Monoclonal Antibody NEO-201 for the Treatment of Human Cancer

Abstract

To provide a method of killing carcinoma cells.SOLUTION: NEO-201 is a humanized IgG1 monoclonal antibody (mAb) that is highly reactive against a majority of tumor tissues from many different carcinomas, including colon, pancreatic, stomach, lung, breast, and uterine cancers, but an overwhelming majority of normal tissues are not recognized by this antibody. Functional assays revealed that NEO-201 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against tumor cells. Furthermore, growth of human pancreatic xenograft tumors in vivo was largely attenuated by treatment with NEO-201 both alone and in combination with human peripheral blood mononuclear cells (PBMC) as an effector cell source for ADCC. In vivo biodistribution studies in human tumor xenograft-bearing mice revealed that NEO-201 preferentially accumulates in the tumor but not organ tissue.SELECTED DRAWING: Figure 1

Description

Technical Field

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

[0002] Array Expression Information This application includes biological sequences listed in a file named "43282o4402.txt", created on Nov. 2, 2018, having a size of 32,563 bytes, which is hereby incorporated by reference in its entirety as part of this disclosure.

Background Art

[0003] Cancer is one of the most frequent causes of death worldwide, and it is predicted that as early as 2025, there will be an estimated 20 million new cases per year (Ferlay et al., 2015). Conventional cancer treatments such as surgery, radiation, and chemotherapy often induce severe side effects, fail to cure the majority of patients with advanced disease, and result in recurrence (Bodey et al., 1996). More recent treatments have been developed mainly to selectively target cancerous cells while sparing normal healthy tissues. Among these, immunotherapy has revolutionized the field of cancer medicine and has become an important treatment option for cancer patients.

[0004] The fundamental principle of cancer immunotherapy is known as immunoediting (Mittal et al., 2014), which is an extrinsic mechanism of cancer suppression that only begins after cellular transformation has occurred and the intrinsic mechanisms of cancer suppression have failed. The immunoediting process occurs in three phases: elimination, equilibrium, and escape. Between each of the elimination and equilibrium phases, the immune rejection response of cancer cells either dominates or balances the growth of cancer cells to control malignant growth. However, in the escape phase, once-suppressed cancer cells may evade immune recognition through insensitivity to immune effector mechanisms and / or induction of immunosuppression in the tumor microenvironment. Cancer cells that have evaded immune recognition can grow and proliferate more freely and become clinically apparent disease (Dunn et al., 2004). The goal of cancer immunotherapy is to keep cancer cells in the elimination and / or equilibrium phases by suppressing tumor growth, delaying tumor recurrence, and prolonging survival by generating and / or amplifying an antitumor immune response (Carter, 2001; Hodge et al., 2006; Vergati et al., 2010; Gabitzsch et al., 2015). Treatment approaches include treating patients with checkpoint inhibitory antibodies, antitumor vaccines, and chimeric antigen receptor (CAR)-T cells, all of which utilize adaptive immunity by T cells. However, innate immunity can also generate and enhance an antitumor response, and monoclonal antibodies (mAbs) targeting tumors can be used to stimulate innate antitumor immunity (Topalian et al., 2011).

[0005] NEO-201 is a novel humanized IgG1 mAb generated against the Hollinshead allogeneic colorectal cancer vaccine platform (Hollinshead et al., 1970; Hollinshead et al., 1972). The immunogenic component of this vaccine was a tumor-associated antigen (TAA) derived from tumor membrane fractions pooled from surgically resected specimens of 79 colorectal cancer patients (Hollinshead et al., 1985). These membrane fractions were semi-purified, screened for delayed-type hypersensitivity (DTH) in colorectal cancer patients versus healthy volunteers, and evaluated in clinical trials of refractory colorectal cancer patients (Hollinshead et al., 1985; Hollinshead, US4810781, 1989; Bristol & Kantor, US Patent No. 7829678, 2010). In these trials, clinical benefit defined by both anti-tumor response and significant prolongation of overall survival in patients who developed a sustained IgG response in addition to a cell-mediated response to the vaccine was reported, suggesting that the vaccine contains an immunogenic component capable of generating anti-tumor antibodies (Hollinshead, 1991). This first colorectal cancer vaccine has been used to generate monoclonal antibodies in mice and previously described ensituximab (NPC-1C / NEO-102) (Luka et al., 2011; Patel et al., 2013; Beg et al., 2016; Kim et al, 2017) and NEO-201. Preliminary studies have suggested 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.

[0006] The human cancer fetal antigen (CEA) family is composed of 29 genes tandemly arranged on chromosome 19q13.2. These genes are classified into two major subfamilies, CEACAM and pregnancy-specific glycoprotein subgroups, based on nucleotide homology. Examples of CEACAM-encoded proteins include CEA (CEACAM5), CEA-related cell adhesion molecule (CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, and CEACAM8). The CEACAM family belongs to the Ig superfamily. Structurally, each human CEACAM contains 108 - 110 amino acids and one N-terminal domain that is homologous to the Ig variable domain, followed by a variable number (0 - 6) of IgC2-type constant-like domains. CEACAM proteins can interact with each other through homo- and hetero-affinity. CEACAM1 is a unique protein within this family because it contains a PD1-like ITIM (immunoreceptor tyrosine-based inhibitory motif) within its cytoplasmic domain. This inhibitory effect is caused by phosphorylation of tyrosine residues by ITIM, and as a result, tyrosine phosphatases -1 and -2 containing Src homology 2 domains are recruited. CEACAM1 protein is expressed in various immune cells such as monocytes, granulocytes, activated T cells, B cells, and NK cells. CEACAM1 occurs as several isoforms, and the two major isoforms are CEACAM1-L and CEACAM1-S, which have long (L) or short (S) cytoplasmic domains, respectively. Expression of CEACAM1-S 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.

[0007] Monoclonal antibodies (mAbs) are composed of a unique antigen-binding region (fragment antigen-binding, Fab) that is specific for a given mAb and a constant region (fragment crystallizable, Fc) that is common to all mAbs of the same isotype. The Fc region can regulate the activity of immune cells by engaging members of the Fc receptor (FcR) family expressed on the surface of specific immune cell types. In particular, human IgG1 mAbs can interact with the Fc gamma receptor IIIa (FcγRIIIa, CD16) expressed on macrophages and NK cells. This interaction stimulates macrophages, leading to the phagocytosis of mAb-opsonized cancer cells, and activates NK cells, causing cancer cells to degranulate and lyse via a mechanism known as antibody-dependent cell cytotoxicity (ADCC). ADCC has been shown to be a major mediator of the in vivo antitumor effect in many preclinical studies and plays an important role in the mechanism of action of several mAbs used in cancer therapy (Seidel et al., 2013). Examples of clinically approved mAbs that can mediate ADCC include trastuzumab, which targets the HER2 receptor in breast cancer (Seidel et al., 2013; Petricevic et al., 2013); rituximab, which targets the pan-B cell marker CD20 in lymphoma (Seidel et al., 2013; Dall’Ozzo et al., 2004); cetuximab, which targets the epidermal growth factor receptor (EGFR) in colorectal and head and neck cancers (Seidel et al., 2013; Levy et al., 2009; Kawaguchi et al., 2007; Lopez-Albaitero et al., 2009); and avelumab, which targets the immunosuppressive ligand PD-L1 in Merkel cell carcinoma and bladder cancer (Boyerinas et al., 2015). Additionally, the Fc region can interact with the C1 complex and activate complement-dependent cytotoxicity (CDC). Here, a proteolytic cascade forms pores in the plasma membrane that cause lysis of the antibody-targeted cells.Even when antitumor CDC has been demonstrated in vitro, it has been debated whether it is important for the clinical efficacy of mAb therapy in cancer (Meyer et al., 2014).

