Monoclonal antibody NEO-201 for treatment of human cancer

Abstract

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

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201880085205.0, whose application date is November 2, 2018 and whose invention name is “Monoclonal Antibody NEO-201 for Treating Human Cancer”.

[0002] Related application disclosure content

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

[0004] Sequence Listing Information

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

[0006] Cancer represents one of the most common causes of death worldwide, with an estimated 20 million new cases per year expected as early as 2025 (Ferlay et al., 2015). Conventional methods for treating cancer such as surgery, radiation and chemotherapy often cause serious side effects and fail to cure most patients with advanced disease, leading to relapse (Bodey et al., 1996). Recent treatment paradigms have been developed to selectively target cancerous cells while largely avoiding normal healthy tissues. Among them, immunotherapy has become an important treatment option for cancer patients because it has revolutionized the field of cancer medicine.

[0007] The underlying principle of cancer immunotherapy is called immunoediting (Mittal et al., 2014), which is an extrinsic mechanism of cancer containment that is initiated only after cell transformation has occurred and the intrinsic mechanisms of cancer containment have failed. The immunoediting process occurs in three stages: elimination, balance, and escape. In the elimination and balance stages, immune rejection of cancer cells is dominant or balanced with cancer cell proliferation to control malignant growth. However, in the escape stage, once kept in check, cancer cells can escape immune recognition due to insensitivity to immune effector mechanisms and / or induction of immune containment in the tumor microenvironment. Cancer cells that escape immune recognition are then able to proliferate more freely and grow into clinically apparent diseases (Dunn et al., 2004). The purpose of tumor immunotherapy is to keep cancer cells in the elimination and / or balance phases by generating and / or increasing anti-tumor immune responses to offset tumor growth, delay tumor recurrence, and prolong survival (Carter, 2001; Hodge et al., 2006; Vergati et al., 2010; Gabitzsch et al., 2015). Treatment approaches include treating patients with checkpoint inhibitory antibodies, anti-tumor vaccines, and chimeric antigen receptor (CAR)-T cells, all of which exploit 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).

[0008] NEO-201 is a novel humanized IgG1 mAb generated against the Hollinshead allogeneic colorectal cancer vaccine platform (Hollinshead et al., 1970; Hollinshead et al., 1972). The immunogenic components of this vaccine are tumor-associated antigens (TAAs) derived from tumor cell membrane fractions pooled from surgically resected specimens of 79 patients with colon cancer (Hollinshead et al., 1985). These cell membrane fractions were semi-purified, screened for delayed-type hypersensitivity (DTH) in patients with colon cancer versus healthy volunteers, and evaluated in clinical trials with refractory colorectal cancer (Hollinshead et al., 1985; Hollinshead, US4810781, 1989; Bristol and Kantor, US Pat. No. 7829678, 2010). These trials reported clinical benefit as defined by anti-tumor responses and significant prolongation of overall survival in patients who developed sustained IgG responses in addition to cell-mediated responses to the vaccine, indicating that the vaccine contained immunogenic components 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 investigational studies 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 the antigen(s) and specific epitope(s) recognized by NEO-201.

[0009] The human carcinoembryonic antigen (CEA) family consists of 29 genes arranged in tandem on chromosome 19q13.2. Based on nucleotide homology, these genes are classified into two major subfamilies, CEACAM and pregnancy-specific 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 contains 108-110 amino acids and is homologous to the Ig variable domain, followed by a varying number (0 to 6) of Ig C2-type constant-like domain. The CEACAM proteins can interact with each other homophilically and heterophilically. 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 with 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 appears as several isoforms, and the two main 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.

[0010] Monoclonal antibodies (mAbs) consist of a unique antigen binding region (antigen binding fragment, Fab) specific for a given mAb and a constant region (crystallizable fragment, Fc) common to all mAbs of the same isotype. The Fc region is able to modulate 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 Fcγ receptor IIIa receptors (FcγRIIIa, CD16) expressed on macrophages and NK cells. This interaction can stimulate macrophages to phagocytose mAb-conditioned cancer cells, and can activate NK cells to degranulate and lyse cancer cells through a mechanism known as antibody-dependent cellular cytotoxicity (ADCC). ADCC has been shown to be a 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 for cancer therapy (Seidel et al., 2013). Examples of clinically approved mAbs that can mediate ADCC include trastuzumab, which targets the HER2 receptor of breast cancer (Seidel et al., 2013; et al., 2013); rituximab, which targets the pan-B cell marker CD20 for lymphoma (Seidel et al., 2013; Dall'Ozzo et al., 2004); cetuximab, which targets the epidermal growth factor receptor (EGFR) for colorectal 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 immunosuppressive ligand PD-L1 for Merkel cell carcinoma and bladder cancer (Boyerinas et al., 2015). In addition, the Fc region can potentially interact with the C1 complex to activate complement-dependent cytotoxicity (CDC), in which a proteolytic cascade culminates in the formation of gaps in the cytoplasmic membrane, resulting in lysis of the cell targeted by the antibody. Even in cases where anti-tumor CDCs have been demonstrated in vitro, whether anti-tumor CDCs are essential for the clinical efficacy of mAb therapy for cancer remains controversial (Meyer et al., 2014).

[0011] 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

[0012] The research described in the examples herein evaluates the in vitro binding characteristics and in vivo activity and positioning of NEO-201 in preclinical models.NEO-201 shows extensive reactivity for a series of human carcinoma cell lines and tumor tissues, but is not observed to bind most healthy tissues.In addition, NEO-201 shows ADCC and CDC activity for human carcinoma cells in vitro, and in vivo alone and in combination with human peripheral blood mononuclear cells (PBMC) as effector cell sources of ADCC to a large extent weakens the growth of human pancreatic xenograft tumors.Finally, the single-dose toxicity study in non-human primates demonstrates the safety and tolerability of NEO-201, because the transient reduction of circulating neutrophils is the only observed side effect.These studies provide the basic principle of the potential clinical utility of NEO-201 as a new therapeutic agent for the treatment of various solid tumors.In addition, the observed CDC activity of the subject antibody opens the opportunity for treating patients with impaired immune function, in which ADCC is not expected to be effective, such as in patients with impaired immune function because of their disease or as the effect of radiation, chemotherapy and other disease treatments.

[0013] The preclinical antitumor activity (Patel et al., 2013) and clinical safety and efficacy (Beg et al., 2016; Kim et al., 2017) of a mAb generated against the Hollinshead allogeneic colorectal cancer vaccine platform, termed ensetaximab (NPC-1C / NEO-102), were previously reported. 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 multiple 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 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 stained positive for NEO-201, including colon, pancreatic, gastric, lung, breast, and uterine tumors. Extended investigation studies using larger sample sizes may reveal that NEO-201 can distinguish between histological and molecular subtypes in various cancers. Interestingly, a higher proportion of tumor tissues reacted with NEO-201 compared to cultured cancer cell lines. This observation may indicate that the targets recognized by NEO-201 are more readily expressed in vivo than in vitro, which would suggest that target expression depends, at least in part, on tumor cell interactions with factors from within the local microenvironment. Experiments are currently underway to further characterize the antigen(s) and epitope(s) recognized by NEO-201 and to determine known or multiple regulatory control mechanisms that control its expression in tumor tissues but not in normal tissues.

[0014] This investigation also revealed that NEO-201 is significantly tumor-specific in its staining spectrum, as the vast majority of healthy normal tissues and normal tissues adjacent to tumor tissues were found to be negative for NEO-201. Although NEO-201 positivity was observed in normal tongue and extracervical tissues, the staining intensity was weak, and the microarray represented only a minimal sample amount (n=2). Further extended analysis of NEO-201 staining in normal tissue samples will be performed to confirm these observations. In addition, 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 indicates that one or more antigens that react with NEO-201 are expressed on these immune cells, and an assessment of the reactivity of NEO-201 with hematopoietic cell types is performed. These encouraging results suggest that 1) NEO-201 may have diagnostic utility in distinguishing cancerous tissue from benign tissue from patient biopsies; and 2) NEO-201 may 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 cancer tumors are being planned.

[0015] 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 conditioned 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 to be 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), with immune cells from donors with a high-affinity V / V genotype exhibiting greater trastuzumab-mediated ADCC activity in vitro (Musolino et al., 2008). The V / V genotype was also shown to be significantly associated with objective response rate and progression-free survival in breast cancer patients treated with trastuzumab (Musolino et al., 2008), providing indirect clinical evidence for the role of ADCC in mAb-based therapies. NEO-201 can mediate ADCC in vitro, as treatment of tumor cells with NEO-201 enhances the cytotoxic activity of NK cells by 2-5 fold, and ADCC activity is retained even at low concentrations of the antibody (0.1 μg / mL). These data raise the possibility that patients with a V / V genotype may gain increased benefit from NEO-201 treatment. Another prospect is to enhance the potential of ADCC activity, and possibly enhance the potential clinical benefits 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 shows enhanced ADCC activity mediated by trastuzumab and cetuximab (Watanabe et al., 2010). Recent preclinical studies of novel fusion protein superagonists (called ALT-803) of IL-15 signaling show greatly enhanced NK cells (and CD8+T cells) proliferation, activation and lytic ability, thereby producing 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 NK cell degranulation, IFN-γ production, and rituximab-mediated ADCC against B cell lymphoma cell lines and primary follicular lymphoma cells in vitro, and combination treatment with ALT-803 and rituximab produced significantly reduced tumor cell burden and improved survival in vivo in two B cell lymphoma models (Rosario et al., 2016).

