a variety of carcinomas and is highly tumor-specific.

[0144] Example 3

[0145] NEO-201 mediates ADCC and CDC to kill tumor cells

[0146] As a humanized IgG1 antibody, NEO-201 is theoretically capable of mediating ADCC to kill tumor cells expressing the NEO-201 antigen. To investigate this potential mechanism of action, an ADCC assay was performed on cell lines that were highly positive for NEO-201 staining, using human natural killer (NK) cells isolated from PBMCs from two different healthy donors (CFPAC-1 and ASPC-1). Treatment with NEO-201 was observed to enhance the killing of CFPAC-1 and ASPC-1 to levels 2 to 6 times greater than the killing of tumor cells treated with control IgG1 ( Figure 3A ). A titration experiment was also performed, and it was revealed that NEO-201 retained the ability to significantly induce ADCC at doses as low as 0.1 μg / mL ( Figure 3B ).

[0147] CDC is a complex cascade of proteolytic cleavage that culminates in the activation of the membrane attack complex, which lyses antibody-bound target cells. Some human IgG1 antibodies are capable of mediating CDC; however, CDC is dependent on the antigen specificity of the antibody. The CDC assay revealed that NEO-201 induced complement-mediated lysis of ASPC-1 cells in a manner dependent on both the mAb dose and the incubation time ( Figure 3C ). In summary, these data indicate that NEO-201 effectively engages innate immune effector mechanisms to specifically lyse antibody-bound tumor cells in vitro.

[0148] Example 4

[0149] NEO-201 alone and in combination with human PBMC effector cells reduces the growth of tumor xenografts

[0150] To determine the potential anti-tumor efficacy of NEO-201, CFPAC-1 cells were grown as tumor xenografts in immunocompromised NU / NU nude mice. These cells were selected based on their high NEO-201 antigen expression levels and high sensitivity to NEO-201-mediated ADCC. Once the CFPAC-1 tumors grew to approximately 100 mm 3For the size, saline, 250 μg of human IgG1, 100 μg of NEO-201, or 250 μg of NEO-201 were injected three times into tumor-bearing mice, followed by three injections of 1.0×10 7 IL-2-activated (200 U / mL) human PBMCs to serve as effector cells mediating ADCC. As Figure 4A shown, compared to the saline + PBMC or human IgG + PBMC control groups, NEO-201 + PBMC induced a significant reduction in tumor growth at two dose levels. While no mice from the control groups were tumor-free at day 36, 1 out of 10 (10%) and 4 out of 10 (40%) mice remaining from the NEO-201 100 μg + PBMC and NEO-201 250 μg + PBMC groups, respectively, had no palpable tumors ( Figure 4B ). Additionally, NEO-201 was administered to another group of mice without the addition of human PBMCs, and a significant reduction in tumor growth relative to the control group was observed ( Figure 4A , 4C). Importantly, monitoring the body weights of tumor-bearing mice revealed no weight loss in any treatment group ( Figure 4D ). Overall, these results indicate that NEO-201 is capable of significantly reducing tumor growth through both ADCC and non-ADCC mechanisms such as CDC without inducing significant toxicity in mice.

[0151] Example 5

[0152] NEO-201 localizes to the xenograft tumor site

[0153] Biodistribution studies were performed in female and male NU / NU nude mice with established CFPAC-1 xenograft tumors using radiolabeled NEO-201. These mice were injected intravenously with the radiolabeled antibody and then blood, organs, and tumors were harvested at different time points post-injection for analysis. Low levels of radioactivity were found in the pancreas, spleen, kidney, liver, stomach, intestine, and lung in male and female mice at all time points ( Figure 5A , 5B). However, the standardized uptake of radioactivity was significantly higher in tumors compared to all other tissues at all time points, and by day 7, the tumor radioactivity had increased stepwise to a level 20 - 30 times higher than that of the blood ( Figure 5A , 5B). Quantitatively similar results were obtained for 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.

[0154] Example 6

[0155] Pharmacokinetics and toxicity evaluation of NEO-201 in non-human primates

[0156] A single-dose study was conducted in purpose-bred cynomolgus monkeys to determine the pharmacokinetics of NEO-201 and associated toxicities. Cynomolgus monkeys were selected because this species is phylogenetically and physiologically closely related to humans and is a species commonly used in non-clinical toxicity evaluations. Female and male animals received a single intravenous infusion of NEO-201 diluted in saline at dose levels of 5 mg / kg, 20 mg / kg, and 49 mg / kg, which were the highest doses achievable per infusion volume. Blood samples were drawn from all animals at different time points before and after injection up to 14 days, and NEO-201 levels in serum preparations were evaluated by ELISA. As depicted in Table 3, quantifiable and dose-dependent serum concentrations of NEO-201 were observed at the last collection time point (14 days post-dose). As expected for intravenous administration, for the majority of animals from all groups, the Tmax value reached a peak by 10 minutes (10 / 12, 83%), except for one male and one female animal from the 5 mg / kg group. Within the dose range evaluated, peak (Cmax) exposure was proportional to the dose; total (AUC) exposure was greater than dose-proportional at the lowest dose and was approximately proportional from 20 mg / kg to 49 mg / kg. The difference in exposure at the lowest dose was attributed to a roughly 2-fold greater mean clearance (CL) and a smaller volume of distribution (Vz). At the higher doses, the mean half-life (HL) was 167 (20 mg / kg) or 170 (49 mg / kg) hours, approximately 3.7-fold greater than at the 5 mg / kg dose (46.2 hours). No gender differences were observed.