[0008] In the prior U.S. Patents 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 of the applicant (each of which is hereby incorporated by reference in its entirety), various anti-cancer antibodies, cancer antigens, and related technologies have been disclosed. SUMMARY OF THE INVENTION

[0009] In the studies described in the examples of this specification, the in vitro binding properties, in vivo activity, and localization of NEO-201 were evaluated in a preclinical model. NEO-201 exhibited broad reactivity against a panel of human cancer cell lines and tumor tissues, but did not bind to the majority of healthy tissues. Furthermore, NEO-201 exhibited both ADCC and CDC activities against human cancer cells in vitro, and NEO-201 significantly attenuated the growth of human pancreatic xenograft tumors in vivo, both alone as an effector cell source for ADCC and in combination with human peripheral blood mononuclear cells (PBMCs). Finally, in a single-dose toxicity study in non-human primates, the only adverse effect observed was a transient decrease in circulating neutrophils, indicating the safety and tolerability of NEO-201. These studies provide a basis for the potential clinical utility of NEO-201 as a novel therapeutic agent for the treatment of various solid tumors. Furthermore, the observed CDC activity of the antibody in question opens the opportunity to treat immunocompromised patients, such as those who are immunocompromised due to disease or as a result of the effects of radiation, chemotherapy, and treatment of other diseases, in whom ADCC is not expected to be effective.

[0010] The present applicants have previously reported the preclinical anti-tumor activity (Patel et al., 2013) as well as the clinical safety and efficacy (Beg et al., 2016; Kim et al., 2017) of a mAb (called ensituximab (NPC-1C / NEO-102)) generated against the Hollinshead syngeneic colorectal cancer vaccine platform. In this report, we describe the characterization of a second tumor antigen-targeted mAb derived from the same vaccine platform called NEO-201. NEO-201 has been shown to stain various human cancer cell lines, such as cells derived from various tumor types, histological subtypes, and mutation profiles, positively in vitro. NEO-201 positivity was observed at a higher frequency in tumor cell lines derived from lung adenocarcinoma versus squamous cell carcinoma, and in HER2-positive breast cancer cell lines versus triple-negative lines. Staining of human tumor samples showed that diverse cancer tissues, such as tumors of the colon, pancreas, stomach, lung, breast, and uterus, were positively stained for NEO-201. An expanded investigation using a larger sample size may reveal that NEO-201 can distinguish between histological and / or molecular subtypes of various cancer tumors. Interestingly, a higher percentage of tumor tissues, as opposed to cultured cancer cell lines, reacted with NEO-201. From this observation, it can be shown that the targets recognized by NEO-201 are more readily expressed in vivo than in vitro. This suggests that target expression is at least partially dependent on the interaction of tumor cells with factors from the local microenvironment. Experiments are currently underway to further clarify the characteristics of the antigen(s) and epitope(s) recognized by NEO-201, and to determine the regulatory mechanism(s) that govern its expression in tumor tissue rather than normal tissue.

[0011] This investigation revealed that the vast majority of healthy normal tissues and normal tissues adjacent to tumor tissues were negative for NEO-201, thus demonstrating that NEO-201 is highly tumor-specific in the staining profile. NEO-201 positivity was observed in normal tongue and cervical tissues, but the staining intensity was weak and represented only the smallest sample size (n = 2) on the microarray. To confirm these observations, a further expanded analysis of NEO-201 staining in normal tissue samples was conducted. Additionally, no macroscopically observable toxicity was induced in mice following NEO-201 administration, and the tolerance was good when administered to non-human primates. The observation of neutrophil depletion in non-human primates suggests that the antigen(s) reacting with NEO-201 are expressed on these immune cells, and the evaluation of the reactivity of NEO-201 with hematopoietic cell types is ongoing. From these promising results, it is suggested that 1) NEO-201 may have diagnostic utility in differentiating between benign and cancerous tissues from patient biopsies, and 2) NEO-201 can effectively target tumors without causing significant toxicity or off-target effects other than neutropenia. Currently, efforts are underway to further evaluate the safety and tolerance of NEO-201, and clinical trials for cancer treatment using NEO-201 are planned.

[0012] The innate immune effector mechanism has been shown to play a major role in promoting and enhancing the host's anti-tumor immunity. It is well known that the Fc portion of human IgG1 mAb can activate innate immunity against opsonized targets and mediate ADCC and / or CDC (Strome et al., 2007; Hayes J, et al., 2017). In particular, the ability to mediate ADCC is considered an important factor in the therapeutic efficacy of various human IgG1 mAbs approved for cancer treatment (Boyerinas et al., 2015; Seidel et al., 2013; Petricevic et al., 2013 Dall’Ozzo et al., 2004; Levy et al., 2009; Kawaguchi et al., 2007; Lopez-Albaitero et al., 2009). Importantly, the V158F polymorphism within the FCGR3A gene (encoding FcγRIIIa) is associated with differences in the affinity of human IgG1 mAbs (Koene et al., 1997; Wu et al., 1997), and immune cells derived from donors with the high-affinity V / V genotype exhibit higher trastuzumab-mediated ADCC activity in vitro (Musolino et al., 2008). The V / V genotype has also been shown to be significantly correlated with objective response rate and progression-free survival in breast cancer patients treated with trastuzumab (Musolino et al., 2008), providing indirect clinical evidence regarding the role of ADCC in mAb-based therapy. Treatment of tumor cells with NEO-201 enhanced the cytotoxic activity of NK cells by 2- to 5-fold and maintained ADCC activity even at low antibody concentrations (0.1 μg / mL), so NEO-201 can mediate ADCC in vitro. These data raise the possibility that patients with the V / V genotype may derive additional benefit from NEO-201 treatment. An additional perspective is that enhancement of NK cell function by cytokine stimulation may improve ADCC activity and potentially the clinical benefits of NEO-201.IL-2 is well known to be a potent activator of NK cells (Hank et al., 1990), and it has been shown that IL-21 enhances the ADCC activity mediated by trastuzumab and cetuximab (Watanabe et al., 2010). In recent preclinical studies using ALT-803, a novel fusion protein superagonist of IL-15 signaling, the proliferation, activation, and lytic ability of NK cells (and CD8+ T cells) have been significantly improved, which has been demonstrated to result in significant antitumor activity in various animal models of cancer (Han et al., 2011; Gomes-Giacoia et al., 2014; Mathios et al., 2016; Rhode et al., 2016; Kim et al., 2016; Felices et al., 2017). Interestingly, ALT-803 substantially enhances in vitro NK cell degranulation, IFN-γ production, and rituximab-mediated ADCC against B cell lymphoma cell lines and primary follicular lymphoma cells, and combination therapy with ALT-803 and rituximab in two B cell lymphoma models in vivo has been found to significantly reduce tumor cell mass and improve survival (Rosario et al., 2016).

[0013] Another innate immune effector mechanism that MAb can engage is the activation of the complement system that promotes CDC, and NEO-201 has been found to have the ability to kill tumor cells via CDC. There is room for debate as to whether CDC contributes to the therapeutic efficacy of MAb, but in at least some specific examples, it has been suggested to be beneficial for cancer treatment (Meyer et al., 2014). Furthermore, several different complement regulatory proteins (CRPs) function to inhibit complement activation, and specific membrane-bound CRPs such as CD46, CD55, and CD59 have been reported to be abnormally expressed in various cancers that are likely to confer resistance to CDC (Seya et al., 1994; Niehans et al., 1996; Donin et al., 2003). In future investigations, a strategy to block CRP will be used to confirm whether it can enhance NEO-201-mediated CDC in resistant tumor cells.

[0014] In the evaluation of NEO-201 in vivo, a significant anti-tumor effect was revealed when administered in combination with activated human immune effector cells. This combination resulted in complete regression even in some mice (5 / 20, 25%) in the two combination groups. Furthermore, NEO-201 was found to preferentially localize in xenograft tumor tissues but not in various healthy tissues. From these data, it was confirmed that the mechanism of action of NEO-201 against tumors is ADCC-dependent lysis of tumor cells by innate immune cells. However, it should be noted that anti-tumor activity was also observed with NEO-201 alone without adding human immune cells to immunodeficient mice. In the ADCC assay, in vitro, treatment of CFPAC-1 tumor cells with NEO-201 did not induce substantial toxicity, so this phenomenon may be specific to the conditions occurring in vivo. One hypothesis regarding NEO-201 activity in the absence of immune effector cells could be the induction of CDC. The CDC activity of NEO-201 was directly demonstrated in further experiments described in Example 3.