[0016] Another innate immune effector mechanism potentially engaged by mAbs is 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 cases (Meyer et al., 2014). In addition, several different complement regulatory proteins (CRPs) act to inhibit complement activation, and some membrane-bound CRPs such as CD46, CD55, CD59 have been reported to be abnormally expressed in various cancers (Seya et al., 1994; Niehans et al., 1996; Donin et al., 2003), which may confer resistance to CDC. Future investigations will determine whether strategies to block CRPs can enhance NEO-201-mediated CDC of resistant tumor cells.

[0017] The evaluation of NEO-201 in vivo reveals a great anti-tumor effect when administered in combination with activated human immune effector cells. This combination even leads to a comprehensive regression of some mice (5 / 20, 25%) from two combination groups. In addition, it is found that NEO-201 preferentially locates to xenograft tumor tissues, but not to various healthy tissues. These data confirm that the mechanism of action of NEO-201 for tumors is the ADCC-dependent dissolution of tumor cells by innate immune cells. However, it should be noted that anti-tumor activity is 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, because CFPAC-1 tumor cells treated with NEO-201 do not induce significant toxicity in an in vitro ADCC assay. A hypothesis of NEO-201 activity in the absence of immune effector cells may be the induction of CDC. The CDC activity of NEO-201 is directly demonstrated in further experiments described in Example 3.

[0018] In summary, this investigation shows that NEO-201 is a significant tumor-specific antibody that can engage innate immune effector mechanisms (including both ADCC and CDC) to kill tumor cells. In addition, NEO-201 demonstrated safety and anti-tumor efficacy in an in vivo xenograft model 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 various carcinomas. The results also support NEO-201 for use in immunocompromised patients (with low NK cell levels), because even in the absence of robust ADCC activity, anti-tumor effects can also be produced by CDC. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A-1D: Flow cytometry of NEO-201 binding to human cancer cell lines. Representative human cancer cell lines with various levels of NEO-201 antigen expression, ( Figure 1 A) Pancreatic CFPAC-1 (high), ( Figure 1 B) NSCLC H441 (intermediate), ( Figure 1 C) breast HCC1937 (low), and ( Figure 1 D) Colon SW1116 (negative). For each cell line, the results are expressed as % NEO-201 positivity and mean fluorescence intensity (MFI). Red, NEO-201 stained cells; black, unstained cells. NEO-201 positivity was defined as % positivity ≥ 10%.

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

[0021] Figures 3A-3C :NEO-201 mediates ADCC and CDC 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 (p<0.05) by T test. ( 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 graph depicts the fold increase (%) in specific lysis of tumor cells treated with NEO-201 versus control cells treated with 10 μg / mL human IgG1. *, statistically significant (p<0.05) by T test. ( Figure 3C ) CDC assay using ASPC-1 cells treated with the indicated doses of NEO-201 for the indicated durations. *, statistically significant (p<0.05) by T-test.

[0022] 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. Mice (n=10 animals / group) were intraperitoneally administered saline solution, human IgG1 (250 μg), or NEO-201 (100 and 250 μg) on ​​days 13, 17, and 20 after tumor cell implantation. Mice were also intraperitoneally administered approximately 1.0×10 7 IL-2 activated PBMC. Figure 4B ) Quantification of the number of mice still bearing palpable tumors at day 36. ( Figure 4C ) Representative images of NEO-201 treated vs. saline treated tumor bearing mice. Figure 4D ) Body weight measurements of tumor-bearing mice at different time points during the study.

[0023] Figure 5A-5B : Biodistribution of NEO-201 in mice bearing CFPAC-1 xenografts. From female ( Figure 5A ) and male ( Figure 5B ) Measurement of normalized radioactivity in the indicated tissues of mice. n=4 animals / time point. Day 1, Day 2, Day 4, and Day 7 represent the amount of time between radiolabeled antibody injection and necropsy.

[0024] Figures 6A-6C : Body weight and neutrophil counts from cynomolgus monkeys treated with NEO-201. ( Fig. 6A ) Percent change in body weight from baseline (Day -1) was measured in monkeys 7 and 14 days after receiving a single dose of NEO-201 at the indicated dose levels. n = 4 animals / group (2 females, 2 males). ( Figure 6B) Percent change from baseline (Day -7) in neutrophil levels in blood from monkeys treated with a single dose of NEO-201 at the indicated dose levels. n = 4 animals / group (2 females, 2 males). ( Figure 6C ) p value of neutrophil levels versus 0 mg / kg control for each dose and time point. *Statistically significant (p<0.05) by T-test.

[0025] Figures 7A-7C : HaNK ADCC assay using NEO-201 (4 hours). Target cells = 3000 cells / well. ( Figure 6A-6B ) H520 lung cancer tumors treated with NEO-201 (upper line, square symbols) or IgG1 negative control (lower line, circle symbols) at 4 h ( Fig. 6A ) or OV90 ovarian cancer ( Figure 6B ) cells as a function of the effector: target (E:T) ratio. The E:T ratio was 6.25:1, 12.5:1, or 25:1. The mAb concentration was 10 μg / mL. The values ​​shown are the mean + / - SD of 3 replicates. Asterisks (*) indicate statistical significance (p<0.01, two-tailed t-test) compared to the IgG negative control. ( Figure 6C ) Specific lysis percentage of lung, (H520, HCC827), breast, (ZR-75-1) and ovarian (OV90) cancer cells treated with NEO-201 (right, light grey bars) or negative control IgG (left, solid black bars) at four hours at a constant E:T ratio of 25:1. mAb concentration was 10 μg / mL. The values ​​shown are the mean + / -SD of 3 replicates. Asterisks (*) indicate statistical significance (p<0.01, two-tailed t-test) compared to IgG negative control.

[0026] 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 medium control for 48 hours and used as effector cells in a 4-hour non-radioactive ADCC assay using a Celigo imaging cytometer. CF-PAC1 (human pancreatic cancer cell line) cells were stained with calcein AM and used as targets at 3,000 cells / well. Results are expressed as specific lysis % (SE).

[0027] Fig. 9: Treatment with ALT-803 enhances 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 % of positive cells (MFI).

[0028] Fig.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). The results are expressed as % of positive cells (MFI).

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

[0030] Fig.12 :Treatment with ALT-803 enhances the ADCC activity of normal donors (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 normal donors with the lowest ADCC activity were treated with ALT-803 (25ng / ml) or culture medium control for 48 hours and used as effector cells in a 4-hour non-radioactive ADCC assay using a Celigo imaging blood cell counter. Anti-CD16 and anti-TIM-3 were used at concentrations of 30μg / ml and 15μg / ml. NK cells were pretreated with anti-CD16 or anti-TIM-3 for 2 hours before adding 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 10μg / ml. E:T=25:1. Results are expressed as % specific lysis (SE). * Statistically significant (p<0.01) compared to no ALT-803 treatment. # Statistically significant (p<0.01) compared to no anti-CD16 and anti-TIM-3 treatment.

[0031] Fig.13 : NK-92 killing assay using NEO-201 (16 hours). Target tumor cells (ASPC-1, BxPC-3, CFPAC-1 or LS174T) were inoculated 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 with effector to target (E:T) ratios of 1.5625:1, 3.125:1, 6.25:1 and 12.5:1. After 16 hours of incubation at 37°C, cell viability was quantified using Celigo imaging blood cell counter and GraphPad Prism 7 software. The live target cells (calcein AM+ / PI-) of each well were counted, and specific lysis was calculated. The results are shown in a graphical manner and are below for each tumor cell type. * Statistically significant (p<0.05). DETAILED DESCRIPTION

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

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

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

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

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

[0037] Prior to or at the time of said administering, the patient may be natural killer ("NK") depleted. Prior to or at the time of said administering, the patient may be severely NK depleted. The patient may have NK cell deficiency (NKD), such as CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD1). The patient may be NK depleted or severely NK depleted due to another therapy, such as cancer therapy (e.g., chemotherapy or radiation therapy). 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).

[0038] The method may include determining whether the patient is NK-depleted prior to or at the time of said administering.

[0039] The method may include determining whether the patient is severely NK-depleted prior to or at the time of administering.

[0040] In the method, prior to or at the time of said administering, NK cells may comprise less than 5% of peripheral blood mononuclear cells (PBMCs) in said individual.

[0041] In the method, prior to or at the time of said administering, NK cells may comprise less than 3% of peripheral blood mononuclear cells (PBMCs) in said individual.

[0042] In the method, prior to or at the time of said administering, less than 70% of PBMC NK cells in said patient may be CD56dimCD16+ NK cells.

[0043] In the method, prior to or at the time of said administering, less than 50% of PBMC NK cells in said patient may be CD56dimCD16+ NK cells.

[0044] 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.

[0045] The NEO-201 antibody may comprise a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38.

[0046] The NEO-201 antibody may comprise a variable light chain sequence that is at least 90% identical to SEQ ID NO:39.

[0047] The NEO-201 antibody may comprise a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38 and a variable light chain sequence that is at least 90% identical to SEQ ID NO:39.

[0048] The NEO-201 antibody may comprise a heavy chain sequence at least 90% identical to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence at least 90% identical to amino acids 20-233 of SEQ ID NO:29.

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

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

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

[0052] The NEO-201 antibody may be conjugated to another moiety.

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

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

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

[0056] The cytokine agonist may include an IL-15 agonist or an IL-15 superagonist.

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

[0058] 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.

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

[0060] In another embodiment, the present disclosure provides a method of killing cancer cells, the method comprising administering an effective amount of NEO-201 antibody to a patient in need thereof, wherein prior to or at the time of said administration, the patient is natural killer cell ("NK") depleted. The NK depletion may include in a sample derived from the patient, such as a blood sample, less than 5% or less than 3% of the patient's peripheral blood mononuclear cells (PBMCs) are NK cells. Alternatively or in addition, prior to or at the time of said administration, less than 70% (or optionally less than 50%) of the PBMC NK cells in the patient may be CD56dimCD16+ NK cells.