[0157] Observations and examinations used to determine toxicity during the 14-day study included 1) regular clinical evaluations; 2) measurement of food consumption and body weight; and 3) urine and blood evaluations, including urinalysis, hematology, coagulation tests, serum chemistry, and toxicokinetics. As Figure 6A shown, no dose-level group experienced a body weight change > 3% compared to their pre-injection weight, and no individual monkey experienced a change > 7%. Food consumption remained unchanged for all animals, but two animals in the 5 mg / kg dose group had low consumption only on day 11. There were no significant changes from baseline (prior to NEO-20 injection) to day 15 in any of serum chemistry, urinalysis, or coagulation tests (see Materials and Methods for details). The main laboratory change in the blood cell count was a decrease in the neutrophil count relative to baseline ( Figure 6B ). The decrease had a variable magnitude, ranging from mild to significant, and a clear dose-response was not evident. For the majority of animals, this was a transient finding as improvement was generally noted by day 8 ( Figure 6B)。By day 15, for the 5 mg / kg group or the 20 mg / kg and 49 mg / kg groups, almost complete or partial recovery of neutrophil counts was observed ( Figure 6B )。The recovery of neutrophil counts by day 15 was reflected in the statistical comparison with the 0 mg / kg animals, which was significantly different at day 2 for all three dose levels (p < 0.05), but not significantly different at days 8 and 15 for two of the three dose groups (p > 0.05)( Figure 6C )。

[0158] Example 7

[0159] Materials and Methods

[0160] Cell Lines and Cultures

[0161] The following human cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA): colon (COLO205, HT-29, LS174T, SW1116, SW1463, SW480, SW620), pancreas (ASPC-1, CFPAC-1, PANC-1), breast (AU-565, BT-474, BT-549, HCC1500, HCC1937, HCC38, MDA-MB-231, MDA-MB-468, SK-BR-3, T-47D, ZR-75-1), and lung (CALU-1, H1703, H226, H441, H520, H522, H596, HCC4006, HCC827, SK-LU-1). All cell cultures were maintained in RPMI 1640, DMEM, or IMDM medium (Corning, Corning, NY) as specified by the supplier for propagation and maintenance. The medium was supplemented with 10% heat-inactivated HyClone fetal bovine serum sourced from the USA (GE Healthcare Life Sciences, Issaquah, WA, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin (Corning Life Science, Manassas, VA, USA). PBMCs from healthy volunteer donors were obtained from the blood bank of the National Institutes of Health Clinical Center (NCT00001846) with appropriate institutional review board approval and informed consent.

[0162] Generation of Humanized NEO-201 Monoclonal Antibody

[0163] The Hollinshead colon cancer-specific vaccine was used as an immunogenic material to generate monoclonal antibodies in mice. Methods for preparing tumor-associated proteins and peptides have been previously described (Hollinshead, US4810781, 1989). Briefly, cancer tissues were minced and used to generate a single-cell suspension, which was then subjected to hypotonic saline membrane extraction, a series of centrifugation steps, and subsequent low-frequency sonication. The resulting membrane-extracted proteins were fractionated on Sephadex G-200 resin or by electrophoresis, then concentrated and quantified (Hollinshead et al., 1970; Hollinshead et al., 1972; Hollinshead et al., 1985). The TAA preparation was blended with complete Freund's adjuvant and injected subcutaneously in BALB / c mice. This was followed by three booster injections in incomplete Freund's adjuvant at 2- to 3-week intervals. The antibody response of mouse sera against the immunizing antigen was tested by ELISA, and mice with an effective response were used to generate immortalized hybridoma cells by fusing mouse B cells from the spleen with the SP2 / 0-Ag14 myeloma cell line and selecting cells that grew and produced mouse immunoglobulin (IgG). From these mouse IgGs, the murine 16C3 clone (m16C3) was selected based on reactivity with colon tumor cell membrane extracts derived from LS174T or HT-29 cells as determined by ELISA. The cDNAs encoding the heavy and light chain IgG1 were determined from RNA isolated from the hybridoma clone 16C3E12 and shown to be unique (Bristol & Kantor, US7829678, 2010). The m16C3 protein sequence was humanized to h16C3 and designated NEO-201. Humanization was performed in silico by replacing the murine sequences outside the complementarity-determining regions (CDRs) of the Fab regions of the heavy and light chain proteins with human Fab sequences and 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 used in other humanized approved mAb products. The amino acid sequences were back-translated into DNA, which was optimized for protein expression in CHO cells. Then the DNA of the heavy and light chains of h16C3 was chemically synthesized, cloned into a mammalian expression plasmid, and transfected into mammalian cell lines (HEK293T and CHO). Several stable CHO cell lines expressing recombinant h16C3 were obtained and banked. The purified recombinant h16C3 was retested in the study, which confirmed that the humanized 16C3 antibody had similar characteristics to the original m16C3 antibody (Bristol and Kantor, US7829678, 2010).