[0015] In summary, this study demonstrated that NEO-201 is a highly tumor-specific antibody that can engage natural immune effector mechanisms such as both ADCC and CDC to kill tumor cells. Furthermore, the safety and anti-tumor efficacy of NEO-201 in an in vivo xenograft model of pancreatic cancer, as well as its tolerability in non-human primates, were demonstrated. These findings form the basis for the clinical development of NEO-201 as a diagnostic and therapeutic agent for patients with various types of cancer. Since anti-tumor effects can occur from CDC even in the absence of strong ADCC activity, these results also support the use of NEO-201 in immunocompromised (low NK cell level) patients.

Brief Description of the Drawings

[0016]

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Mode for Carrying Out the Invention

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

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

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

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

[0021] This antibody can mediate complement-mediated cytotoxicity (CDC), thereby killing the cancer tumor cells of the patient.

[0022] The patient may have depleted natural killer (“NK”) cells before or at the time of administration. The patient may have severe NK depletion before or at the time of administration. The patient may have a natural killer cell deficiency (NKD), such as CNKD (e.g., CNKD1, CNKD2), or FNKD (e.g., FNKD1). The patient may have NK depletion or severe NK depletion as a result of another treatment, such as a 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., radicicola, 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel), and / or immunomodulatory agents (e.g., lenalidomide).

[0023] This method may include determining whether the patient has depleted NK cells before or at the time of administration.

[0024] This method may include determining whether the patient has severe NK depletion before or at the time of administration.

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

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

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

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

[0029] The NEO-201 antibody can contain at least 1, 2, 3, 4, 5, or all 6 of the CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO: 29.

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

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

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

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

[0034] The NEO-201 antibody can contain all 6 CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO: 29.

[0035] The NEO-201 antibody can contain a human IgG1 constant domain.

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

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

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

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

[0040] This method may further comprise administering to the patient an effective amount of a complement regulatory protein (CRP) antagonist to enhance or stimulate the killing of cancer cells. The CRP antagonist can antagonize one or more of CD46, CD55, or CD59. The CRP antagonist can include an antibody or an antigen-binding fragment thereof.

[0041] The cytokine agonist can include an IL-15 agonist or an IL-15 superagonist.

[0042] The cytokine agonist can include a complex consisting of an IL-15 variant (IL-15N72D) bound to an IL-15 receptor α / IgG1 Fc fusion protein such as ALT-803.

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

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

[0045] In another embodiment, the present disclosure provides a method of killing cancer tumor cells, comprising administering an effective amount of the NEO-201 antibody to a patient in need thereof, wherein the patient has depleted natural killer (“NK”) cells before or at the time of administration. NK depletion can be a patient having less than 5% or less than 3% NK cells among peripheral blood mononuclear cells (PBMCs) in a patient-derived sample, such as a blood sample. Alternatively or additionally, in this method, less than 70% (optionally less than 50%) of the patient's PBMC NK cells can be CD56dimCD16+ NK cells before or at the time of administration.

[0046] In another embodiment, the present disclosure provides a method of treating cancer tumor, comprising administering an effective amount of the NEO-201 antibody to a patient in need thereof, wherein the patient has depleted natural killer (“NK”) cells before or at the time of administration.

[0047] In another embodiment, the present disclosure provides a method of preventing recurrence of cancer tumor, comprising administering an effective amount of the NEO-201 antibody to a patient in need thereof, wherein the patient has depleted natural killer (“NK”) cells before or at the time of administration.

[0048] In another embodiment, the present disclosure provides a method of reducing the tumor mass of a patient having cancer tumor, comprising administering an effective amount of the NEO-201 antibody to a patient in need thereof, wherein the patient has depleted natural killer (“NK”) cells before or at the time of administration.

[0049] In the foregoing method, the antibody mediates CDC, whereby, for example, even if there is no effective ADCC due to NK depletion in the patient, it can thereby kill cancer tumor cells in the patient. The patient can be severely NK-depleted at the time of administration. Optionally, the method further includes determining, for example, during the period at or before administration, such as 1 week or 2 weeks before, whether the patient is NK-depleted or severely NK-depleted. The NK depletion or severe NK depletion state may be inferred from the patient's medical history, such as the prior or concurrent use of another therapy that depletes NK cells. For example, the patient has received or is receiving cancer therapy such as radiotherapy or chemotherapy. The cancer therapy may include administering one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, suberoylanilide-hydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, aphidicolin, mitomycin, methotrexate, etoposide), GSK inhibitors (e.g., LiCl, BIO, SB21), BET inhibitors (e.g., JQ1), HSP90 inhibitors (e.g., radicicola, 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel), and / or immunomodulatory agents (e.g., lenalidomide).

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

[0051] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the NEO-201 antibody may include at least 1, 2, 3, 4, 5, or all 6 of the CDR sequences included in SEQ ID NO: 28 and SEQ ID NO: 29.

[0052] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the NEO-201 antibody can comprise a variable heavy chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to SEQ ID NO: 38. The variable heavy chain having the above percent sequence identity can comprise all three CDR sequences contained in SEQ ID NO: 38.

[0053] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the NEO-201 antibody can comprise a variable light chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to SEQ ID NO: 39. The variable light chain can comprise all three CDR sequences contained in SEQ ID NO: 39.

[0054] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the NEO-201 antibody can comprise a variable heavy chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to SEQ ID NO: 38, and a variable light chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to SEQ ID NO: 39. The variable light chain can comprise all three CDR sequences contained in SEQ ID NO: 39, and the variable heavy chain having the percent sequence identity can comprise all three CDR sequences contained in SEQ ID NO: 38.

[0055] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the NEO-201 antibody may include a heavy chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to amino acids 20 to 470 of SEQ ID NO: 28, and a light chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to amino acids 20 to 233 of SEQ ID NO: 29. The light chain may include all three CDR sequences included in SEQ ID NO: 29, and the variable heavy chain having percent sequence identity may include all three CDR sequences included in SEQ ID NO: 28.

[0056] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the NEO-201 antibody may include the heavy chain variable region sequence included in SEQ ID NO: 28 and the light chain variable region sequence included in SEQ ID NO: 29.

[0057] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the NEO-201 antibody may include a heavy chain sequence including amino acids 20 to 470 of SEQ ID NO: 28 and a light chain sequence including amino acids 20 to 233 of SEQ ID NO: 29.

[0058] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the NEO-201 antibody includes a human IgG1 constant domain.

[0059] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the NEO-201 antibody can be humanized.

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

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

[0062] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the method may further comprise administering to the patient an effective amount of a complement regulatory protein (CRP) antagonist to enhance or stimulate the killing of cancer cells. The CRP antagonist can antagonize one or more of CD46, CD55, or CD59. The CRP antagonist can include an antibody or an antigen-binding fragment thereof. The cytokine agonist can include an IL-15 agonist or an IL-15 superagonist. The cytokine agonist can include a complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor α / IgG1 Fc fusion protein. The cytokine agonist can include ALT-803.

[0063] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the effective dosage of the NEO-201 antibody is reduced as compared to treatment with the NEO-201 antibody alone without a cytokine agonist.

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

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

[0066] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the cancer can be pancreatic cancer.

[0067] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the cancer can be ovarian cancer.

[0068] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the cancer can be gastric cancer.

[0069] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the cancer can be lung cancer.

[0070] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the cancer can be breast cancer.

[0071] In a preferred embodiment of the invention that can be used with any of the foregoing or following embodiments, the cancer can be uterine cancer.

[0072] Definitions

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

[0074] As used in the description of the present specification and throughout the following claims, unless the context clearly dictates otherwise, the meanings of "a," "an," and "the" include plural referents.

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

[0076] As used herein, the term "NK depletion" or "natural killer cell depletion" refers to a patient having a low natural killer (NK) cell level as compared to the normal range. NK cells are cytotoxic innate immune lymphocytes. Typically, NK cells constitute 5 - 20% of peripheral blood mononuclear cells (PBMCs) in a healthy individual. A patient having NK cells that constitute less than 5% of PMBCs is said to have NK depletion. Further, if the NK cells constitute less than 3% of PMBCs, the patient is said to have severe NK cell depletion. Further, in a normal individual, up to 90% of PBMC NK cells are CD56 dim CD16 + NK cells, which are considered to be the most cytotoxic subset. If less than 70% of PBMC NK cells are CD56 dim CD16 + NK cells, the patient is said to have NK depletion. Further, if less than 50% of PBMC NK cells are CD56 dim CD16 + NK cells, the patient is said to have severe NK depletion. A given patient may be said to have NK depletion or severe NK depletion based on meeting one or both of these individual criteria. Generally speaking, the status of a patient as having NK depletion or severe NK depletion is determined by examining a sample taken from the patient, e.g., a blood sample, e.g., a sample obtained and examined within one or two weeks prior. The status of a patient as having NK depletion or severe NK depletion may also be inferred from the diagnosis of a disease associated with such depletion of NK cells and / or a course of treatment.