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

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

[0063] In another embodiment, the present disclosure provides a method of reducing tumor burden in a patient having a cancer, the method comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody, wherein prior to or at the time of said administering, the patient is natural killer cell ("NK") depleted.

[0064] In the aforementioned method, the antibody can mediate CDC, thereby killing 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 may be severely NK depleted. Optionally, the method also includes, for example, determining whether the patient is NK depleted or severely NK depleted within a certain period of time at the time of the administration or before the administration, such as within the previous 1 or 2 weeks. NK depleted or severe NK depleted state can also be inferred from the patient's medical history, such as before or simultaneously with the use of another therapy that depletes NK cells. For example, the patient has undergone or is undergoing cancer therapy, such as radiotherapy or chemotherapy. The cancer therapy may include administration of one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, 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).

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

[0066] In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise at least one, two, three, four, five or all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0067] In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise a variable heavy chain sequence that is at least 80%, at least 85%, at least 90%, or most preferably at least 95% identical to SEQ ID NO: 38. The variable heavy chain having said percentage of sequence identity may comprise all 3 of the CDR sequences contained in SEQ ID NO: 38.

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

[0069] In a preferred embodiment of the invention that can be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise a variable heavy chain sequence that is at least 80%, at least 85%, at least 90%, or most preferably at least 95% identical to SEQ ID NO: 38 and a variable light chain sequence that is at least 80%, at least 85%, at least 90%, or most preferably at least 95% identical 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 said percentages of sequence identity may comprise all 3 of the CDR sequences contained in SEQ ID NO: 38.

[0070] In a preferred embodiment of the invention that can be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise a heavy chain sequence that is at least 80%, at least 85%, at least 90%, or most preferably at least 95% identical to amino acids 20-470 of SEQ ID NO: 28 and a light chain sequence that is at least 80%, at least 85%, at least 90%, or most preferably at least 95% identical 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 said percentages of sequence identity may comprise all 3 of the CDR sequences contained in SEQ ID NO: 28.

[0071] In a preferred embodiment of the invention, which may be used with any of the preceding 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.

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

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

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

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

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

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

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

[0079] In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the cancer may express the NEO-201 antigen. The expression of the NEO-201 antigen may be determined by detecting the NEO-201 antigen in a sample of the cancer. The detection may be performed by techniques including, but not limited to, 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 may 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.

[0080] In a preferred embodiment of the present invention, which may be used with any of the preceding or following embodiments, the cancer may comprise colon cancer.

[0081] In a preferred embodiment of the present invention, which may be used with any of the preceding or following embodiments, the cancer may comprise pancreatic cancer.

[0082] In a preferred embodiment of the present invention, which may be used with any of the preceding or following embodiments, the cancer may comprise ovarian cancer.

[0083] In a preferred embodiment of the present invention, which may be used with any of the preceding or following embodiments, the cancer may comprise gastric cancer.

[0084] In a preferred embodiment of the present invention, which may be used with any of the preceding or following embodiments, the cancer may comprise lung cancer.

[0085] In a preferred embodiment of the present invention, which may be used with any of the preceding or following embodiments, the cancer may comprise breast cancer.

[0086] In a preferred embodiment of the present invention, which may be used with any of the preceding or following embodiments, the cancer may comprise uterine cancer.

[0087] definition

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

[0089] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0090] As used herein, "amino acid" refers in a broad sense to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function 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 that are subsequently modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon, a carboxyl group, an amino group, and an R group bound to hydrogen, for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to naturally occurring amino acids.

[0091] As used herein, the term "NK depletion" or "natural killer cell depletion" means that the patient has low levels of natural killer (NK) cells relative to the normal range. NK cells are cytotoxic innate immune lymphocytes. Typically, NK cells account for 5%-20% of peripheral blood mononuclear cells (PBMCs) in healthy individuals. Patients with NK cells that account for less than 5% of PMBCs are called NK depleted. In addition, if NK cells account for less than 3% of PMBCs, the patient is called severely NK cell depleted. In addition, in normal individuals, up to 90% of PBMC NK cells are CD56 dim CD16 + NK cells, and these are considered the most toxic subset. If less than 70% of PBMC NK cells are CD56 dim CD16 + If less than 50% of PBMC NK cells are CD56 dim CD16 +NK cells, the patient is referred to as severe NK depletion. A given patient may be referred to as NK depletion or severe NK depletion based on meeting one or two of these individual criteria. In general, the patient's status is determined to be NK depletion or severe NK depletion by testing a sample obtained from the patient, such as a blood sample, for example, a sample obtained and tested within one or two weeks before. The patient's status may also be inferred to be NK depletion or severe NK depletion from disease diagnosis and / or a treatment process associated with this depletion of NK cells.

[0092] NK depletion type 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 functions in peripheral blood lymphocytes; functional NKD (FNKD), which is characterized by the presence of NK cells in peripheral blood lymphocytes, with defective NK cell activity. In both CNKD and FNKD, NK cell abnormality is a major immunodeficiency, which causes insufficient ADCC response. CNKD and FNKD can be further subdivided based on patient characteristics, such as the identity of one or more pathogenic genes and other patient characteristics. CNKD includes CNKD subtype 1 (CNKD1), which is autosomal dominant and related to defects in the GATA2 gene; and CNKD subtype 2 (CNKD2), which is autosomal recessive and related to defects in the MCM4 gene. FNKD includes FNKD1, which is autosomal recessive and related to defects in the FCCR3A gene.

[0093] The term "antibody" as used herein refers in a broad sense to any polypeptide chain-containing molecular structure having a specific shape that cooperates 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, and IgD from all sources (e.g., humans, rodents, rabbits, cows, sheep, pigs, dogs, chickens) 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, IgNARs (single-chain antibodies obtained from sharks), small molecule immunopharmaceuticals (SMIPs), and antibody fragments (e.g., Fab, Fab', F(ab')2). Many antibody coding sequences have been described; and other antibody coding sequences can be produced 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.

[0094] "NEO-201 antibody" refers to a heavy chain and a light chain containing SEQ ID NOs: 28 and 29 or the variable regions contained therein, optionally together with the constant regions, and fragments and variants thereof. Such variants include sequences containing one, two, three, four, five or preferably all six of the CDR sequences contained in SEQ ID NOs: 28 and 29, i.e., 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 a humanized antibody. The antibody may be expressed as containing one or more leader sequences, which may be removed during the expression and / or processing and secretion of the antibody. The antibody may be presented in a monovalent, divalent or higher multivalent form, including but not limited to bispecific or multispecific antibodies containing the NEO-201 antibody sequence and binding fragments of different antibodies. Typically, the antibody specifically binds to cancer cells and competes with an antibody comprising a variable heavy chain of SEQ ID NO: 38 and a variable light chain of SEQ ID NO: 39 or a heavy chain of SEQ ID NO: 28 and a light chain of SEQ ID NO: 29 for binding to cancer cells. One or more of those CDR sequences contained in SEQ ID NO: 28 and / or SEQ ID NO: 29 may be substituted with a variant sequence, such as a light chain CDR1 of SEQ ID NO: 1 or 4; a light chain CDR2 of SEQ ID NO: 2 or 5; a light chain CDR3 of SEQ ID NO: 3 or 6; a heavy chain CDR1 of SEQ ID NO: 7; a heavy chain CDR2 of SEQ ID NO: 8, 10, 30 or 31; a heavy chain CDR3 of SEQ ID NO: 9 or 11 or SEQ ID NO: 30-31. The light chain may comprise a CDR contained in a light chain sequence of SEQ ID NO: 14, 16, 17, 18, 19, 20, 21 or 29. The heavy chain may comprise the CDRs contained in the heavy chain sequence of SEQ ID NO: 15, 22, 23, 24, 25, 26, 27 or 29.The antibody may comprise a variable heavy chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:38 and / or a variable light chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:39, optionally wherein the heavy and / or light chain sequences contain one, two, three, four, five or preferably all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29, i.e., 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.

[0095] As used herein, "antigen" refers in a broad sense to a molecule or a portion of a molecule that can be bound by an antibody, which in turn can induce an animal to produce an antibody that can bind to an epitope of the antigen. An antigen may have one epitope, or more than one epitope. The specific reactions mentioned herein indicate that an 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. Antigens may be tumor-specific (e.g., expressed by neoplastic cells of pancreatic and colon cancers.)

[0096] "Cancer" as used herein refers broadly to any neoplastic disease characterized by abnormal and uncontrolled cell division, giving rise to malignant growth or tumors, whether invasive or metastatic.

[0097] As used herein, "chimeric antibody" refers broadly to antibody molecules in which the constant region, or a portion thereof, is altered, substituted or exchanged so 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 an entirely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that confers new properties to the chimeric antibody; or the variable region, or a portion thereof, is altered, substituted or exchanged with a variable region having a different or altered antigenic specificity.

[0098] As used herein, "conservatively modified variants" apply to amino acid sequences and nucleic acid sequences, and with respect to specific nucleic acid sequences, refer in a broad sense to conservatively modified variants, to those nucleic acids encoding identical or substantially identical amino acid sequences, or to substantially identical sequences when the nucleic acid does not encode an amino acid sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. Such nucleic acid variations are "silent variations," which are a type of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also illustrates each possible silent variation of the nucleic acid. The skilled artisan will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule.