[0164] The NEO-201 antibody sequence used in these examples is contained in the following figure:

[0165]

[0166] In each sequence, the boundaries between the leader sequence, variable region, and constant region are delimited by a forward slash (" / "), and the CDR sequences are shown as bold, underlined text. The antibody sequences used contain the indicated variable and constant regions. These include the heavy chain CDR1 of SEQ ID NO:32, the heavy chain CDR2 of SEQ ID NO:33, the heavy chain CDR3 of SEQ ID NO:34, the light chain CDR1 of SEQ ID NO:35, the light chain CDR2 of SEQ ID NO:36, and the light chain CDR3 of SEQ ID NO:37.

[0167] Flow cytometry

[0168] The binding of NEO-201 to human cancer cell lines was analyzed by flow cytometry. Cells (1.0×10 6 cells) were incubated with LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) in 1X phosphate-buffered saline (PBS) at 4°C for 30 minutes per 1 μL tested to complete live cell versus dead cell discrimination. The 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) at 4°C for 30 minutes. After staining, the cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis of cell fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). A staining value >10% positive was considered positive for NEO-20 expression. Positive cell lines were ranked according to their quantified expression levels (positive % × MFI) and then classified into low (<200), medium (200 - 1000), and high (<1000) expression groups.

[0169] Immunohistochemistry (IHC)

[0170] Tissue microarrays of colon samples (CO808, CO951) were obtained from USBiomax (Rockville, MD), and AccuMax tissue microarrays of colon (A303(I)), pancreas (A207(II), A307), stomach (A209), lung (A206(V), A306), breast (A202(VI), A712), uterus (A212), ovary (A212, A213(II)), prostate (A302(IV)) and various normal (A103(VII)) samples were obtained from Accurate Chemical and Scientific Corporation (Westbury, NY). NEO-201 was biotinylated using a biotin-protein labeling kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. The slides were baked at 60 °C for 20 minutes, deparaffinized with xylene, and rehydrated with a series of gradient ethanol. Then the slides were peroxidase-blocked for 2 minutes using Peroxidazed I solution (Biocare Medical, Concord, CA), avidin-blocked for 10 minutes using avidin solution (Biocare Medical, Concord, CA), biotin-blocked for 10 minutes using biotin solution (Biocare Medica, Concord, CA), and protein-blocked for 10 minutes using CAS-block tissue chemistry reagent (Thermo Fisher Scientific, Waltham, MA). Then the slides were incubated with either 10 μg / mL biotinylated human IgG1κ (Ancell, Bayport, MN) or biotinylated NEO-201 diluted in 1X PBS with a negative control at room temperature for 2 hours. Detection was performed for 30 minutes with a 1:300 Dako streptavidin-HRP conjugate (Agilent Technologies, Santa Clara, CA), incubated with DAB peroxidase substrate (Thermo Fisher Scientific, Waltham, MA) for 1 - 3 minutes, and counterstained with hematoxylin. The cellular staining intensity of each microarray tissue spot was evaluated by light microscopy using the following scale: 0 (negative), ± (ambiguous), 1+ (weak), 2+ (moderate), 3+ (strong). If the tissue spot contained cells with staining intensity ≥ +1, the tissue spot was recorded as positive.

[0171] Antibody-dependent cell cytotoxicity (ADCC) assay

[0172] ADCC assays were performed using a modification of the previously described procedure (Boyerinas et al., 2015). NK cells were negatively selected from normal human donor PBMCs using the EasySep Human NK Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's protocol. The purified NK cells were incubated overnight in RPMI-1640 medium supplemented with L-glutamine, 10% FBS, and antibiotics. On the day of the assay, target cells (CFPAC-1, ASPC-1) were labeled with 10 μM calcein AM cell-permeable dye (Termo Fisher Scientific, Waltham, MA, USA) for 30 minutes and then seeded in triplicate at 3.0×10 3 cells / well into black-walled flat-bottom 96-well culture plates (#655090 Greiner bio-one, Germany). The tumor cells were then treated with 10 μg / mL of human IgG1 isotype control antibody (Thermo Fisher Scientific, Waltham, MA, USA) or NEO-201 (unless otherwise stated), and then NK cells were added at effector-to-target (E:T) ratios of 12.5:1 and 25:1. After incubation at 37 °C for 4 hours, 10 μg / mL propidium iodide (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well, and the plates were imaged and analyzed using a Celigo imager (Nexcelom Bioscence LLC, Lawrence, MA, USA). Live target cells (calcein AM+ / PI-) in each well were counted, and specific ADCC lysis was calculated as follows: % specific lysis = 100 – [(average live target count 实验值 / average live target count 对照 ) x 100].