[0077] NK depletion includes subjects having natural killer cell deficiency (NKD). Exemplary NKD states include classical NKD (CNKD) characterized by the absence of NK cells and their function among peripheral blood lymphocytes, and functional NKD (FNKD) characterized by the presence of NK cells within peripheral blood lymphocytes and having a defect in NK cell activity. In both CNKD and FNKD, the abnormality of NK cells is the major immunological defect, which results in an insufficient ADCC response. CNKD and FNKD can be further classified more finely based on patient characteristics such as the identity of the causative gene(s) or other patient characteristics. Examples of CNKD include CNKD subtype 1 (CNKD1) which is autosomal dominant and associated with a defect in the GATA2 gene, and CNKD subtype 2 (CNKD2) which is autosomal recessive and associated with a defect in the MCM4 gene. An example of FNKD is FNKD1 which is autosomal recessive and associated with a defect in the FCCR3A gene.

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

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

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

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

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

[0083] As used herein, "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, it broadly refers to conservatively modified variants that encode the same or essentially the same amino acid sequence, or, when the nucleic acid does not encode an amino acid sequence, refers to essentially the same sequence. Due to the degeneracy of the genetic code, a large number of nucleic acids that are functionally identical encode any given protein. Such nucleic acid diversity is "silent variation" and is a type of conservatively modified variation. Every possible silent variation of the nucleic acids encoding the polypeptides described herein is described. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except for 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.

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

[0085] As used herein, "control amount" broadly refers to a marker and can be any amount or range of amounts that is compared to the test amount of the marker. For example, the control amount of a marker can be the amount of the marker in a patient having a particular disease or condition or in a human not having such a disease or condition. The control amount can be either an absolute amount (e.g., micrograms / ml) or a relative amount (e.g., relative intensity of a signal).

[0086] As used herein, "differentially present" broadly refers to a difference in the amount or quality of a marker present in a sample taken from a patient having a disease or condition, as compared to an equivalent sample taken from a patient not having one of the diseases or conditions. For example, when measured by a hybridization / or NAT-based assay, a nucleic acid fragment may optionally be present differently between two samples if, for example, the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample. A polypeptide is present differently between two samples if the amount of the polypeptide in one sample is substantially different from the amount of the polypeptide in the other sample. It should be noted that such markers can be considered to be present differently if the marker is detectable in one sample and not detectable in the other sample. Optionally, a relatively small amount of upregulation can serve as a marker.

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

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

[0089] As used herein, "effective amount" broadly refers to an amount of a compound, antibody, antigen, or cell that is sufficient to effect such treatment of a disease when administered to a patient for treating the disease. An effective amount may be an amount effective for prevention and / or prophylaxis. An effective amount may be an amount effective for reducing the occurrence of signs / symptoms, preventing the occurrence of signs / symptoms, reducing the severity of the occurrence of signs / symptoms, eliminating the occurrence of signs / symptoms, delaying the development of the occurrence of signs / symptoms, preventing the development of the occurrence of signs / symptoms, and / or resulting in the prevention of the occurrence of signs / symptoms. An "effective amount" may vary depending on the disease and its severity, as well as the age, weight, medical history, sensitivity, and existing condition of the patient being treated. The term "effective amount" is synonymous with "therapeutically effective amount" for the purposes of the present invention.

[0090] As used herein, the term "expression vector" broadly refers to any recombinant expression system for the constitutive or inducible expression of the nucleic acid sequences of the present invention in vitro or in vivo in any cell, such as prokaryotic, yeast, fungal, plant, insect or mammalian cells. This term includes linear or circular expression systems. This term includes expression systems that remain episomal or are integrated into the host cell genome. The expression system may be capable of self-replicating or not self-replicating, i.e., driving only transient expression in the cell. This term includes recombinant expression cassettes containing only the minimal elements necessary for the transcription of recombinant nucleic acids.

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

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

[0093] As used herein, "high affinity" means at least 10 -8 M, more preferably at least 10 -9 M, even more preferably at least 10 -10Refers broadly to an antibody having a KD of M. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for the IgM isotype refers to an antibody having a KD of at least 10 -7 M, more preferably at least 10 -8 M.

[0094] As used herein, "homology" refers broadly 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 not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The term "sequence identity" can be used synonymously with "homology".

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

[0096] As used herein, "hybridization" refers broadly to the physical interaction of complementary (such as partially complementary) polynucleotide strands by the formation of hydrogen bonds between complementary nucleotides when the strands are arranged antiparallel to each other.

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

[0098] As used herein, "immunoassay" broadly refers to an assay that specifically binds an antigen using an antibody. An immunoassay can be characterized by using the specific binding properties of a particular antibody to isolate, target, and / or quantify an antigen.

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

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

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

[0102] As used herein, "mammal" broadly refers to any and all warm-blooded vertebrates of the mammalian class, such as humans, characterized by a body hair covering of the skin and, in females, milk-producing mammary glands for raising offspring. Examples of mammals include, but are not limited to, alpaca, armadillo, capybara, cat, camel, chimpanzee, chinchilla, cow, dog, goat, gorilla, hamster, horse, human, squirrel monkey, llama, mouse, non-human primates, pig, rat, sheep, vole, lizard, and bat. Mammals include, but are not limited to, Bovidae, Canidae, Equidae, Felidae, Muridae, Ovine, Suidae, Primates, and Rodents. Mammals also include any and all those listed in the World Mammals maintained by the National Museum of Natural History, Smithsonian Institution (Washington DC).

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

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

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

[0106] As used herein, "patient" broadly refers to any animal in need of treatment for either reducing a disease state or preventing the occurrence or recurrence of a disease state. Also, "patient" as used herein broadly refers to any animal that has risk factors, medical history, susceptibility, symptoms, signs, and has or has had a disease diagnosed 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 companion animal, livestock animal, farm animal, exotic animal, or zoo animal. The term "subject" can be used synonymously with the term "patient".

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

[0108] As used herein, "promoter" broadly refers to an array of nucleic acid sequences that direct transcription of nucleic acids. When used herein, promoters include, for example, in the case of type II polymerase promoters, the necessary nucleic acid sequences near the transcription start site, such as the TATA element. Also included as promoters are, optionally, distal enhancer or repressor elements that can be located at a distance of up to 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 the control of environmental or developmental regulation.

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

[0110] As used herein, "prevention" broadly refers to a course of treatment in which signs and / or symptoms are absent in the patient, in remission, or were previously present in the patient. Prevention includes preventing a disease that occurs after treatment of a patient's disease. Further, prevention includes treating patients who are potentially at risk of developing a disease, particularly patients who are predisposed to the disease (e.g., members of a patent population, patients having risk factors, or patients at risk of developing the disease).

[0111] As used herein, "recombinant" refers broadly to a product, such as a cell, or a nucleic acid, protein, or vector, that 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 is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene that is not found within the natural (non-recombinant) form of the cell, or alternatively expresses a native gene that is abnormally expressed, expressed less, or not expressed at all.

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

[0113] 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 traditional Watson-Crick or other non-traditional types. The binding free energy of a nucleic acid molecule having a complementary sequence thereto is sufficient to drive related functions 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, for example, Turner, et al. (1987) CSH Symp. Quant. Biol. LII:123-33; Frier, et al. (1986) PNAS 83:9373-77; Turner, et al. (1987) J. Am. Chem. Soc. 109:3783-85. Percent complementarity refers to the percentage of contiguous residues within 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, or 10 out of 10 are about 50%, 60%, 70%, 80%, 90%, and 100% complementary, inclusive). "Fully complementary" or 100% complementarity broadly refers to all of the contiguous residues of a nucleic acid sequence that hydrogen bond with an equal number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" refers to a polynucleotide strand that exhibits at least about 90% complementarity, excluding regions of the polynucleotide strand such as overhangs that are selected to be non-complementary. For specific binding, a degree of complementarity sufficient to avoid non-specific binding of the oligomeric compound to non-target sequences is required under the conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, or the conditions under which the assay is performed in the case of in vitro assays. Non-target sequences can typically differ by at least five nucleotides only.