[0099] As used herein, "complementarity determining region," "hypervariable region" or "CDR" refers broadly to one or more hypervariable or complementarity determining regions (CDRs) found in the variable region of the light chain or heavy chain of an antibody. See Kabat, et al. (1987) " Sequences of Proteins of Immunological Interest "National Institutes of Health, Bethesda, MD. These statements include those by Kabat, et al. (1983)" Sequences of Proteins of Immunological Interest "U.S. Department of Health and Human Services defines a hypervariable region or a hypervariable loop in the 3-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 the 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 select amino acids that have been described as selectivity determining regions (SDRs), which represent the key contact residues used by the CDRs in antibody-antigen interactions. Kashmiri (2005) Methods 36:25–34.

[0100] As used herein, "control amount" refers in a broad sense to any amount or range of amounts of a marker to be compared to 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 who does not have the 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).

[0101] As used herein, "differentially present" refers in a broad sense to the difference in the amount or quality of a marker present in a sample taken from a patient suffering from one of the diseases or disorders, compared to a comparable sample taken from a patient not suffering from the disease or disorder. For example, if the amount of nucleic acid fragments in one sample is significantly different from the amount of nucleic acid fragments in another sample, the nucleic acid fragments may optionally be differentially present between the two samples, such 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 a 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, it can be considered that such a marker is differentially present. Optionally, a relatively low amount of upregulation may serve as a marker.

[0102] As used herein, "diagnosis" refers in a broad sense to identifying the presence or nature of a pathological disorder. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of sick individuals that test positive (the percentage of "true positives"). Sick individuals that are not detected by the assay are "false negatives." Subjects who are not sick and test negative in the assay are referred to as "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the ratio of those that test positive but do not suffer from the disease. Although a particular diagnostic method may not provide a clear diagnosis of a disorder, it is qualified if the method provides a positive indication that helps in diagnosis.

[0103] As used herein, "diagnosis" refers in a broad sense 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 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 physically removed from the subject.

[0104] As used herein, "effective amount" refers in a broad sense to the amount of compounds, antibodies, antigens or cells that are sufficient to achieve such treatment of the disease when applied to a patient for the treatment of a disease. An effective amount can be an amount that is effective for preventing and treating (prophylaxis), and / or an amount that is effective for preventing (prevention). An effective amount can be an amount that is effectively reduced, effectively preventing the occurrence of signs / symptoms, reducing the severity of the occurrence of signs / symptoms, eliminating the occurrence of signs / symptoms, slowing down the development of the occurrence of signs / symptoms, preventing the development of the occurrence of signs / symptoms and / or effectively preventing and treating the occurrence of signs / symptoms. "Effective amount" can vary depending on the disease and severity thereof of the patient to be treated, as well as age, weight, medical history, susceptibility and pre-existing illness. For purposes of the present invention, the term "effective amount" is synonymous with "therapeutically effective amount".

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

[0106] As used herein, "framework region" or "FR" refers broadly to one or more framework regions within the variable region of the light chain 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 interposed between the CDRs within the variable regions of the light and heavy chains of antibodies.

[0107] As used herein, "heterologous" refers broadly to portions of a nucleic acid, indicating that the nucleic acid comprises two or more subsequences that are not found in the same qualitative relationship to each other. For example, nucleic acids are often produced recombinantly so that two or more sequences from unrelated genes are arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that a protein comprises two or more subsequences that are not found in the same qualitative relationship to each other (e.g., a fusion protein).

[0108] As used herein, "high affinity" broadly refers to an antibody that has at least 10 –8 M, more preferably at least 10–9 M and even more preferably at least 10 –10 However, for other antibody isotypes, "high affinity" binding may vary. For example, for an IgM isotype, "high affinity" binding refers to a KD of at least 10 –7 M, more preferably at least 10 –8 Antibodies for KD of M.

[0109] As used herein, "homology" refers in a broad sense 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 sequences are identical. Partially homologous nucleic acid or amino acid sequences are nucleic acid or amino acid sequences that are not identical to a reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The term "sequence identity" can be used interchangeably with "homology".

[0110] As used herein, "host cell" refers in a broad sense to a cell that contains an expression vector and supports replication or expression of the expression vector. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insects (e.g., SF9), amphibians or mammalian cells such as CHO, HeLa, HEK-293, for example, cultured cells, explants and in vivo cells.

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

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

[0113] As used herein, "immunoassay" refers broadly to an assay using an antibody that specifically binds to an antigen. An immunoassay can be characterized by using the specific binding properties of a particular antibody to isolate, target and / or quantify the antigen.

[0114] As used herein, "isolated" refers in a broad sense to a material that is removed from its original environment in which it naturally occurs and thus artificially altered from its natural environment. An isolated material can be, for example, an exogenous nucleic acid contained in a vector system, an exogenous nucleic acid contained in a host cell, or any material that has been removed from its original environment and thus artificially altered (e.g., an "isolated antibody").

[0115] As used herein, "label" or "detectable moiety" refers broadly to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[0116] As used herein, "low stringency", "medium stringency", "high stringency" or "very high stringency conditions" refer broadly to conditions for nucleic acid hybridization and washing. Guidance for conducting hybridization reactions can be found in Ausubel, et al. (2002). ShortProtocols inMolecular Biology (5 th Ed.) John Wiley & Sons, NY. Exemplary specific hybridization conditions include, but are not limited to: (1) low stringency hybridization conditions of 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least two washes in 0.2X SSC, 0.1% SDS at 50°C (for low stringency conditions, the temperature of the washes can be increased to 55°C); (2) moderate stringency hybridization conditions of 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions of 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and (4) very high stringency hybridization conditions of 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2X SSC, 1% SDS at 65°C.

[0117] As used herein, "mammal" refers in a broad sense to any and all warm-blooded vertebrates of the class Mammalia, including humans, characterized by having hair covering the skin and, in females, milk-producing mammary glands for rearing young. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammals include, but are not limited to, bovine, canine, equine, feline, murine, ovine, swine, primates, and rodent species. Mammals also include any and all mammals listed on the World's Mammal Species maintained by the National Museum of Natural History, Smithsonian Institution, Washington, D.C.

[0118] As used herein, "nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide oligonucleotide 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 specific nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0119] As used herein, the term "operably linked" refers in a broad sense to joining two DNA fragments so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0120] As used herein, "paratope" refers broadly to a portion of an antibody that recognizes an antigen (e.g., the antigen binding site of an antibody.) A paratope 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 an antibody. See Goldsby, et al. Antigens (Chapter 3) Immunology (5th ed.) New York: WH Freeman and Company, pp. 57–75.

[0121] As used herein, "patient" refers in a broad sense to any animal that needs treatment to alleviate a disease state or prevent the occurrence or recurrence of a disease state. In addition, as used herein, "patient" refers in a broad sense to any animal that has previously diagnosed risk factors, medical history, susceptibility, symptoms, signs, or is at risk of a disease or is 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".

[0122] "Polypeptide," "peptide," and "protein" are used interchangeably herein and in a broad sense refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Polypeptides may 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.

[0123] As used herein, "promoter" refers in a broad sense to a nucleic acid sequence that directs the transcription of a nucleic acid. As used herein, "promoter" includes essential nucleic acid sequences near the transcription start site, such as the TATA element in the case of a type II polymerase promoter. The promoter also optionally includes a distal enhancer or repressor element, which may be located as far as several thousand base pairs 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 regulation.

[0124] As used herein, a "prophylactic effective amount" refers in a broad sense to an amount of a compound that is sufficient to achieve such prevention or treatment of the disease or recurrence when administered to a patient for the purpose of preventing or treating a disease or preventing the recurrence of a disease. A prophylactic effective amount may be an amount that is effective to prevent the occurrence of signs and / or symptoms. A "prophylactic effective amount" may vary depending on the disease and its severity as well as the age, weight, medical history, susceptibility to the disorder, pre-existing disorders of the patient to be treated.

[0125] As used herein, "preventing" broadly refers to a course of treatment in which signs and / or symptoms are absent from a patient, are in remission, or were previously present in a patient. Preventing includes preventing a disease from occurring following treatment of a patient's disease. In addition, prevention includes treating a patient who may potentially develop a disease, particularly a patient who is susceptible to the disease (e.g., a member of a patient population, a patient with risk factors, or a patient at risk of developing a disease).

[0126] As used herein, "recombinant" with respect to a product refers broadly 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 the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell or expresses native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

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

[0128] As used herein, "specifically hybridizable" and "complementary" refer broadly to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence, either by the traditional Watson-Crick type or other non-traditional types. The binding free energy of a nucleic acid molecule to its complementary sequence is sufficient to allow the relevant function of the nucleic acid, such as RNAi activity. Determination of the binding free energy 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 percent 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 refers broadly 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" refers to polynucleotide chains that exhibit at least about 90% complementarity, excluding regions of the polynucleotide chains that are selected to be non-complementary, such as overhangs. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions where specific binding is desired (i.e., under physiological conditions in the case of an in vivo assay or therapeutic treatment, or under the conditions under which the assay is performed in the case of an in vitro assay). Non-target sequences can typically differ by at least 5 nucleotides.

[0129] As used herein, "signs" of disease refer broadly to any abnormality detectable upon examination of a patient that is indicative of disease; in contrast to symptoms, which are subjective indicators of disease.

[0130] As used herein, "solid support," "support," and "substrate" refer broadly to any material that provides a solid or semisolid structure to which another material can attach, including but not limited to smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) as well as textured and porous materials.