[0173] Complement-dependent cytotoxicity (CDC) assay

[0174] CDC assays were performed using a modification of the previously described procedure (Konishi et al., 2008). ASPC-1 target cells were labeled with calcein AM as described above and seeded at 5.0×10 3Cells per well were seeded into black-bottom 96-well plates. The cells were then treated with 0.5 or 5.0 μg / mL NEO-201 at 37 °C for 15 minutes to condition the cells, and then purified rabbit complement (MP Biomedicals, Santa Ana, CA) was added to each well at a 1:8 dilution. After incubation at 37 °C for 30, 60, or 120 minutes, propidium iodide was added, the plates were imaged and analyzed using a Celigo imaging cytometer, and specific lysis was calculated as described above for ADCC activity.

[0175] Xenograft antitumor assay

[0176] Tumors were established in 6-week-old female athymic NU / NU nude mice (Charles River Laboratories International, Wilmington, MA) by subcutaneous implantation of a suspension of cultured tumor cells in 1X PBS into the right flank of the mice. Once the tumors reached approximately 100 mm 3 in size, the mice were sorted by tumor volume and randomly divided into 5 groups (n = 10 animals). On days 13, 17, and 20 post-implantation, the mice were injected intraperitoneally with either vehicle alone (saline solution), human IgG1 (250 μg), or NEO-201 (100 and 250 μg). The mice also received intraperitoneal injections of approximately 1.0 x 10 7 human PBMCs activated with IL-2 (treated overnight at 200 U / mL in culture) as a source of immune effector cells on days 14, 18, and 21. 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 according to the formula (width 2 × length) / 2 = mm 3 , where the width is the shorter of the two measurements. The mice were also weighed once a week as a measure of overall health. Mice with tumor volumes > 2000 mm 3 were euthanized according to IACUC guidelines.

[0177] Biodistribution analysis

[0178] 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 subcutaneously in the flank with 4.0 × 10 6A suspension of 200 μL of CFPAC-1 cells in 1X PBS. On day 14 after transplantation, mice were injected intravenously with 20 μCi of 125 I-labeled NEO-201, and then necropsied 1, 2, 4, or 7 days later. Blood, tumor tissue, and visceral organs (lungs, kidneys, liver, spleen, pancreas, intestine, and stomach) were harvested 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 the tissue cpm values were normalized relative to the blood cpm values.

[0179] Single-dose toxicity study in cynomolgus monkeys

[0180] A single-dose toxicity study was conducted in purpose-bred cynomolgus monkeys to test the pharmacokinetics and toxicity of NEO-201 after a single dose of NEO-201. The duration of the study was 15 days from dose administration, with an additional 14 days of quarantine before dose administration to acclimatize the monkeys to the study facility. NEO-201 diluted in saline solution was administered to eight male and eight female animals (2 animals / sex / group) at dose levels of 0 mg / kg, 5 mg / kg, 20 mg / kg, and 49 mg / kg by slow intravenous infusion (approximately 30 minutes ± 5 minutes infusion) using an infusion pump and a plastic disposable syringe with a catheter extension tube. The dose levels were the highest concentrations achievable with the antibody. Blood samples were drawn from all animals receiving NEO-201 at the following time points: pre-dose, 10 minutes, 1, 2, 4, 6, 24, 48, 72, 96, 168, and 336 hours. Serum was prepared from blood samples for pharmacokinetic and toxicological analysis. Whole blood was used for cell analysis. NEO-201 levels in serum were measured by ELISA using a human therapeutic IgG1 ELISA kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions.

[0181] Laboratory tests included hematology and coagulation (baseline (BL), Day 2, Day 8, Day 15): CBC and differential, activated partial thromboplastin time, fibrinogen, and prothrombin time; serum chemistry (BL, Day 2, Day 8, Day 15): albumin, alkaline phosphatase, ALT, AST, total bilirubin, calcium, total cholesterol, creatine kinase, creatinine, glucose, inorganic phosphorus, total protein, triglycerides, sodium, potassium, chloride, globulin, albumin / globulin ratio, BUN; urinalysis (BL, Day 15): color, clarity, glucose, ketones, occult blood, protein, bilirubin, nitrite, pH, urobilinogen, leukocytes, volume, specific gravity; bioanalysis (using ELISA) - (BL, 10 minutes, 1, 2, 4, 6 hours, 24, 48, 72, 96, 168, and 336 hours) from Groups 2 to 4 using Phoenix WinNonlin version 6.1 software (Certara USA, Princeton, NJ). Animal body weight measurements were recorded (BL, 7, and 14), and neutrophil counts were evaluated (BL, Day 2, Day 8, Day 15).

[0182] Statistical analysis

[0183] Data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA). Comparisons between two groups were performed by the T-test, and p < 0.05 was considered statistically significant. Graphs depict the mean ± SD from representative experiments performed in triplicate.

[0184] Example 8

[0185] ALT-803 enhances ADCC mediated by NEO-201

[0186] ALT-803 is a novel IL-15 superagonist complex consisting of an IL-15 mutant (IL-15N72D) conjugated to an IL-15 receptor α / IgG1 Fc fusion protein. This example tests the ability of ALT-803 to modulate ADCC mediated by NEO-201.