[0114] As used herein, "sign" of a disease broadly refers to any abnormality that indicates an objective sign of a disease, as opposed to symptoms, which are subjective signs of the disease, and which are detectable by examination of a patient.

[0115] As used herein, "solid support", "support", and "substrate" broadly refer to, but are not limited to, smooth supports (e.g., the surfaces of metals, glass, plastics, silicon, and ceramics), and any substance that provides a solid or semi-solid structure to which another substance such as a texture and a porous material can be adhered.

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

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

[0118] "Treatment method", "therapeutic", "treat", or "treatment", as used herein, broadly refers to treating a disease, halting or reducing the development of a disease or its clinical symptoms, and / or alleviating a disease and causing regression of the disease or its clinical symptoms. Treatment methods include bringing about prevention, treatment, repair, reduction, alleviation, and / or mitigation of a disease, disease signs, and / or symptoms. Treatment methods include reduction of signs and / or symptoms in a patient having ongoing disease signs and / or symptoms (e.g., tumor growth, metastasis). Treatment methods also include "prevention". The term "reduced" broadly refers to a clinically significant reduction of signs and / or symptoms for the purposes of treatment methods. Treatment methods include treatment of signs and / or symptoms of relapse or recurrence (e.g., tumor growth, metastasis). Treatment methods include, but are not limited to, precluding 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. Treatment methods include treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing relapse or recurrence of signs and / or symptoms (e.g., tumor growth, metastasis).

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

[0120] As used herein, "vector" broadly refers to a plasmid, cosmid, phagemid, phage DNA, or other DNA molecule that can replicate autonomously within a host cell and is characterized by one or a few restriction endonuclease recognition sites. At such recognition sites, such DNA sequences can be cleaved in a determinable manner without loss of the essential biological function of the vector, and DNA can be inserted into the site to effect its replication and cloning. The vector may further contain a marker suitable for use in identifying cells transformed with the vector.

[0121] Techniques and procedures are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook, et al. (2001) Molec. Cloning: Lab. Manual [3rd Ed] Cold Spring Harbor Laboratory Press. Standard techniques may 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 the manufacturer's specifications, or as commonly accomplished in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques of these, are well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, preparation, formulation, and delivery of pharmaceuticals, and treatment of patients.

[0122] Examples

[0123] The invention generally described herein will be more readily understood by reference to the following examples. These are included for purposes of illustration only of certain aspects and embodiments of the invention and are not intended to limit the invention.

[0124] Example 1

[0125] NEO-201 binds to various human cancer cell lines.

[0126] Flow cytometry analysis was used to profile a panel of human cancer cell lines for NEO-201 binding. The staining profiles are summarized in Table 1, and representative histograms from cell lines with high, medium, low, and negative staining are shown in FIGS. 1A - C. In the evaluation of NEO-201 binding activity, it was revealed that the positive rates were high in 3 / 6 (50%) of the colon cancer cell lines and 4 / 5 (80%) of the pancreatic cancer cell lines. When profiling non-small cell lung cancer (NSCLC) cell lines of various histological subtypes, it was found 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 positive. Screening of breast cancer cell lines was also performed. Among the cell lines expressing either estrogen receptor (ER) or progesterone receptor (PR), 2 / 4 (50%) were positively stained for NEO-201, whether alone or in combination with HER2. Among the HER2+ cell lines, 3 / 4 (75%) were recognized by NEO-201, whether alone or in combination with ER or PR. However, NEO-201 staining was found at low levels in only 1 / 4 (25%) of the triple-negative breast cancer cell lines. Overall, 15 / 30 (50%) of the tumor cell lines examined were recognized by NEO-201. These data indicate that NEO-201 is reactive against a wide range of in vitro cultured tumor cell lines and that there can be clear differences in antibody reactivity based on tumor subtype.

[0127] Example 2

[0128] NEO-201 tissue staining has high tumor specificity

[0129] Immunohistochemistry was used to investigate NEO-201 reactivity from human tumor samples using tissue microarrays representing dozens of samples for each cancer type. As shown in Figure 2A, immunoreactivity (7,829,678) with NEO-201 was not present at all in normal colon, pancreas, and lung tissues, but the positive rate was high in tumor tissues derived from these organs. Surprisingly, since the surrounding stromal cells were not stained, the staining was seen only on tumor cells (Figure 2A). In IHC staining and microarray samples, NEO-201 was determined to be highly reactive against colon cancer (72%), pancreatic cancer (80%), gastric cancer (71%), lung cancer (61%), breast cancer (55%), and uterine cancer (54%). Furthermore, a fairly small number of ovarian cancer (26%) samples also showed positive staining, but no staining was observed in prostate cancer tissue (Figure 2B). Overall, 258 / 345 (74.7%) of the sampled tumor tissues were positively stained for NEO-201. Importantly, the reactivity of NEO-201 was almost completely absent in normal healthy tissues (Table 2), as well as in normal tumor-adjacent tissues except for some uterine and ovarian samples (Figure 2C). However, in this series of uterine and ovarian tissues, the number of tissues was limited (5 and 9 samples, respectively). In summary, these data indicate that NEO-201 recognizes tumor tissues from various cancer tumors and has high tumor specificity.

[0130] Experimental Example 3

[0131] NEO-201 kills tumor cells by mediating ADCC and CDC

[0132] NEO-201, as a humanized IgG1 antibody, is theorized to be able to mediate ADCC to kill tumor cells expressing the NEO-201 antigen. To investigate this potential mechanism of action, an ADCC assay using human natural killer (NK) cells isolated from PBMCs of two different healthy donors was performed on cell lines with high positive rates for NEO-201 staining (CFPAC-1 and ASPC-1). Treatment with NEO-201 was observed to increase the killing of both CFPAC-1 and ASPC-1 cells to levels 2 - 6 times higher than the killing of control IgG1-treated tumor cells (Figure 3A). A titration assay was also performed, and it was revealed that NEO-201 retains the ability to significantly induce ADCC at low doses of about 0.1 μg / mL (Figure 3B).

[0133] CDC is a proteolytic cleavage complex cascade that leads to the activation of the membrane attack complex to lyse antibody-bound target cells. Certain human IgG1 antibodies can mediate CDC, but CDC depends on the antigen specificity of the antibody. In the CDC assay, it was revealed that NEO-201 induces complement-mediated lysis of ASPC-1 cells in a manner dependent on both the mAb dose and the incubation time (Figure 3C). Collectively, these data indicate that NEO-201 effectively engages natural immune effector mechanisms and specifically lyses antibody-bound tumor cells in vitro.

[0134] Example 4

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

[0136] To determine the potential anti-tumor effect of NEO-201, CFPAC-1 cells were grown as tumor xenografts in immunodeficient NU / NU nude mice. These cells were selected based on their high expression level of the NEO-201 antigen and high sensitivity to NEO-201-mediated ADCC. When the size of the CFPAC-1 tumor reached approximately 100 mm 3Once it had grown to this stage, tumor-bearing mice were injected three times with saline, 250 μg of human IgG1, 100 μg of NEO-201, or 250 μg of NEO-201, and then 1.0 x 107 7 IL-2-activated (200 U / mL) human PBMCs were injected three times to function as ADCC-mediated effector cells. As shown in Figure 4A, NEO-201 + PBMC induced a substantial reduction in tumor growth at both dose levels compared to either the saline + PBMC or human IgG + PBMC control groups. By day 36, no tumors were present in any of the control group mice, whereas palpable tumors remained in none of the mice treated with 100 μg of NEO-201 + PBMC and in 4 out of 10 (40%) of the mice treated with 250 μg of NEO-201 + PBMC (Figure 4B). Furthermore, when NEO-201 was administered to another group of mice without adding human PBMCs, a significant decrease in tumor growth was observed compared to the control group (Figure 4A, C). Importantly, monitoring the body weights of the tumor-bearing mice revealed that there was no weight loss in any of the treatment groups (Figure 4D). In summary, these results indicate that NEO-201 can significantly reduce tumor growth in mice without inducing significant toxicity via both the ADCC mechanism and non-ADCC mechanisms (such as CDC).