[0131] As used herein, "subject" refers in a broad sense to a subject suitable for treatment according to the present invention, including but not limited to avian and mammalian subjects, and preferably mammals. Mammals of the present invention include but are not limited to dogs, cats, cattle, goats, horses, sheep, pigs, rodents (e.g., rats and mice), lagomorphs, primates, and humans. Any mammalian subject that needs to be treated according to the present invention is suitable. Human subjects of both sexes and at any stage of development (i.e., neonates, infants, teenagers, youth, adults) can be treated according to the present invention. The present invention can also be carried out in animal subjects, particularly mammalian subjects, such as mice, rats, dogs, cats, cattle, goats, sheep, and horses for veterinary purposes and for drug screening and drug development purposes. "Subject" can be used interchangeably with "patient".

[0132] As used herein, "symptoms" of a disease refer broadly to any pathological phenomenon or deviation from normal structure, function, or sensation experienced by a patient and indicative of the disease.

[0133] As used herein, "therapy", "therapeutic", "treating" or "treatment" refers in a broad sense to treating a disease, curbing or reducing the development of a disease or its clinical symptoms, and / or alleviating a disease, thereby causing the disease or its clinical symptoms to regress. Therapy encompasses prophylaxis, treatment, remediation, reduction, alleviation and / or alleviation of a disease, signs and / or symptoms of a disease. Therapy encompasses alleviation of signs and / or symptoms of a patient with developing signs and / or symptoms of a disease (e.g., tumor growth, metastasis). Therapy also encompasses "prevention and treatment". For therapeutic purposes, the term "reduction" refers in a broad sense to a clinically significant reduction in signs and / or symptoms. Therapy includes treating recurrent or recurrent signs and / or symptoms (e.g., tumor growth, metastasis). Therapy encompasses, but is not limited to, excluding the appearance of signs and / or symptoms at any time, as well as reducing existing signs and / or symptoms and eliminating existing signs and / or symptoms. Therapy includes treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing recurrence or recurrence of signs and / or symptoms (eg, tumor growth, metastasis).

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

[0135] As used herein, "vector" refers in a broad sense to a plasmid, cosmid, phagemid, phage DNA or other DNA molecule capable of autonomous replication in a host cell and characterized by one or a small number of restriction endonuclease recognition sites, at which such DNA sequences can be cut in a determinable manner without losing the basic biological function of the vector, and into which DNA can be inserted to bring about its replication and cloning. The vector may also contain a marker suitable for use in the identification of cells transformed with the vector.

[0136] The techniques and procedures are generally performed according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout the present specification. See, for example, Sambrook, et al. (2001) Molec.Cloning:Lab.Manual [3rd ed.] Cold Spring Harbor Laboratory Press. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. Nomenclature used with respect to and laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0137] Example

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

[0139] Example 1

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

[0141] Flow cytometry analysis was used to profile NEO-201 binding to 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 Figure 1A-1C shows. Evaluation of the binding activity of NEO-201 revealed that 3 / 6 (50%) colon cancer cell lines and 4 / 5 (80%) pancreatic cancer cell lines were highly positive. When the non-small cell lung cancer (NSCLC) cell lines of various histological subtypes were analyzed, 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. In cell lines expressing estrogen receptors (ER) or progesterone receptors (PR), 2 / 4 (50%) were positive for NEO-201 staining, whether used alone or in combination with HER2. In HER2+ cell lines, 3 / 4 (75%) were recognized by NEO-201, whether used alone or in combination with ER or PR. However, NEO-201 staining was found at low levels on only 1 / 4 (25%) of triple-negative breast cancer cell lines. In total, 15 / 30 (50%) of the tumor cell lines tested were recognized by NEO-201. These data demonstrate that NEO-201 is reactive against a broad range of in vitro cultured tumor cell lines and suggest that significant differences in antibody reactivity can occur based on tumor subtype.

[0142] Example 2

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

[0144] Using tissue microarrays representing many samples of each cancer type, immunohistochemical studies were used to investigate NEO-201 reactivity from human tumor samples. Figure 2A As 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. Strikingly, staining was found only on tumor cells, as surrounding stromal cells were not stained ( Figure 2A IHC staining of microarray samples determined that NEO-201 was highly reactive against colon cancer (72%), pancreatic cancer (80%), gastric cancer (71%), lung cancer (61%), breast cancer (55%), and uterine cancer (54%). In addition, a relatively small number of ovarian cancer samples (26%) also showed positive staining, but in prostate cancer tissues ( Figure 2B ). Overall, 258 / 345 (74.7%) of the sampled tumor tissues stained positive for NEO-201. Importantly, NEO-201 reactivity was almost completely absent from normal healthy tissues (Table 2) and from normal tumor-adjacent tissues (except for some uterine and ovarian samples) ( Figure 2C). However, the number of tissues in this panel of uterine and ovarian tissues was limited (5 and 9 samples, respectively). In summary, these data indicate that NEO-201 recognizes tumor tissue from a wide variety of carcinomas and is highly tumor specific.

[0145] Example 3

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

[0147] As a humanized IgG1 antibody, NEO-201 is theoretically capable of mediating ADCC to kill tumor cells expressing the NEO-201 antigen. To investigate this potential mechanism of action, cell lines that stained highly positive for NEO-201 were subjected to ADCC assays utilizing human natural killer (NK) cells (CFPAC-1 and ASPC-1) isolated from PBMCs from two different healthy donors. It was observed that treatment with NEO-201 enhanced killing of CFPAC-1 and ASPC-1 to levels 2- to 6-fold greater than killing of control IgG1-treated tumor cells ( Figure 3A Titration experiments were also performed and revealed that NEO-201 retained significant ADCC induction ability at doses as low as 0.1 μg / mL ( Figure 3B ).

[0148] CDC is a complex cascade of proteolytic cleavages that culminates in the activation of the membrane attack complex, which lyses the antibody-bound target cell. Certain human IgG1 antibodies are capable of mediating CDC, however, CDC is dependent on the antigenic specificity of the antibody. The CDC assay revealed that NEO-201 induced complement-mediated lysis of ASPC-1 cells in a manner that depended on both mAb dose and incubation time ( Figure 3C ). Taken together, these data demonstrate that NEO-201 effectively engages innate immune effector mechanisms to specifically lyse antibody-bound tumor cells in vitro.

[0149] Example 4

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

[0151] To determine the potential 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 CFPAC-1 tumors grew to approximately 100 mm 3The tumor-bearing mice were injected three times with saline, 250 μg human IgG1, 100 μg NEO-201, or 250 μg NEO-201, followed by three injections of 1.0 × 10 7 IL-2 activated (200 U / mL) human PBMCs to act as effector cells for mediating ADCC. Figure 4A As shown, NEO-201+PBMC induced a significant reduction in tumor growth at both dose levels compared to saline+PBMC or human IgG+PBMC control groups. While no mice from the control group were tumor-free on day 36, 1 of 10 (10%) and 4 of 10 (40%) mice from the NEO-201 100 μg+PBMC and NEO-201 250 μg+PBMC groups, respectively, were free of palpable tumors ( Figure 4B In addition, another group of mice was given NEO-201 without the addition of human PBMCs, and a significant reduction in tumor growth relative to the control group was observed ( Figure 4A Importantly, monitoring of the body weight of tumor-bearing mice revealed no weight loss in any of the treatment groups ( Figure 4D ). Overall, these results indicate that NEO-201 is able to significantly reduce tumor growth through both ADCC and non-ADCC mechanisms (such as CDC) without inducing significant toxicity in mice.

[0152] Example 5

[0153] NEO-201 localizes to xenograft tumor sites

[0154] Biodistribution studies were performed using radiolabeled NEO-201 in female and male NU / NU nude mice with established CFPAC-1 xenograft tumors. The mice were injected intravenously with the radiolabeled antibody, and then blood, organs, and tumors were harvested at various time points after injection for analysis. Low levels of radioactivity ( ) were found in the pancreas, spleen, kidney, liver, stomach, intestine, and lungs in male and female mice at all time points. Figure 5A , 5B). However, the normalized uptake of radioactivity was significantly higher in tumors compared to all other tissues at all time points, with tumor radioactivity rising progressively to levels 20-30 times higher than that of blood by day 7 ( 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.

[0155] Example 6

[0156] Evaluation of NEO-201 pharmacokinetics and toxicity in nonhuman primates

[0157] Single-dose studies were conducted in purposefully bred cynomolgus monkeys to determine NEO-201 pharmacokinetics and related toxicity.Choose cynomolgus monkeys because this species is closely related to humans in terms of phylogeny and physiology, and is a species commonly used for nonclinical toxicity evaluation.Female and male animals received a single intravenous infusion of 5mg / kg, 20mg / kg and 49mg / kg dose levels of NEO-201 diluted in saline, which is the highest dose that can be achieved per infusion volume.Blood samples were drawn from all animals at different time points before and after injection until 14 days, and the NEO-201 levels of serum preparations were assessed by ELISA.As depicted in Table 3, quantifiable and dose-dependent serum concentrations of NEO-201 were observed by the last collection time point (14 days after the dose).As expected for intravenous administration, for most animals from all groups, the Tmax value reached a peak value by 10 minutes (10 / 12, 83%), except for one male and one female animal from the 5mg / kg group, respectively. Peak (Cmax) exposure was proportional to dose over the dose range evaluated; total (AUC) exposure was greater than dose proportional at the lowest dose and was roughly proportional from 20 mg / kg to 49 mg / kg. The difference in exposure at the lowest dose was attributed to an approximately 2-fold greater mean clearance (CL) and a smaller volume of distribution (Vz). At higher doses, the mean half-life (HL) was 167 (20 mg / kg) or 170 (49 mg / kg) hours, approximately 3.7 times greater than at the 5 mg / kg dose (46.2 hours). No gender differences were observed.