[0187] Methods

[0188] NK cells were isolated from normal donors and treated with different concentrations of ALT-803 for 48 hours before being used as effector cells, and a human cancer cell line expressing the NEO-201 antigen was used as the target in an in vitro non-radioactive ADCC assay. The ability of ALT-803 to affect the phenotype of NK cells and regulate NK cell gene expression was evaluated by flow cytometry and by using Nanostring analysis, respectively.

[0189] Results

[0190] Treatment with ALT-803 significantly enhanced the ADCC activity mediated by NEO-201 against NEO-201-positive cancer cells (Figure 8, Figure 11). The effect of ALT-803 was dose-dependent and achieved statistical significance at all doses tested compared to vehicle control treatment. Treatment of NK cells with ALT-803 enhanced the ADCC activity also from donors with minimal ADCC activity and reduced the effective dose of NEO-201 required for the initial ADCC response compared to untreated NK cells (Figure 12). In addition, the ADCC activity could be blocked by using anti-CD16 and anti-TIM3 blocking antibodies (Figure 12).

[0191] Phenotypic analysis of NK cells treated with 25 ng / ml of ALT-803 for 48 hours showed that ALT-803 enhanced the expression of TIM3 and NKG2D and the mean fluorescence intensity (MFI) of granzyme B and CD107a in CD16 / CD56-positive NK cells (Figure 9).

[0192] Nanostring analysis of human NK cells treated with different concentrations of ALT-803 for 48 hours showed that ALT-803 was able to regulate the mRNA expression of 62 genes (a 1.6 log2 fold change compared to vehicle control was considered significant).

[0193] ALT-803 treatment upregulated the mRNA expression of 43 genes (including NK activating receptors, factors involved in NK cell activity, cytokines and their receptors), and downregulated the mRNA expression of 19 genes (including NK inhibitory receptors and factors involved in apoptosis activation).

[0194] Thus, ALT-803 enhanced the ADCC activity mediated by NEO-201 against human cancer cells. The enhancement of ADCC activity may be partially due to the increased expression of TIM3, NKG2D, granzyme B- and CD107a-positive NK cells, and due to the regulation of transcripts involved in NK activation and cytotoxicity.

[0195] In summary, treatment of NK cells isolated from normal donors with ALT-803 enhanced NEO-201-mediated ADCC activity. Phenotypic analysis of ALT-803-treated NK cells isolated from normal donors demonstrated that ALT-803 enhanced the expression of TIM-3 and NKG2D on CD16 / CD56-positive NK cells. Treatment of normal NK cells with ALT-803 also increased the MFI of granzyme B in CD16 / CD56-positive NK cells. Treatment of normal NK cells with ALT-803 also increased the MFI of CD107a in CD16 / CD56-positive NK cells in one of the two donors tested. TIM-3 is an inducible human NK cell receptor that enhances interferon-γ production. It is also a maturation marker. The enhancement of NEO-201-mediated ADCC activity 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 enhanced NEO-201-mediated ADCC activity at lower concentrations. When NK cells were treated with ALT-803, lower concentrations of Mab could be used to mediate ADCC activity and equivalent levels of cytotoxicity could be produced compared to NK cells using higher concentrations of NEO-201 without ALT-803 treatment. This result suggests that smaller doses of Mab could be combined with ALT-803 for use in clinical trials for the treatment of cancer.

[0196] Example 9

[0197] NEO-201 enhances NK cell-dependent killing of tumor cells by blocking the inhibitory CEACAM5 / CEACAM1 immune checkpoint pathway.

[0198] Immunotherapy using checkpoint-blocking antibodies targeting effector cell inhibitory receptors such as PD-1 and CTLA-4 has elicited some remarkable 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, and it can inhibit T cell function similar to PD-1 and CTLA-4. CEACAM1 is also an effective inhibitor of natural killer (NK) cell function; binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling by NKG2D, which prevents NK cell lysis and allows tumor cells to escape NK killing.

[0199] NEO-201 binds to members of the CEACAM family and can activate innate immune mechanisms such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to kill tumor cells. This study was designed to determine whether NEO-201 blocks the CEACAM1 inhibitory pathway to restore the anti-tumor functionality of NK cells.

[0200] Methods

[0201] In vitro assays using human tumor cell lines were performed to identify CEACAM family members bound by NEO-201. Functional assays were performed to evaluate the ability of NEO-201 to enhance the in vitro killing of tumor cells by the NK cell line NK-92, which expresses CEACAM1 and lacks CD16, and its ability to mediate ADCC.

[0202] Cytotoxicity assays were performed using a modification of a previously described procedure (David et al., 2017). Briefly, target cells derived from pancreatic (ASPC-1, BxPC-3, CFPAC-1) and colon (LS174T) carcinomas were labeled with 10 μM calcein AM cell-permeable dye (Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes and then seeded in triplicate at 3.0 × 10 3 cells / well into black-walled flat-bottom 96-well culture plates. The 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 then 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 incubation at 37 °C for 16 hours, propidium iodide (PI; Thermo Fisher Scientific, Waltham, MA, USA) was added to each well at a final concentration of 1.67 μg / mL, and the plates were centrifuged, imaged using a Celigo imaging cytometer (Nexcelom Bioscence LLC, Lawrence, MA, USA), and analyzed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). Live target cells (calcein AM+ / PI-) in each well were counted, and specific lysis was calculated as follows: Specific lysis % = 100 – [(mean live target count 实验值 / mean live target count 对照 ) x 100].