[0137] Example 5

[0138] NEO-201 localizes to the xenograft tumor site

[0139] Biodistribution studies were performed using radiolabeled NEO-201 in female and male NU / NU nude mice bearing established CFPAC-1 xenograft tumors. These mice were injected intravenously with the radiolabeled antibody, and blood, organs, and tumors were collected at various time points after injection for analysis. Low levels of radioactivity were detected in the pancreas, spleen, kidney, liver, stomach, intestine, and lung of both male and female mice at all time points (Figure 5A, B). However, the normalized radioactivity uptake was significantly higher in tumors compared to all other tissues at all time points, and the radioactivity in tumors gradually increased to levels 20 - 30 times higher than that in blood by day 7 (Figure 5A, B). Quantitatively similar results were obtained in both female and male mice. These results indicate that NEO-201 localizes preferentially to malignant tissues expressing the target antigen and does not accumulate in normal tissues.

[0140] Example 6

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

[0142] To determine the pharmacokinetics and related toxicities of NEO-201, single-dose studies were conducted in cynomolgus monkeys for breeding purposes. Cynomolgus monkeys were selected because they are closely related to humans both phylogenetically and physiologically and are a species commonly used in nonclinical toxicity evaluations. Male and female animals were injected once intravenously with NEO-201 diluted in saline at doses of 5 mg / kg, 20 mg / kg, and 49 mg / kg. This was the highest achievable dose per injection volume. Blood samples were collected from all animals at various time points up to 14 days maximum before and after injection, and serum preparations were evaluated for NEO-201 levels by ELISA. As shown in Table 3, quantifiable, dose-dependent serum concentrations of NEO-201 were observed at the last collection time point (14 days post-dose). As predicted for intravenous administration, the Tmax values were such that the majority of animals (10 / 12, 83%) in all groups reached peak levels in 10 minutes, except for one male and one female in the 5 mg / kg group. Over the dose range evaluated, peak (Cmax) exposure was dose-proportional. Total (AUC) exposure was greater than dose-proportional at the lowest dose and approximately proportional from about 20 mg / kg to 49 mg / kg. The difference in exposure at the lowest dose was due to being approximately two-fold greater than the mean clearance (CL) and a smaller volume of distribution (Vz). The mean half-life (HL) was 167 (20 mg / kg) hours or 170 (49 mg / kg) hours at the high doses and was approximately 3.7-fold longer than the 5 mg / kg dose (46.2 hours). No gender differences were observed.

[0143] The following observations and tests are included to determine toxicity during the 14-day study: 1) regular clinical evaluations; 2) measurement of food intake and body weight; and 3) urine and blood tests (such as urine tests, hematology, coagulation tests, serum chemistry, and toxicokinetics). As shown in Figure 6A, none of the dosing groups experienced a change in body weight exceeding 3% from the pre-injection body weight, and none of the individual monkeys experienced a change exceeding 7%. Food intake did not change for all animals except for two in the 5 mg / kg dose group with low food intake only on day 11. There were no significant changes in any of the serum chemistry, urine tests, or coagulation tests from baseline (before NEO-201 injection) until day 15 (see Materials and Methods for details). The main change in blood cell counts in the laboratory was a decrease in the number of neutrophils compared to baseline (Figure 6B). The decreases were of various degrees from mild to marked, and no clear dose-response was evident. For most animals, this was a transient finding as improvement was usually observed by day 8 (Figure 6B). By day 15, a near-total or partial recovery of the neutrophil count was observed in the 5 mg / kg group or the 20 mg / kg and 49 mg / kg groups, respectively (Figure 6B). The recovery of the neutrophil count by day 15 is reflected in the statistical comparison with the 0 mg / kg animals. The 0 mg / kg animals had a significant difference on day 2 at all three dosing levels (p < 0.05), but there was no significant difference on days 8 and 15 for two of the three dosing groups (p > 0.05) (Figure 6C).

[0144] Example 7

[0145] Materials and Methods

[0146] Cell Lines and Cultures

[0147] The following human cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA): colon (COLO 205, HT-29, LS174T, SW1116, SW1463, SW480, SW620), pancreas (ASPC-1, CFPAC-1, PANC-1), breast (AU-565, BT-474, BT-549, HCC1500, HCC1937, HCC38, MDA-MB-231, MDA-MB-468, SK-BR-3, T-47D, ZR-75-1), and lung (CALU-1, H1703, H226, H441, H520, H522, H596, HCC4006, HCC827, SK-LU-1). All cell cultures were maintained in RPMI 1640, DMEM, or IMDM culture medium (Corning, Corning, NY) as specified by the supplier for propagation and maintenance. The culture medium was supplemented with 10% defined heat-inactivated HyClone fetal bovine serum (GE Healthcare Life Sciences, Issaquah, WA, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin (Corning Life Science, Manassas, VA, USA). PBMCs from healthy volunteer donors were obtained from the National Institutes of Health Clinical Center Blood Bank (NCT00001846) under the approval of the appropriate institutional review board and informed consent.

[0148] Generation of humanized NEO-201 monoclonal antibody

[0149] The Hollinshead colon cancer-specific vaccine was used as an immunogenic substance to generate monoclonal antibodies in mice. Methods for preparing tumor-associated proteins and peptides have been described previously (Hollinshead, US4810781, 1989). Briefly, cancer tissue was minced and used to generate a single cell suspension, which was then subjected to hypotonic saline membrane extraction, a series of centrifugation steps, and subsequent low-frequency sonication. The resulting membrane-extracted proteins were fractionated by Sephadex G-200 resin or electrophoresis, and then concentrated and quantified (Hollinshead et al, 1970; Hollinshead et al., 1972; Hollinshead et al., 1985). The TAA preparation was mixed with complete Freund's adjuvant and injected subcutaneously into BALB / c mice. This was followed by three booster injections with incomplete Freund's adjuvant at 2- to 3-week intervals. Mouse sera were tested by ELISA for antibody responses to the immunizing antigen, and mice with strong responses were used to fuse mouse B cells from the spleen with the SP2 / 0-Ag14 myeloma cell line, grow them, and select cells that produced mouse immunoglobulin (IgG) to generate immortalized hybridoma cells. From these mouse IgGs, the mouse 16C3 clone (m16C3) was selected based on its reactivity with colon tumor cell membrane extracts derived from LS174T or HT-29 cells as determined by ELISA. The cDNAs encoding the heavy and light chain IgG1 were determined from RNA isolated from the hybridoma clone 16C3 E12 and shown to be unique (Bristol&Kantor, US7829678, 2010). The m16C3 protein sequence was humanized as h16C3 and named NEO-201. Humanization was performed in silico by replacing the mouse sequences outside the complementarity-determining regions (CDRs) of the Fab regions of both the heavy and light chain proteins with human Fab sequences, while retaining three mouse CDR sequences from each chain. The Fc regions of the heavy and light chains were selected from the human IgG1 isotype used in other humanized approved mAb products. The amino acid sequence was reverse-translated into DNA optimized for protein expression in CHO cells.Next, the DNA of the heavy and light chains of h16C3 was chemically synthesized, cloned into a mammalian expression plasmid, and transfected into mammalian cell lines (HEK293T and CHO). Several stable CHO cell lines expressing recombinant h16C3 were induced and preserved. The purified recombinant h16C3 was retested in studies to confirm that the humanized 16C3 antibody has the same characteristics as the original m16C3 antibody (Bristol&Kantor, US7829678, 2010).

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

Number

[0151] The boundaries between the expression leader sequence, variable region, and constant region are delimited by a slash (" / ") in each sequence, and the CDR sequences are indicated by bold underlined strings. The antibody sequences used included the indicated variable and constant regions. These regions include heavy chain CDR1 of SEQ ID NO: 32, heavy chain CDR2 of SEQ ID NO: 33, heavy chain CDR3 of SEQ ID NO: 34, light chain CDR1 of SEQ ID NO: 35, light chain CDR2 of SEQ ID NO: 36, and light chain CDR3 of SEQ ID NO: 37.