[0158] Observations and examinations used to determine toxicity during the 14-day study included 1) periodic 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. Fig. 6A As shown, no dose level group experienced a body weight change of >3% compared to their pre-injection weight, and no individual monkey experienced a change of >7%. Food consumption remained constant for all animals, but two animals in the 5 mg / kg dose group had only low consumption on Day 11. There were no significant changes from baseline (pre-NEO-201 injection) to Day 15 in any of the serum chemistry, urinalysis, or coagulation tests (see Materials and Methods for details). The main laboratory change in blood cell counts was a decrease in neutrophil counts relative to baseline ( Figure 6B The decrease was of variable magnitude, ranging from mild to significant, and a clear dose response was not evident. For most animals, this was a transient finding, as improvements were usually noted by day 8 ( Figure 6BBy day 15, almost complete or partial recovery of neutrophil counts was observed for the 5 mg / kg group or the 20 mg / kg and 49 mg / kg groups, respectively ( Figure 6B The recovery of neutrophil counts by day 15 was reflected in statistical comparisons to 0 mg / kg animals, which were significantly different (p<0.05) for all three dose levels on day 2, but not significantly different (p>0.05) on days 8 and 15 for two of the three dose groups ( Figure 6C ).

[0159] Example 7

[0160] Materials and Methods

[0161] Cell lines and culture

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

[0163] Generation of humanized NEO-201 monoclonal antibody

[0164] Hollinshead colon cancer specific vaccine is used as immunogenic material to produce monoclonal antibodies in mice. Methods for preparing tumor-related proteins and peptides have been previously described (Hollinshead, US4810781, 1989). In brief, cancer tissue is minced and used to produce a single cell suspension, which is then subjected to hypotonic saline membrane extraction, a series of centrifugation steps, and subsequent low-frequency ultrasonic treatment. The resulting membrane-extracted protein is 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). TAA preparations are blended with complete Freund's adjuvant and injected subcutaneously in BALB / c mice. This is followed by 3 booster injections in incomplete Freund's adjuvant at intervals of 2-3 weeks. The antibody response to the immune antigen of mouse serum is tested by ELISA, and the mouse B cell from spleen is fused with SP2 / 0-Ag14 myeloma cell line and the cell of selecting growth and producing mouse immunoglobulin (IgG) will be used to produce the hybridoma cell of immortalization with effective response.From these mouse IgG, based on the reactivity of colon tumor cell membrane extracts derived from LS174T or HT-29 cells as determined by ELISA, mouse 16C3 clone (m16C3) is selected.The cDNA encoding heavy chain and light chain IgG1 is determined from the RNA separated from hybridoma clone 16C3E12 and is shown to be unique (Bristol & Kantor, US7829678,2010).The m16C3 protein sequence is humanized to h16C3 and is designated as NEO-201.The mouse sequence outside the complementary determining region (CDR) of the Fab region of heavy chain and light chain protein is replaced with human Fab sequence and three mouse CDR sequences from each chain are retained and humanized in computer. The Fc region of heavy and light chains was selected from the human IgG1 isotype used in other humanized approved mAb products. The amino acid sequence was back-translated into DNA, which was optimized for protein expression in CHO cells. The DNA of heavy and light chain h16C3 was then chemically synthesized, cloned into mammalian expression plasmids and transfected into mammalian cell lines (HEK293T and CHO). Several stable CHO cell lines expressing recombinant h16C3 were obtained and stored in storage. 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).

[0165] The NEO-201 antibody sequence used in these examples is contained in the following diagram: H16C3-Abb* Heavy Chain

[0166]

[0167] H16C3-Abb* light chain

[0168]

[0169] The leader sequence is expressed in each sequence, the boundary between the variable region and the constant region is defined by a forward slash (" / "), and the CDR sequences are shown in bold, underlined text. The antibody sequences used include the variable and constant regions shown. These include the heavy chain CDR1 of SEQ ID NO:32, the heavy chain CDR2 of SEQ ID NO:33, the heavy chain CDR3 of SEQ ID NO:34, the light chain CDR1 of SEQ ID NO:35, the light chain CDR2 of SEQ ID NO:36, and the light chain CDR3 of SEQ ID NO:37.

[0170] Flow cytometry

[0171] The binding of NEO-201 to human cancer cell lines was analyzed by flow cytometry. Cells (1.0 × 10 6 ) were incubated with LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) in 1X phosphate buffered saline (PBS) per test for 30 minutes to complete the distinction between live cells and dead cells. The cells were then centrifuged, washed twice with cold PBS, and then stained with Pacific Blue conjugated NEO-201 antibodies (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). BD FACSuite software (BD Biosciences, San Jose, CA, USA) was used for analysis of cell fluorescence. Staining values>10% positive were considered positive for NEO-201 expression. Positive cell lines were ranked according to their quantified expression levels (% positive x MFI) and then classified into low (<200), medium (200-1000) and high (<1000) expression groups.

[0172] Immunohistochemistry (IHC)

[0173] 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. Slides were baked at 60°C for 20 minutes, dewaxed with xylene, and rehydrated with a graded ethanol series. The slides were then peroxidized for 2 minutes using Peroxidazed I solution (Biocare Medical, Concord, CA), avidin for 10 minutes using avidin solution (Biocare Medical, Concord, CA), biotin for 10 minutes using biotin solution (Biocare Medica, Concord, CA), and protein for 10 minutes using CAS-blocked histochemical reagents (Thermo Fisher Scientific, Waltham, MA). The slides were then incubated for 2 hours at room temperature with 10 μg / mL of biotinylated human IgG1κ (Ancell, Bayport, MN) or biotinylated NEO-201 diluted in 1X PBS with negative controls. Detection was performed for 30 minutes using 1:300 Dako streptavidin-HRP conjugate (Agilent Technologies, Santa Clara, CA), incubated for 1-3 minutes with DAB peroxidase substrate (Thermo Fisher Scientific, Waltham, MA), and counterstained with hematoxylin. The cell staining intensity of each microarray tissue spot was evaluated by optical microscopy using the following scale: 0 (negative), ± (equivocal), 1+ (weak), 2+ (moderate), 3+ (intense). If a tissue spot contained cells with staining intensity ≥ +1, the tissue spot was recorded as positive.

[0174] Antibody-dependent cellular cytotoxicity (ADCC) assay

[0175] ADCC assays were performed using a modification of a previously described procedure (Boyerinas et al., 2015). Negative selection of NK cells from normal human donor PBMCs was performed using the EasySep Human NK Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's protocol. Purified NK cells were incubated overnight in RPMI-1640 medium supplemented with L-glutamine, 10% FBS, and antibiotics. On the day of the assay, target cells (CFPAC-1, ASPC-1) were labeled with 10 μM calcein AM cell permeable dye (Termo Fisher Scientific, Waltham, MA, USA) for 30 minutes and then lysed at 3.0×10 3 Cells / well were seeded in triplicate into black-walled flat-bottom 96-well culture plates (#655090 Greiner bio-one, Germany). Tumor cells were then treated with 10 μg / mL of human IgG1 isotype control antibody (Thermo Fisher Scientific, Waltham, MA, USA) or NEO-201 (unless otherwise stated), and NK cells were then added at effector to target (E:T) ratios of 12.5:1 and 25:1. After incubation for 4 hours at 37°C, 10 μg / mL of propidium iodide (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well, and the plates were imaged and analyzed using a Celigo imager (Nexcelom Bioscence LLC, Lawrence, MA, USA). The 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].

[0176] Complement-dependent cytotoxicity (CDC) assay

[0177] CDC assays were performed using a modification of a previously described procedure (Konishi et al., 2008). ASPC-1 target cells were labeled with Calcein AM as described above and cultured at 5.0 × 10 3Cells / well were seeded into black-bottom 96-well plates. The cells were then treated with 0.5 or 5.0 μg / mL NEO-201 for 15 minutes at 37°C to condition the cells, and purified rabbit complement (MP Biomedicals, Santa Ana, CA) was then 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 blood cell counter, and specific lysis was calculated as described above for ADCC activity.

[0178] Xenograft antitumor assay

[0179] Tumors were established in 6-week-old female athymic NU / NU nude mice (Charles River Laboratories International, Wilmington, MA) by implanting a suspension of cultured tumor cells in 1X PBS subcutaneously in the right flank of the mice. Once tumors reached approximately 100 mm 3 The mice were sorted by tumor volume and randomly divided into 5 groups (n=10 animals) based on the size of the tumor. The mice were injected intraperitoneally with vehicle alone (saline solution), human IgG1 (250 μg), or NEO-201 (100 and 250 μg) on ​​days 13, 17, and 20 after implantation. The mice also received approximately 1.0×10 7 One group of mice was injected intraperitoneally with human PBMC activated with IL-2 (treated overnight in culture at 200 U / mL) as a source of immune effector cells. One group of mice was similarly treated with NEO-201 but did not receive human PBMC. Tumors were measured every 2-3 days with a digital caliper, and tumor volume (width) was calculated according to the following formula 2 × length) / 2 = mm 3 , where width is the shorter of the two measurements. Mice were also weighed weekly as a general measure of overall health. Tumor volumes >2000 mm 3 The mice were sacrificed according to IACUC guidelines.

[0180] Biodistribution analysis

[0181] 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 6On day 14 after transplantation, mice were injected intravenously with 20 μCi of 125 I-labeled NEO-201, and then autopsy was performed after 1, 2, 4 or 7 days. Blood, tumor tissue and internal organs (lung, kidney, liver, spleen, pancreas, intestine and stomach) were harvested at each time point (n=4 animals), all tissues were weighed, and the radioactivity in the tissue was measured with a γ counter. The data for each mouse was first calculated as cpm / mg tissue, and then the tissue cpm value was standardized relative to the blood cpm value.