[0203] Results

[0204] It was found that NEO-201 reacted with different variants of CEACAM5 and CEACAM6, but not with CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell line cells expressed different levels of the native forms of CEACAM5 / 6 versus the NEO-201-reactive variant forms of these molecules. Functionally, NEO-201 treatment enhanced the cytolytic activity of NK-92 cells against NEO-201+ tumor cells expressing CEACAM5, but not against NEO-201+ cells expressing only CEACAM6 (Figure 13).

[0205] Conclusion

[0206] NEO-201 reacts with tumor-associated variants of CEACAM5 / 6 and is able to block the interaction between tumor cell CEACAM5 and NK cell CEACAM1 to reverse CEACAM1-dependent inhibition of NK cell cytotoxicity.

[0207] Abbreviations

[0208] Antibody-dependent cell cytotoxicity (ADCC), area under the plasma concentration-time curve from time 0 to infinity (AUCinf), area under the dose-normalized plasma concentration-time curve from time 0 to infinity (AUCinf / D), baseline (BL), complement-dependent cell cytotoxicity (CDC), clearance (CL), observed maximum plasma concentration (Cmax), dose-normalized measured maximum plasma concentration (Cmax / D), estrogen receptor (ER), half-life (HL), immunohistochemistry (IHC), natural killer cell (NK), non-small cell lung cancer (NSCLC), peripheral blood mononuclear cell (PBMC), progesterone receptor (PR), tumor-associated antigen (TAA), time to observed maximum plasma concentration (Tmax), volume of distribution (Vz).

[0209] References

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[0259] Table 1: Flow cytometry analysis of the binding of NEO-201 to cultured tumor cell lines derived from various types of solid tumors. The percentage of positive cells and the mean fluorescence intensity (MFI) values are detailed for each cell line. NEO-201-positive cell lines are shown in bold. NEO-201 positivity was defined as positive % > 10%.

[0260]

[0261]

[0262] Table 2: IHC profiles of NEO-201 staining in normal human microarray tissues.

[0263]

[0264]

[0265] Table 3: Pharmacokinetic results of single-dose NEO-201 administration in cynomolgus monkeys. Eight male and eight female animals (2 animals / sex / group) were intravenously injected with 0 mg / kg (saline solution) or 5 mg / kg, 20 mg / kg, or 49 mg / kg of NEO-201. Blood samples were drawn from all animals receiving NEO-201 at different time points (before dose, 10 minutes, 1, 2, 4, 6, 24, 48, 72, 96, 168, and 336 hours after dose), and pharmacokinetic measurements were obtained from serum preparations by ELISA. The values in the table represent the mean from 2 animals / sex / group (M, F) or from all 4 animals (all).

[0266] Abbreviations: Area Under the Plasma Concentration-Time Curve from Time 0 to Infinity (AUCinf); Dose-Normalized Area Under the Plasma Concentration-Time Curve from Time 0 to Infinity (AUCinffD); Clearance (CL); Observed Maximum Plasma Concentration (Cmax); Dose-Normalized Measured Maximum Plasma Concentration (Cmax / D); Half-Life (HL); Time of Observed Maximum Plasma Concentration (Tmax); Volume of Distribution (Vz).