[0152] Flow cytometry

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

[0154] Immunohistochemistry (IHC)

[0155] Tissue microarrays of colon samples (CO808, CO951) were obtained from US Biomax (Rockville, MD), and AccuMax tissue microarrays of colon (A303(I)), pancreas (A207(II), A307), stomach (A209), lung (A206(V), A306), breast (A202(VI), A712), uterus (A212), ovary (A212, A213(II)), prostate (A302(IV)), and various normal (A103(VII)) samples were obtained from Accurate Chemical and Scientific Corporation (Westbury, NY). NEO-201 was biotinylated using a biotin protein labeling kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. The slides were baked at 60 °C for 20 minutes, deparaffinized with xylene, and rehydrated in a graded ethanol series. The slides were subjected to peroxidase blocking for 2 minutes using peroxidase I solution (Biocare Medical, Concord, CA), avidin blocking for 10 minutes using avidin solution (Biocare Medical, Concord, CA), biotin blocking for 10 minutes using biotin solution (Biocare Medica, Concord, CA), and protein blocking for 10 minutes using CAS-Block histochemical reagent (Thermo Fisher Scientific, Waltham, MA). Next, the slides were incubated at room temperature for 2 hours with either biotinylated human IgG1 kappa (Ancell, Bayport, MN) as a negative control or 10 μg / mL biotinylated NEO-201 diluted in 1XPBS. Detection was carried out by incubating with Dako streptavidin-HRP conjugate (Agilent Technologies, Santa Clara, CA) at 1:300 for 30 minutes and DAB peroxidase substrate (Thermo Fisher Scientific, Waltham, MA) for 1 - 3 minutes, and counterstaining with hematoxylin for validation. Each microarray tissue spot was evaluated by light microscopy for cell staining intensity using the following scale: 0 (negative), ± (border region), 1+ (weak), 2+ (moderate), 3+ (strong).Tissue spots containing cells stained with an intensity of +1 or higher were recorded as positive.

[0156] Antibody-dependent cell-mediated cytotoxicity (ADCC) assay

[0157] The ADCC assay was performed using a modification of the above-described procedure (Boyerinas et al., 2015). According to the manufacturer's protocol, 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). The purified NK cells were incubated overnight in RPMI-1640 medium supplemented with L-glutamine, 10% FBS, and antibiotics. On the day of the assay, target cells (CFPAC-1, ASPC-1) were labeled with 10 μM calcein AM cell-permeable dye (Termo Fisher Scientific, Waltham, MA, USA) for 30 minutes and then seeded at 3.0 x 10 3 cells / well in triplicate into black-wall flat-bottom 96-well culture plates (#655090 Greiner bio-one, Germany). Subsequently, the tumor cells were treated with 10 μg / mL of human IgG1 isotype control antibody (Thermo Fisher Scientific, Waltham, MA, USA) or NEO-201, and NK cells were added at effector-to-target (E:T) ratios of 12.5:1 and 25:1 unless otherwise specified. After incubation at 37°C for 4 hours, 10 μg / mL of propidium iodide (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well, and the plates were imaged and analyzed using a Celigo Imaging Cytometer (Nexcelom Bioscence LLC, Lawrence, MA, USA). Live target cells (calcein AM / PI-) were counted in each well, and specific ADCC lysis was calculated as follows: Specific lysis rate % = 100 - [(average number of live targets experimental / average number of live targets control ) x 100].

[0158] Complement-dependent cytotoxicity (CDC) assay

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

[0160] Xenograft antitumor assay

[0161] Tumors were established in 6-week-old female athymic NU / NU nude mice (Charles River Laboratories International, Wilmington, MA) by subcutaneous transplantation of a culture tumor cell-containing 1X PBS suspension into the right flank of the mice. When the tumor size reached approximately 100 mm 3 , the mice were sorted by tumor volume and randomly divided into 5 groups (n = 10 animals). Next, the mice were intraperitoneally injected with vehicle only (saline), human IgG1 (250 μg), or NEO-201 (100 μg and 250 μg) on days 13, 17, and 20 after transplantation. The mice also received an intraperitoneal injection of approximately 1.0 x 10 7 human PBMCs activated with IL-2 (200 U / mL, treated overnight in culture) on days 14, 18, and 21 as a source of immune effector cells. One group of mice was treated similarly with NEO-201 but did not receive an administration of human PBMCs. Tumors were measured every 2 - 3 days with digital calipers, and tumor volume was calculated using the formula (width 2x length) / 2 = mm 3 Calculated by. Here, the width was taken as the shorter value of the two measured values. The mice were also weighed weekly as a general measure of health. In accordance with IACUC guidelines, mice with a tumor volume exceeding 2000 mm 3 were sacrificed.

[0162] Biodistribution analysis

[0163] Using previously described procedures (Patel et al., 2013), in vivo distribution studies were evaluated in tumor-bearing mice using radiolabeled NEO-201 (Comparative Biosciences, Sunnyvale, CA). Briefly, a 200 μL 1X PBS suspension containing 4.0 x 10 6 CFPAC-1 cells was subcutaneously injected into the flanks of male and female athymic NU / NU nude mice (Charles River Laboratories International, Wilmington, MA). On day 14 after engraftment, the mice were intravenously injected with 20 μCi of 125 I-labeled NEO-201 and sacrificed 1, 2, 4, or 7 days later. Blood, tumor tissue, and internal organs (lung, kidney, liver, spleen, pancreas, intestine, and stomach) were collected at each time point (n = 4 animals), the weight of all tissues was measured, and the radioactivity in the tissues was measured using a gamma counter. The data for each mouse were first calculated as cpm / mg tissue and then the tissue cpm values were normalized to the blood cpm values.

[0164] Single-dose toxicity study in cynomolgus monkeys

[0165] To test NEO-201 for its pharmacokinetics and toxicity after single administration, a single-dose toxicity study was conducted in cynomolgus monkeys for breeding purposes. The study period was 15 days from dosing, and the monkeys were further isolated for 14 days prior to dosing to acclimatize them to the laboratory. Using a plastic disposable syringe with an infusion pump and catheter extension tube, NEO-201 diluted with saline was slowly intravenously infused (infusion over approximately 30 minutes ± 5 minutes) at dose levels of 0 mg / kg, 5 mg / kg, 20 mg / kg, and 49 mg / kg (which was the highest achievable concentration of the antibody) to 8 male and female animals (2 animals / sex / group). Blood samples were collected from all animals that received NEO-201 at the following time points: pre-dose, 10 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 24 hours, 48 hours, 72 hours, 96 hours, 168 hours, and 336 hours. Serum was prepared from the 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.

[0166] Clinical examinations included hematology and coagulation (baseline (BL), days 2, 8, and 15); CBC and differential, activated partial thromboplastin time, fibrinogen, and prothrombin time; serum chemistry (BL, days 2, 8, 15): albumin, alkaline phosphatase, ALT, AST, total bilirubin, calcium, total cholesterol, creatine kinase, creatinine, glucose, inorganic phosphorus, total protein, triglyceride, sodium, potassium, chloride, globulin, albumin / globulin ratio, BUN; urine examination (BL, day 15): color, transparency, glucose, ketone, occult blood, protein, bilirubin, nitrite, pH, urobilinogen, white blood cells, volume, specific gravity; bioanalysis (using ELISA) - (BL, 10 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 24 hours, 48 hours, 72 hours, 96 hours, 168 hours, and 336 hours), including groups 2 to 4, using Phoenix WinNonlin version 6.1 software (Certara USA, Princeton, NJ). Animal body weight measurements were recorded (BL, 7, and 14), and neutrophil counts were evaluated (BL, days 2, 8, 15).

[0167] Statistical Analysis

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

[0169] Example 8

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

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

[0172] Method

[0173] NK cells were isolated from normal donors and treated with various concentrations of ALT-803 for 48 hours before being used as effector cells. Human cancer cell lines expressing the NEO-201 antigen were utilized as targets in an in vitro non-radioactive ADCC assay. The ability of ALT-803 to affect the phenotype of NK cells and regulate their gene expression was evaluated using flow cytometry and Nanostring analysis, respectively.