[0182] Single-dose toxicity study in cynomolgus monkeys

[0183] Single-dose toxicity studies were conducted in purposefully 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 the dose, and another 14 days of isolation before the dose was applied to adapt the monkey to the research room. Eight male and eight female animals (2 animals / sex / group) were given NEO-201 diluted in saline solution using an infusion pump and a plastic disposable syringe with a catheter extension tube by slow intravenous infusion (approximately 30 minutes ± 5 minutes infusion) at a dose level of 0mg / kg, 5mg / kg, 20mg / kg and 49mg / kg, and the dose level was the highest concentration that the antibody could reach. Blood samples were drawn in all animals receiving NEO-201 at the following time points: before the 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.

[0184] 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 assessed (BL, day 2, day 8, day 15).

[0185] Statistical analysis

[0186] Data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA). Comparisons between two groups were performed by T-test, and p<0.05 was considered statistically significant. The graphs depict mean ± SD from a representative experiment performed in triplicate.

[0187] Example 8

[0188] ALT-803 enhances ADCC mediated by NEO-201

[0189] ALT-803 is a novel IL-15 superagonist complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor alpha / IgG1 Fc fusion protein. This example tests the ability of ALT-803 to modulate ADCC mediated by NEO-201.

[0190] method

[0191] NK cells were isolated from normal donors and treated with different concentrations of ALT-803 for 48 hours before use as effector cells, and a human carcinoma cell line expressing the NEO-201 antigen was used as a target in an in vitro non-radioactive ADCC assay. The ability of ALT-803 to affect the phenotype of NK cells and to modulate NK cell gene expression was evaluated by flow cytometry and by using Nanostring analysis, respectively.

[0192] result

[0193] Treatment with ALT-803 significantly enhanced ADCC activity mediated by NEO-201 against NEO-201-positive cancer cells ( Figure 8 , Fig.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 ADCC activity also from donors with minimal ADCC activity and reduced the effective dose of NEO-201 required to initiate an ADCC response compared to untreated NK cells ( Fig.12 In addition, ADCC activity can be blocked by using anti-CD16 and anti-TIM3 blocking antibodies ( Fig.12 ).

[0194] 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 ( Fig. 9 ).

[0195] Nanostring analysis of human NK cells treated with various concentrations of ALT-803 for 48 hours showed that ALT-803 was able to modulate the mRNA expression of 62 genes (a 1.6 log2 fold change compared to vehicle control was considered significant).

[0196] 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 the activation of apoptosis.

[0197] Thus, ALT-803 enhances ADCC activity mediated by NEO-201 against human cancer cells. The enhancement of ADCC activity may be due in part to increased expression of TIM3, NKG2D, granzyme B, and CD107a-positive NK cells, as well as to the regulation of transcripts involved in NK activation and cytotoxicity.

[0198] In summary, treatment of NK cells isolated from normal donors with ALT-803 can enhance ADCC activity mediated by NEO-201. Phenotypic analysis of NK cells isolated from normal donors treated with ALT-803 demonstrated that ALT-803 can enhance the expression of TIM-3 and NKG2D on CD16 / CD56 positive NK cells. Treatment of normal NK cells with ALT-803 can also increase the MFI of granzyme B in CD16 / CD56 positive NK cells. Treatment of normal NK cells with ALT-803 can also increase 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 gamma production. It is also a mature marker. The enhancement of ADCC activity mediated by NEO-201 after treatment with ALT-803 may be due in part to increased expression of TIM-3 positive, NKG2D positive, granzyme B positive and CD107a positive NK cells, although this theory is not intended to be limiting. Treatment of NK cells with ALT-803 can enhance ADCC activity mediated by lower concentrations of NEO-201. When NK cells are treated with ALT-803, lower concentrations of Mab can be used to mediate ADCC activity and can produce equivalent levels of cytotoxicity compared to NK cells treated with higher concentrations of NEO-201 without ALT-803 treatment. This result suggests that smaller doses of Mab can be used in combination with ALT-803 in clinical trials for the treatment of cancer.

[0199] Example 9

[0200] NEO-201 enhances NK cell-dependent killing of tumor cells by blocking the inhibitory CEACAM5 / CEACAM1 immune checkpoint pathway.

[0201] Immunotherapy using checkpoint blocking antibodies targeting effector cell inhibitory receptors such as PD-1 and CTLA-4 has elicited some significant and durable responses in several tumor types. Carcinoembryonic antigen-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 a potent 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 cytolysis and allows tumor cells to evade NK killing.

[0202] NEO-201 binds to members of the CEACAM family and can activate innate immune mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to kill tumor cells. This study was designed to determine whether NEO-201 blocks the CEACAM1 inhibitory pathway to restore the anti-tumor functionality of NK cells.

[0203] method

[0204] 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 in vitro killing of tumor cells by the NK cell line NK-92 (which expresses CEACAM1 and lacks CD16) and to mediate ADCC.

[0205] Killing 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 min and then incubated at 3.0 × 10 3 Cells / well were seeded in triplicate into black-walled flat-bottom 96-well culture plates. Tumor cells were then treated with 10 μg / mL of human IgG1 isotype control antibody (Thermo Fisher Scientific, Waltham, MA, USA) or NEO-201, 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 16 hours of incubation at 37°C, propidium iodide (PI; Thermo Fisher Scientific, Waltham, MA, USA) was added to each well at a final concentration of 1.67 μg / mL, and the plate was centrifuged, imaged using a Celigo imaging blood cell counter (Nexcelom Bioscence LLC, Lawrence, MA, USA), and analyzed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). The 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 / Average live target count 对照 )x 100].

[0206] result

[0207] NEO-201 was found to react with different variants of CEACAM5 and CEACAM6, but not with CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell lines expressed different levels of native forms of CEACAM5 / 6 versus 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 ( Fig.13 ).

[0208] in conclusion

[0209] 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.

[0210] abbreviation

[0211] Antibody-dependent cellular 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 cytotoxicity (CDC), Clearance (CL), Maximum observed plasma concentration (Cmax), Dose-normalized maximum measured plasma concentration (Cmax / D), Estrogen receptor (ER), Half-life (HL), Immunohistochemistry (IHC), Natural killer cells (NK), Non-small cell lung cancer (NSCLC), Peripheral blood mononuclear cells (PBMC), Progesterone receptor (PR), Tumor-associated antigen (TAA), Time to maximum observed plasma concentration (Tmax), Volume of distribution (Vz).

[0212] References

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[0262] Table 1: Flow cytometry analysis of NEO-201 binding to cultured tumor cell lines derived from various types of solid tumors. The percentage of positive cells and mean fluorescence intensity (MFI) values ​​are detailed for each cell line. NEO-201 positive cell lines are shown in bold. NEO-201 positivity is defined as % positive > 10%.

[0263]

[0264]

[0265] Table 2: IHC profile of NEO-201 staining of normal human microarray tissues.

[0266]

[0267]

[0268] Table 3: Pharmacokinetic results of single-dose NEO-201 administration in cynomolgus monkeys. Eight male and eight female animals (2 animals / sex / group) were injected intravenously 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 (pre-dose, 10 minutes, 1, 2, 4, 6, 24, 48, 72, 96, 168 and 336 hours after dose), and pharmacokinetic measurements from serum preparations were obtained by ELISA. The values ​​in the table represent the average values ​​from 2 animals / sex / group (M, F) or from all 4 animals (all).

[0269] Abbreviations: 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); clearance (CL); maximum observed plasma concentration (Cmax); dose-normalized maximum measured plasma concentration (Cmax / D); half-life (HL); time of maximum observed plasma concentration (Tmax); volume of distribution (Vz).

[0270]

[0271] The present invention relates to the following embodiments:

[0272] 1. A method for killing cancer cells, the method comprising administering an effective amount of NEO-201 antibody to a patient in need thereof.

[0273] 2. A method for treating cancer, comprising administering an effective amount of NEO-201 antibody to a patient in need thereof.

[0274] 3. A method for preventing cancer recurrence, the method comprising administering an effective amount of NEO-201 antibody to a patient in need thereof.

[0275] 4. A method of reducing tumor burden in a patient suffering from a cancer, the method comprising administering to the patient in need thereof an effective amount of the NEO-201 antibody.

[0276] 5. The method of any one of the preceding claims, wherein the antibody mediates complement-mediated cytotoxicity (CDC), thereby killing cancer cells in the patient.

[0277] 6. The method of any of the preceding claims, wherein prior to or at the time of said administering, said patient is natural killer cell ("NK") depleted.

[0278] 7. The method of any one of the preceding claims, wherein prior to or at the time of said administering, said patient is severely NK-depleted.

[0279] 8. The method of claim 6, further comprising determining whether the patient is NK-depleted prior to or at the time of administering.

[0280] 9. The method of claim 6, further comprising determining whether the patient is severely NK-depleted before or during the administering.

[0281] 10. The method of any of the preceding claims, wherein the patient has an NK cell deficiency (NKD), optionally comprising CNKD (eg, CNKD1, CNKD2) or FNKD (eg, FNKD1).

[0282] 11. The method of any of the preceding claims, wherein the patient is NK-depleted or severely NK-depleted as a result of another therapy.

[0283] 12. The method of any one of the preceding claims, wherein the patient is undergoing cancer therapy.

[0284] 13. The method of any one of the preceding claims, wherein the patient is undergoing chemotherapy or radiotherapy.

[0285] 14. The method of claim 13, wherein the chemotherapy comprises administration of one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, 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).

[0286] 15. The method of any one of the preceding claims, wherein prior to or at the time of said administering, NK cells comprise less than 5% of peripheral blood mononuclear cells (PBMCs) in said individual.

[0287] 16. The method of any one of the preceding claims, wherein prior to or at the time of said administering, NK cells comprise less than 3% of peripheral blood mononuclear cells (PBMCs) in said individual.