[0267] Sequence Listing <110> Precision Biologics, Inc. Philip M. Arlen Kwong Y. Tsang <120> Monoclonal Antibody NEO-201 for the Treatment of Human Cancers <130> 43282.4402 <140> TBD <141> Filed As A Continuation <150> 62 / 592,778 <151> 2017-11-30 <150> 62 / 581,380 <151> 2017-11-03 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> House Mouse (Mus musculus) <400> 1 Gly Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn 1 5 10 <210> 2 <211> 7 <212> PRT <213> House Mouse (Mus musculus) <400> 2 Gly Ala Ser Asn Leu Ala Asp 1 5 <210> 3 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 3 Gln Asn Val Leu Ser Ser Pro Tyr Thr 1 5 <210> 4 <211> 11 <212> PRT <213> Human (Homo sapiens) <400> 4 Gln Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> Human (Homo sapiens) <400> 5 Gly Ala Ser Asn Leu Ala Thr 1 5 <210> 6 <211> 9 <212> PRT <213> Human (Homo sapiens) <400> 6 Gln Gln Val Leu Ser Ser Pro Tyr Thr 1 5 <210> 7 <211> 10 <212> PRT <213> House mouse (Mus musculus) <400> 7 Gly Tyr Thr Phe Thr Asp Tyr Ala Met His 1 5 10 <210> 8 <211> 17 <212> PRT <213> House mouse (Mus musculus) <400> 8 Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe Lys 1 5 10 15 Gly <210> 9 <211> 12 <212> PRT <213> Mus musculus <400> 9 Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr 1 5 10 <210> 10 <211> 17 <212> PRT <213> Homo sapiens <400> 10 Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe Gln 1 5 10 15 Gly <210> 11 <211> 12 <212> PRT <213> Homo sapiens <400> 11 Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr 1 5 10 <210> 12 <211> 965 <212> DNA <213> Mus musculus <400> 12 gcggggcagc ctcacacaga acacacacag atatgggtgt acccactcag ctcctgttgc 60 tgtggcttac agtcgtagtt gtcagatgtg acatccagat gactcagtct ccagcttcac 120 tgtctgcatc tgtgggagaa actgtcacca tcacatgtgg agcaagtgag aatatttacg 180 gtgctttaaa ttggtatcag cggaaacagg gaaaatctcc tcagctcctg atttatggcg 240 caagtaattt ggcagatggc atgtcatcga ggttcagtgg cagtggatct ggtagacagt 300 attctctcaa gatcagtagc ctgcatcctg acgatgttgc aacgtattac tgtcaaaatg 360 tattaagtag tccgtacacg ttcggagggg ggaccaagct ggaaataaaa cgggctgatg 420 ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct ggaggtgcct 480 cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag tggaagattg 540 atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac agcaaagaca 600 gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa cgacataaca 660 gctatacctg tgaggccact cacaagacac caacttcacc cattgtcaag agcttcaaca 720 ggaatgagtg ttagagacaa aggtcctgag acgccaccac cagctcccca gctccatcct 780 atcttccctt ctaaggtctt ggaggcttcc ccacaagcga cctaccactg ttgcggtgct 840 ccaaacctcc tccccacctc cttctcctcc tcctcccttt ccttggcttt tatcatgcta 900 atatttgcag aaaatattca ataaagtgag tctttgcaca aaaaaaaaaa aaaaaaaaaa 960 aaaaa 965 <210> 13 <211> 1575 <212> DNA <213> Mus musculus <400> 13 acgcgggaca cagtagtctc tacagtcaca ggagtacaca ggacattgcc atgggttgga 60 gctgtatcat cttctttctg gtagcaacag ctacaggtgt gcactcccag gtccagctgc 120 agcagtctgg gcctgaggtg gtgaggcctg gggtctcagt gaagatttcc tgcaagggtt 180 ccggctacac attcactgat tatgctatgc actgggtgaa gcagagtcat gcaaagagtc 240 tcgagtggat tggacttatt agtacttaca gtggtgatac aaagtacaac cagaacttta 300 agggcaaggc cacaatgact gtagacaaat cctccaacac agcctatatg gaacttgcca 360 gattgacatc tgaggattct gccatctatt actgtgcaag aggggattat tccggtagta 420 ggtactggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca gccaaaacga 480 cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac tccatggtga 540 ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc tggaactctg 600 gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac ctctacactc 660 tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc acctgcaacg 720 ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg gattgtggtt 780 gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc cccccaaagc 840 ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg gtagacatca 900 gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag gtgcacacag 960 ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc agtgaacttc 1020 ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc aacagtgcag 1080 ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg aaggctccac 1140 aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc agtctgacct 1200 gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg aatgggcagc 1260 cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct tacttcgtct 1320 acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc acctgctctg 1380 tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac tctcctggta 1440 aatgatccca gtgtccttgg agccctctgg ccctacagga ctttgacacc tacctccacc 1500 cctccctgta taaataaagc acccagcact gcctcgggac cctgcataaa aaaaaaaaaa 1560 aaaaaaaaaa aaaaa 1575 <210> 14 <211> 107 <212> PRT <213> Mus musculus <400> 14 Leu Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr 1 5 10 15 Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn 20 25 30 Trp Tyr Gln Arg Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile Tyr Gly 35 40 45 Ala Ser Asn Leu Ala Asp Gly Met Ser Ser Arg Phe Ser Gly Ser Gly 50 55 60 Ser Gly Arg Gln Tyr Ser Leu Lys Ile Ser Ser Leu His Pro Asp Asp 65 70 75 80 Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ser Pro Tyr Thr Phe 85 90 95 Gly Gly Gly Thr Lys Leu Glu Ile Lys Lys Gly 100 105 <210> 15 <211> 116 <212> PRT <213> Mus musculus <400> 15 Leu Glu Glu Ser Gly Pro Glu Val Val Arg Pro Gly Val Ser Val Lys 1 5 10 15 Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala Met His 20 25 30 Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile Gly Leu Ile 35 40 45 Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe Lys Gly Lys 50 55 60 Ala Thr Met Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg Gly 85 90 95 Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Arg 115 <210> 16 <211> 107 <212> PRT <213> Mus musculus <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Asp Gly Met Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Ser Leu Lys Ile Ser Ser Leu His Pro 65 70 75 80 Asp Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Asp Gly Met Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 18 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Asp Gly Met Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Thr Gly Met Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Thr Gly Met Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Val Leu Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 21 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Asn Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Asp Gly Met Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 22 <211> 121 <212> PRT <213> Mus musculus <400> 22 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 23 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized Antibody Sequence <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 24 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 24 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 25 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 25 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 26 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 26 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Val His Ala Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 28 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* heavy chain) <220> <221> MISC_FEATURE <222> (1)..(19) <223> Leader sequence <220> <221> MISC_FEATURE <222> (20)..(140) <223> Variable region <220> <221> MISC_FEATURE <222> (141)..(470) <223> Constant region <220> <221> MISC_FEATURE <222> (45)..(54) <223> CDR1 <220> <221> MISC_FEATURE <222> (70)..(85) <223> CDR2 <220> <221> MISC_FEATURE <222> (118)..(129) <223> CDR3 <400> 28 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu 50 55 60 Glu Trp Met Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn 65 70 75 80 Gln Asn Phe Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ala Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> 29 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* light chain) <220> <221> MISC_FEATURE <222> (1)..(19) <223> Leader sequence <220> <221> MISC_FEATURE <222> (20)..(127) <223> Variable region <220> <221> MISC_FEATURE <222> (128)..(233) <223> Constant region <220> <221> MISC_FEATURE <222> (43)..(53) <223> CDR1 <220> <221> MISC_FEATURE <222> (69)..(75) <223> CDR2 <220> <221> MISC_FEATURE <222> (108)..(116) <223> CDR3 <400> 29 Met Gly Val Pro Thr Gln Leu Leu Leu Leu Trp Leu Thr Val Val Val 1 5 10 15 Val Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile 35 40 45 Tyr Gly Ala Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys 50 55 60 Leu Leu Ile Tyr Gly Ala Ser Asn Leu Ala Thr Gly Met Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser 85 90 95 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Val Leu Ser 100 105 110 Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 30 <211> 17 <212> PRT <213> Homo sapiens <400> 30 Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Ser Gln Lys Phe Gln 1 5 10 15 Gly <210> 31 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <400> 31 Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 32 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* heavy chain CDR1) <400> 32 Gly Tyr Thr Phe Thr Asp Tyr Ala Met His 1 5 10 <210> 33 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* heavy chain CDR2) <400> 33 Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe Gln Gly 1 5 10 15 <210> 34 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* heavy chain CDR3) <400> 34 Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr 1 5 10 <210> 35 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* light chain CDR1) <400> 35 Gln Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn 1 5 10 <210> 36 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* light chain CDR2) <400> 36 Gly Ala Ser Asn Leu Ala Thr 1 5 <210> 37 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* light chain CDR3) <400> 37 Gln Gln Val Leu Ser Ser Pro Tyr Thr 1 5 <210> 38 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* heavy chain variable) <220> <221> MISC_FEATURE <222> (26)..(35) <223> CDR1 <220> <221> MISC_FEATURE <222> (51)..(66) <223> CDR2 <220> <221> MISC_FEATURE <222> (99)..(110) <223> CDR3 <400> 38 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence (h16C3-Abb* light chain) <220> <221> MISC_FEATURE <222> (24)..(34) <223> CDR1 <220> <221> MISC_FEATURE <222> (50)..(56) <223> CDR2 <220> <221> MISC_FEATURE <222> (89)..(97) <223> CDR3 <400> 39 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Thr Gly Met Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Val Leu Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105