[0174] Results

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

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

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

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

[0179] Therefore, ALT-803 enhances the ADCC activity against human cancer tumor cells mediated by NEO-201. The enhancement of ADCC activity may be partly due to the increase in the expression of TIM3, NKG2D, granzyme B, and CD107a-positive NK cells, as well as the regulation of transcripts involved in NK activation and cytotoxicity.

[0180] In summary, the ADCC activity mediated by NEO-201 can be enhanced by treating NK cells isolated from normal donors with ALT-803. Phenotypic analysis of ALT-803-treated NK cells isolated from normal donors showed that ALT-803 can enhance the expression of TIM-3 and NKG2D in CD16 / CD56-positive NK cells. Treatment of normal NK cells with ALT-803 also increases the MFI of granzyme B in CD16 / CD56-positive NK cells. Treatment of normal NK cells with ALT-803 increases 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 maturation marker. The enhancement of ADCC activity mediated by NEO-201 after treatment with ALT-803 may be partly due to the increased expression of TIM-3-positive, NKG2D-positive, granzyme B-positive, and CD107a-positive NK cells, although this theory is not intended to be limiting. Treatment of NK cells with ALT-803 can enhance the ADCC activity mediated by low concentrations of NEO-201. Low concentrations of Mab can be used to mediate ADCC activity when NK cells are treated with ALT-803 and can achieve equivalent levels of cytotoxicity compared to NK cells without ALT-803 treatment with high concentrations of NEO-201. This result suggests that in clinical trials for cancer treatment, lower doses of Mab can be used in combination with ALT-803.

[0181] Example 9

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

[0183] In immunotherapy using checkpoint-blocking antibodies targeting effector cell inhibitory receptors such as PD-1 and CTLA-4, several dramatic and durable responses are elicited in some tumor types. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell surface protein expressed by immune cells and tumor cells, and 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, thereby preventing NK cell lysis and enabling tumor cells to avoid NK killing.

[0184] NEO-201 can bind to members of the CEACAM family and activate innate immune mechanisms such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cell cytotoxicity (CDC) to kill tumor cells. This investigation was designed to determine whether NEO-201 blocks the CEACAM1 inhibitory pathway and restores antitumor function to NK cells.

[0185] Methods

[0186] An in vitro assay using human tumor cell lines was performed to identify members of the CEACAM family bound by NEO-201. A functional assay was performed to evaluate the ability of NEO-201 to enhance the in vitro killing of tumor cells by the NK cell line NK-92, which expresses CEACAM1, lacks CD16, and has no ability to mediate ADCC.

[0187] The killing assay was performed using a modification of the procedure described above (David et al., 2017). Briefly, target cells derived from pancreatic cancer (ASPC-1, BxPC-3, CFPAC-1) and colon cancer (LS174T) were labeled with 10 μM calcein AM cell-permeable dye (Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes and then triplicated at 3.0 x 103 Cells / well were seeded in a black-walled, flat-bottom 96-well culture plate. Subsequently, tumor cells were treated with 10 μg / mL of human IgG1 isotype control antibody (Thermo Fisher Scientific, Waltham, MA, USA) or NEO-201, and natural killer (NK) cell line NK-92 was added at effector-to-target (E:T) ratios of 1.5625:1, 3.125:1, 6.25:1, and 12.5:1. After incubation at 37 °C for 16 hours, propidium iodide (PI; Thermo Fisher Scientific, Waltham, MA, USA) was added to each well at a final concentration of 1.67 μg / mL, the plate was centrifuged, imaged using a Celigo Imaging Cytometer (Nexcelom Bioscence LLC, Lawrence, MA, USA), and analyzed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). Live target cells (calcein AM / PI-) were counted in each well, and specific lysis was calculated as follows: Specific lysis rate % = 100 - [(average number of live targets experimental / average number of live targets control ) x 100].

[0188] Results

[0189] 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 line cells expressed different levels of the negative forms of CEACAM5 / 6 and the NEO-201-reactive variant forms of these molecules. Functionally, treatment with NEO-201 enhanced the cytolytic activity of NK-92 cells against NEO-201+ tumor cells expressing CEACAM5 but not against NEO-201+ cells expressing only CEACAM6 (Figure 13).

[0190] Conclusions

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

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

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Table 1

Table 2

Table 3

Claims

Claim 1 A pharmaceutical composition for killing cancer tumor cells, treating cancer tumors, preventing recurrence of cancer tumors, or reducing tumor burden in a patient in need thereof, comprising an effective amount of NEO-201 antibody, wherein: (a) the antibody mediates antibody-dependent cell cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC), thereby killing the cancer tumor cells of the patient; (b) the antibody comprises (i) a heavy chain variable region sequence contained in SEQ ID NO: 28 and a light chain variable region sequence contained in SEQ ID NO: 29, and (ii) a human IgG1 constant domain; and (c) the patient has been determined to have depleted natural killer ("NK") cells before or at the time of administration of the pharmaceutical composition, and further, (d) the cancer tumor is characterized by cells that are reactive to complement-mediated activation and complement-mediated lysis by NEO-201. A pharmaceutical composition. Claim 2 The pharmaceutical composition according to claim 1, wherein the patient has been determined to have severe NK depletion before or at the time of administration. Claim 3 The pharmaceutical composition according to claim 1 or 2, comprising: (a) the patient optionally has NK cell deficiency (NKD) including CNKD (e.g., CNKD1, CNKD2), or FNKD (e.g., FNKD1); (b) the patient has depleted NK cells as a result of another treatment method or has severely depleted NK cells; (c) the patient is receiving cancer treatment; or (d) the patient is receiving chemotherapy or radiotherapy, and optionally, the chemotherapy comprises administering one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin A, suberoylanilide-hydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, aphidicolin, mitomycin, methotrexate, etoposide), GSK inhibitors (e.g., LiCl, BIO, SB21), BET inhibitors (e.g., JQ1), HSP90 inhibitors (e.g., radicicol, 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel), and / or immunomodulatory drugs (e.g., lenalidomide). Claim 4 The pharmaceutical composition according to any one of claims 1 to 3, comprising: before or at the time of said administration, (a) NK cells constitute less than 5% of peripheral blood mononuclear cells (PBMCs) in said patient; (b) NK cells constitute less than 3% of peripheral blood mononuclear cells (PBMCs) in said patient; (c) less than 70% of the patient's PBMC NK cells are CD56 ｄｉｍ CD16 ＋ NK cells; or (d) less than 50% of the patient's PBMC NK cells are CD56 ｄｉｍ CD16 ＋ NK cells.

5. The pharmaceutical composition according to any one of claims 1 to 4, comprising: The NEO-201 antibody comprises a heavy chain sequence having the sequence of SEQ ID NO: 28 and a light chain sequence having the sequence of SEQ ID NO:

29.

6. The pharmaceutical composition according to any one of claims 1 to 5, for administration in combination with: (a) an effective amount of a cytokine agonist for enhancing or stimulating the killing of said cancer cells, provided that optionally, said cytokine agonist is interleukin-2 (IL-2), interleukin 21 (IL-21), ALT-803, an IL-15 inhibitor, a checkpoint inhibitor, anti-PD1, anti-PD-L1, anti-CTLA-4, anti-41BB, anti-OX40, anti-Tim-3, or a combination thereof, an IL-15 agonist or IL-15 superagonist, or a complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor α / IgG1 Fc fusion protein (a complex such as ALT-803), comprising; or (b) an effective amount of a complement regulatory protein (CRP) antagonist for enhancing or stimulating the killing of said cancer cells, Said CRP antagonist optionally antagonizes one or more of CD46, CD55, or CD59, and said CRP antagonist optionally comprises an antibody or an antigen-binding fragment thereof.

7. The pharmaceutical composition according to claim 6, wherein said effective amount of said NEO-201 antibody is reduced as compared to treatment with said NEO-201 antibody alone without said cytokine agonist.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein said cancer is colon cancer, pancreatic cancer, ovarian cancer, gastric cancer, lung cancer, breast cancer, or uterine cancer.

9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the cytokine agonist is an IL-15 agonist or IL-15 superagonist.

10. The pharmaceutical composition according to claim 9, wherein the IL-15 agonist or IL-15 superagonist comprises ALT-803.

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