[0288] 17. The method of any of the preceding claims, wherein prior to or at the time of said administering, less than 70% of the PBMC NK cells in said patient are CD56 dim CD16 + NK cells.

[0289] 18. The method of any of the preceding claims, wherein prior to or at the time of said administering, less than 50% of the PBMC NK cells in said patient are CD56 dimCD16 + NK cells.

[0290] 19. The method of any of the preceding claims, wherein the NEO-201 antibody comprises at least one, two, three, four, five, or all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0291] 20. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38.

[0292] 21. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable light chain sequence that is at least 90% identical to SEQ ID NO:39.

[0293] 21. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38 and a variable light chain sequence that is at least 90% identical to SEQ ID NO:39.

[0294] 22. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence of SEQ ID NO: 38 and a variable light chain of SEQ ID NO: 39.

[0295] 23. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a heavy chain sequence at least 90% identical to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence at least 90% identical to amino acids 20-233 of SEQ ID NO:29.

[0296] 24. The method of claim 22 or 23, wherein the NEO-201 antibody comprises all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0297] 25. The method of any of the preceding claims, wherein the NEO-201 antibody comprises the heavy chain variable region sequence contained in SEQ ID NO:28 and the light chain variable region sequence contained in SEQ ID NO:29.

[0298] 26. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a heavy chain sequence comprising amino acids 20-470 of SEQ ID NO:28 and a light chain sequence comprising amino acids 20-233 of SEQ ID NO:29.

[0299] 27. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a human IgG1 constant domain.

[0300] 28. The method of any of the preceding claims, wherein the NEO-201 antibody is humanized.

[0301] 29. The method of any of the preceding claims, wherein the NEO-201 antibody is conjugated to another moiety.

[0302] 30. The method of any of the preceding claims, wherein the NEO-201 antibody is conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

[0303] 31. The method of any of the preceding claims, further comprising administering to the patient an effective amount of a cytokine agonist to enhance or stimulate killing of cells of the cancer.

[0304] 32. The method of claim 31, wherein the cytokine agonist comprises interleukin 2 (IL-2), interleukin 21 (IL-21), ALT-803, IL-15 inhibitors, checkpoint inhibitors, anti-PD1, anti-PDL1, anti-CTLA-4, anti-41BB, anti-OX40, anti-Tim-3, or a combination thereof.

[0305] 33. The method of any of the preceding claims, further comprising administering to the patient an effective amount of a complement regulatory protein (CRP) antagonist to enhance or stimulate killing of cells of the cancer.

[0306] 34. The method of claim 33, wherein the CRP antagonist antagonizes one or more of CD46, CD55, or CD59.

[0307] 35. The method of claim 33 or 34, wherein the CRP antagonist comprises an antibody or an antigen-binding fragment thereof.

[0308] 36. The method of claim 31, wherein the cytokine agonist comprises an IL-15 agonist or an IL-15 superagonist.

[0309] 37. The method of claim 31, wherein the cytokine agonist comprises a complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor alpha / IgG1 Fc fusion protein.

[0310] 38. The method of claim 37, wherein the cytokine agonist comprises ALT-803.

[0311] 39. The method of any one of claims 31-38, wherein the effective dose of the NEO-201 antibody is reduced compared to treatment with the NEO-201 antibody alone in the absence of the cytokine agonist.

[0312] 40. The method of any of the preceding claims, wherein the cancer comprises colon cancer.

[0313] 41. The method of any of the preceding claims, wherein the cancer comprises pancreatic cancer.

[0314] 42. The method of any one of the preceding claims, wherein the cancer comprises ovarian cancer.

[0315] 43. The method of any of the preceding claims, wherein the cancer comprises gastric cancer.

[0316] 44. The method of any one of the preceding claims, wherein the cancer comprises lung cancer.

[0317] 45. The method of any one of the preceding claims, wherein the cancer comprises breast cancer.

[0318] 46. ​​The method of any of the preceding claims, wherein the cancer comprises uterine cancer.

Claims

1. Use of an antibody in the preparation of a medicament for killing cancer cells in a cancer patient, wherein the patient has been determined to be natural killer cell ("NK") depleted, such that NK cells account for less than 5% of the patient's peripheral blood mononuclear cells (PBMCs), and / or wherein the patient has been determined to have NK cell deficiency (NKD), and wherein the use of the antibody stimulates ADCC- and CDC-mediated cancer cell killing in the NK cell depleted or NK cell deficient patient; The antibody comprises: (i) a heavy chain comprising amino acids 20-470 of SEQ ID NO:28 and a light chain comprising amino acids 20-233 of SEQ ID NO:29, or a heavy chain variable region of SEQ ID NO:38 and a light chain variable region of SEQ ID NO:39, and (ii) a human IgG1 constant region.

2. Use of an antibody in the preparation of a medicament for treating a cancer in a patient in need thereof, wherein the patient has been determined to be natural killer cell ("NK") depleted, such that NK cells account for less than 5% of the patient's peripheral blood mononuclear cells (PBMCs), and / or wherein the patient has been determined to have NK cell deficiency (NKD), and wherein the use of the antibody stimulates ADCC- and CDC-mediated killing of cancer cells in the NK cell-depleted or NK cell-deficient patient; The antibody comprises: (i) a heavy chain comprising amino acids 20-470 of SEQ ID NO:28 and a light chain comprising amino acids 20-233 of SEQ ID NO:29, or a heavy chain variable region of SEQ ID NO:38 and a light chain variable region of SEQ ID NO:39, and (ii) a human IgG1 constant region.

3. Use of an antibody in the preparation of a medicament for preventing cancer recurrence in a patient in need thereof, wherein the patient has been determined to be natural killer cell ("NK") depleted, such that NK cells account for less than 5% of the patient's peripheral blood mononuclear cells (PBMCs), and / or wherein the patient has been determined to have NK cell deficiency (NKD), and wherein the use of the antibody stimulates ADCC- and CDC-mediated cancer cell killing in the NK cell-depleted or NK cell-deficient patient; The antibody comprises: (i) a heavy chain comprising amino acids 20-470 of SEQ ID NO:28 and a light chain comprising amino acids 20-233 of SEQ ID NO:29, or a heavy chain variable region of SEQ ID NO:38 and a light chain variable region of SEQ ID NO:39, and (ii) a human IgG1 constant region.

4. Use of an antibody in the preparation of a medicament for reducing tumor burden in a patient with a cancer tumor in a patient in need thereof, wherein the patient has been determined to be natural killer cell ("NK") depleted, such that NK cells account for less than 5% of the patient's peripheral blood mononuclear cells (PBMCs), and / or wherein the patient has been determined to have NK cell deficiency (NKD), and wherein the use of the antibody stimulates ADCC- and CDC-mediated killing of cancer cells in the NK cell-depleted or NK cell-deficient patient; The antibody comprises: (i) a heavy chain comprising amino acids 20-470 of SEQ ID NO:28 and a light chain comprising amino acids 20-233 of SEQ ID NO:29, or a heavy chain variable region of SEQ ID NO:38 and a light chain variable region of SEQ ID NO:39, and (ii) a human IgG1 constant region.

5. The use according to any one of claims 1 to 4, wherein the patient has been determined to be severely NK-depleted before or at the time of the administration.

6. The use of any one of claims 1-4, wherein the patient has been determined to have an NK cell deficiency (NKD), including CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD1).

7. The use according to any one of claims 1 to 4, wherein the patient is NK-depleted or severely NK-depleted due to another therapy.

8. The use according to claim 7, wherein the other therapy is chemotherapy or radiotherapy.

9. The use of claim 8, wherein the chemotherapy comprises one or more proteasome inhibitors, histone deacetylase inhibitors, genotoxic agents, GSK inhibitors, BET inhibitors, HSP90 inhibitors, microtubule assembly inhibitors and / or immunomodulatory drugs.

10. The use according to any one of claims 1 to 4, wherein before or during administration of the medicament, NK cells account for less than 5% of peripheral blood mononuclear cells (PBMC) in the patient.

11. The use according to any one of claims 1 to 4, wherein before or during administration of the drug, NK cells account for less than 3% of peripheral blood mononuclear cells (PBMC) in the patient.

12. The use of any one of claims 1 to 4, wherein before or during administration of the drug, less than 70% of the PBMC NK cells in the patient are CD56 dim CD16 + NK cells.

13. The use of any one of claims 1 to 4, wherein before or during administration of the drug, less than 50% of the PBMC NK cells in the patient are CD56 dim CD16 + NK cells.

14. The use of any one of claims 1-4, wherein the NEO-201 antibody comprises a heavy chain comprising amino acids 20-470 of SEQ ID NO: 28 and a light chain comprising amino acids 20-233 of SEQ ID NO: 29, and a human IgG1 constant domain.

15. The method of any one of claims 1 to 4, wherein the drug further comprises a cytokine agonist to enhance or stimulate the killing of cancer cells.

16. The use of claim 15, wherein the cytokine or cytokine agonist comprises interleukin 2 (IL-2), interleukin 21 (IL-21), IL-15 agonist, optionally ALT-803, checkpoint inhibitors, anti-PD1, anti-PDL1, anti-CTLA-4, anti-41BB, anti-OX40, anti-Tim-3 or a combination thereof.

17. The method according to any one of claims 1 to 4, wherein the drug further comprises a complement regulatory protein (CRP) antagonist to enhance or stimulate the killing of cancer cells.

18. The use of claim 17, wherein the CRP antagonist antagonizes one or more of CD46, CD55 or CD59.

19. The use according to claim 17 or 18, wherein the CRP antagonist comprises an antibody or an antigen-binding fragment thereof.

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