Claims

1. Use of a combination of an antibody and an IL-15 superagonist for the preparation of a medicament for killing cancer cells in a patient suffering from a cancer tumor, wherein the combined use of the antibody and the IL-15 superagonist synergistically enhances or stimulates CDC-mediated killing of cancer cells; wherein the antibody comprises: (i) a heavy chain CDR1 of SEQ ID NO: 32, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 34, and a light chain CDR1 of SEQ ID NO: 35, a light chain CDR2 of SEQ ID NO: 36, and a light chain CDR3 of SEQ ID NO: 37, and (ii) a human IgG1 constant region; wherein the IL-15 superagonist comprises a complex consisting of an IL-15 mutant IL-15N72D bound to an IL-15 receptor α / IgG1 Fc fusion protein; wherein the cancer tumor is selected from the group consisting of: colon cancer, pancreatic cancer, ovarian cancer, gastric cancer, lung cancer, breast cancer, and uterine cancer, and the cancer cells express the antigen to which the antibody binds.

2. The use according to claim 1, wherein the IL-15 superagonist comprises ALT-803.

3. The use according to claim 1, wherein the effective dose of the antibody is reduced compared to the use of the antibody in the absence of the IL-15 superagonist.

4. The use according to any one of claims 1-3, wherein the cancer tumor comprises colon cancer.

5. The use according to any one of claims 1-3, wherein the cancer tumor comprises pancreatic cancer.

6. The use according to any one of claims 1-3, wherein the cancer tumor comprises ovarian cancer.

7. The use according to any one of claims 1-3, wherein the cancer tumor comprises gastric cancer.

8. The use according to any one of claims 1-3, wherein the cancer tumor comprises lung cancer.

9. The use according to any one of claims 1-3, wherein the cancer tumor comprises breast cancer.

10. The use according to any one of claims 1-3, wherein the cancer tumor comprises uterine cancer.

11. The use according to any one of claims 1-3, wherein the antibody comprises a heavy chain consisting of amino acids 20-470 of SEQ ID NO: 28 and a light chain consisting of amino acids 20-233 of SEQ ID NO:

29.

12. The use according to any one of claims 1-3, wherein the antibody comprises the heavy chain variable region of SEQ ID NO: 38 and the light chain variable region of SEQ ID NO: 39.

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