Monoclonal antibody NEO-201 for treating human carcinoma
阅读说明：本技术 用于治疗人癌瘤的单克隆抗体neo-201 (Monoclonal antibody NEO-201 for treating human carcinoma ) 是由 P·M·阿兰 K·Y·曾 于 2018-11-02 设计创作，主要内容包括：NEO-201是针对来自许多不同癌瘤的大多数肿瘤组织具有高反应性的人源化IgG1单克隆抗体(mAb),所述癌瘤包括结肠癌、胰腺癌、胃癌、肺癌、乳腺癌和子宫癌,但绝大多数正常组织不被这种抗体识别。功能测定揭示NEO-201能够介导针对肿瘤细胞的抗体依赖性细胞毒性(ADCC)和补体依赖性细胞毒性(CDC)两者。此外,通过单独使用NEO-201以及与作为ADCC的效应细胞来源的人外周血单核细胞(PBMC)组合使用NEO-201进行治疗,体内人胰腺异种移植物肿瘤的生长大大减弱。在携带人肿瘤异种移植物的小鼠中的体内生物分布研究揭示,NEO-201优先在肿瘤而不是器官组织中积聚。在非人灵长类动物中的单剂量毒性研究证明了NEO-201的安全性和耐受性,因为循环嗜中性粒细胞的短暂减少是所观察到的唯一相关副作用。(NEO-201 is a humanized IgG1 monoclonal antibody (mAb) with high reactivity against most tumor tissues from many different carcinomas, including colon, pancreatic, gastric, lung, breast and uterine cancers, but the vast majority of normal tissues are not recognized by this antibody. Functional assays revealed that NEO-201 was able to mediate both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against tumor cells. Furthermore, the growth of human pancreatic xenograft tumors in vivo was greatly reduced by treatment with NEO-201 alone and in combination with human Peripheral Blood Mononuclear Cells (PBMCs) as effector cell source for ADCC. In vivo biodistribution studies in mice bearing human tumor xenografts revealed that NEO-201 preferentially accumulates in tumors rather than organ tissues. Single dose toxicity studies in non-human primates demonstrated the safety and tolerability of NEO-201, as the only relevant side effect observed was a transient reduction in circulating neutrophils.)
1. A method of killing cancer cells, comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody.
2. A method of treating cancer comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody.
3. A method of preventing cancer recurrence comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody.
4. A method of reducing tumor burden in a patient having a carcinoma, comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody.
5. The method of any one of the preceding claims, wherein the antibody mediates complement-mediated cytotoxicity (CDC) killing of cancerous cells in the patient.
6. The method of any one of the preceding claims, wherein prior to or at the time of the administering, the patient is natural killer cell ("NK") depleted.
7. The method of any one of the preceding claims, wherein prior to or at the time of the administration, the patient is severe NK-attenuating.
8. The method of claim 6, further comprising determining whether said patient is NK attenuating prior to or at the time of said administering.
9. The method of claim 6, further comprising determining whether said patient is severe NK attenuating prior to or at the time of said administering.
10. The method of any one of the preceding claims, wherein the patient has an NK cell deficiency (NKD), optionally including CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD 1).
11. The method of any one of the preceding claims, wherein the patient is NK-diminished or severe NK-diminished as a result of another therapy.
12. The method of any one of the preceding claims, wherein the patient is undergoing cancer therapy.
13. The method of any one of the preceding claims, wherein the patient is undergoing chemotherapy or radiation therapy.
14. The method of claim 13, wherein the chemotherapy comprises administration of one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin a, suberoylanilide-hydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, aphidicolin, mitomycin, methotrexate, etoposide), licgsk inhibitors (e.g., LiCl, BIO, SB21), BET inhibitors (e.g., JQ1), HSP90 inhibitors (e.g., radicicolin), 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel), and/or immunomodulatory drugs (e.g., lenalidomide).
15. The method of any one of the preceding claims, wherein NK cells comprise less than 5% of Peripheral Blood Mononuclear Cells (PBMCs) in the individual prior to or at the time of the administration.
16. The method of any one of the preceding claims, wherein NK cells comprise less than 3% of Peripheral Blood Mononuclear Cells (PBMCs) in the individual prior to or at the time of the administration.
17. The method of any one of the preceding claims, wherein less than 70% of PBMC NK cells in the patient prior to or at the time of the administration are CD56dimCD16+NK cells.
18. The method of any one of the preceding claims, wherein less than 50% of PBMC NK cells in the patient prior to or at the time of the administration are CD56dimCD16+NK cells.
19. The method of any of the preceding claims, wherein the NEO-201 antibody comprises at least one, two, three, four, five or all six of the CDR sequences contained in SEQ ID NO 28 and SEQ ID NO 29.
20. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity to SEQ ID NO 38.
21. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a variable light chain sequence having at least 90% identity to SEQ ID NO 39.
22. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity to SEQ ID NO 38 and a variable light chain sequence having at least 90% identity to SEQ ID NO 39.
23. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises the variable heavy chain sequence of SEQ ID NO 38 and the variable light chain of SEQ ID NO 39.
24. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a heavy chain sequence having at least 90% identity to amino acids 20-470 of SEQ ID NO 28 and a light chain sequence having at least 90% identity to amino acids 20-233 of SEQ ID NO 29.
25. The method of claim 22 or 23, wherein said NEO-201 antibody comprises all six of the CDR sequences contained in SEQ ID No. 28 and SEQ ID No. 29.
26. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises the heavy chain variable region sequence contained in SEQ ID NO 28 and the light chain variable region sequence contained in SEQ ID NO 29.
27. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a heavy chain sequence comprising amino acids 20-470 of SEQ ID NO 28 and a light chain sequence comprising amino acids 20-233 of SEQ ID NO 29.
28. The method of any one of the preceding claims, wherein the NEO-201 antibody comprises a human IgG1 constant domain.
29. The method of any one of the preceding claims, wherein the NEO-201 antibody is humanized.
30. The method of any one of the preceding claims, wherein the NEO-201 antibody is conjugated to another moiety.
31. The method of any one of the preceding claims, wherein the NEO-201 antibody is conjugated to another cytotoxic moiety, label, radioactive moiety or affinity tag.
32. The method of any one of the preceding claims, further comprising administering to the patient an effective amount of a cytokine agonist to enhance or stimulate killing of the cells of the carcinoma.
33. The method of claim 31, wherein the cytokine agonist comprises interleukin 2(IL-2), interleukin 21(IL-21), ALT-803, an IL-15 inhibitor, a checkpoint inhibitor, anti-PD 1, anti-PDL 1, anti-CTLA-4, anti-41 BB, anti-OX 40, anti-Tim-3, or a combination thereof.
34. The method of any one of the preceding claims, further comprising administering to the patient an effective amount of a Complement Regulatory Protein (CRP) antagonist to enhance or stimulate killing of the cells of the carcinoma.
35. The method of claim 33, wherein the CRP antagonist antagonizes one or more of CD46, CD55, or CD 59.
36. The method of claim 33 or 34, wherein the CRP antagonist comprises an antibody or antigen-binding fragment thereof.
37. The method of claim 31, wherein the cytokine agonist comprises an IL-15 agonist or an IL-15 superagonist.
38. The method of claim 31, wherein the cytokine agonist comprises a complex consisting of an IL-15 mutant that binds to an IL-15 receptor alpha/IgG 1 Fc fusion protein (IL-15N 72D).
39. The method of claim 37, wherein the cytokine agonist comprises ALT-803.
40. The method of any one of claims 31-38, wherein the effective dose of the NEO-201 antibody is reduced as compared to treatment with the NEO-201 antibody alone in the absence of the cytokine agonist.
41. The method of any one of the preceding claims, wherein the cancer comprises colon cancer.
42. The method of any one of the preceding claims, wherein the cancer comprises pancreatic cancer.
43. The method of any one of the preceding claims, wherein the cancer comprises ovarian cancer.
44. The method of any one of the preceding claims, wherein the cancer comprises gastric cancer.
45. The method of any one of the preceding claims, wherein the cancer comprises lung cancer.
46. The method of any one of the preceding claims, wherein the cancer comprises breast cancer.
47. The method of any one of the preceding claims, wherein the cancer comprises uterine cancer.
Background
Cancer represents one of the most common causes of death worldwide, with an estimated 2000 new cases being expected annually as early as 2025 (Ferlay et al, 2015). Conventional methods of treating cancer, such as surgery, radiation and chemotherapy, often induce severe side effects and fail to cure most patients with advanced disease, resulting in relapse (Bodey et al, 1996). Recent therapeutic modalities have been developed to selectively target cancerous cells while largely sparing normal healthy tissue. Among them, immunotherapy has become an important treatment option for cancer patients because it drastically changes the cancer medical field.
The underlying principle of cancer immunotherapy is known as immunoediting (Mittal et al, 2014), which is the extrinsic mechanism of cancer suppression that is initiated only after cellular transformation has occurred and the intrinsic mechanism of cancer suppression has failed. The immunoediting process occurs in three phases: elimination, balance and avoidance. In the elimination and equilibration stages, immune rejection of cancer cells dominates or balances cancer cell proliferation, respectively, to control malignant growth. However, in the escape phase, once kept under examination, cancer cells may escape immune recognition due to insensitivity to immune effector mechanisms and/or induction of immune suppression in the tumor microenvironment. Cancer cells that escape immune recognition are then able to proliferate more freely and grow into a clinically significant disease (Dunn et al, 2004). The goal of tumor immunotherapy is to maintain cancer cells in the elimination phase and/or equilibrium phase by generating and/or augmenting anti-tumor immune responses to counteract tumor growth, delay tumor recurrence, and prolong survival (Carter, 2001; Hodge et al, 2006; Vergati et al, 2010; Gabitzsch et al, 2015). Therapeutic approaches include treatment of patients with checkpoint inhibitory antibodies, anti-tumor vaccines and Chimeric Antigen Receptor (CAR) -T cells, all of which utilize adaptive immunity of T cells. However, innate immunity can also produce and enhance anti-tumor responses, and tumor-targeting monoclonal antibodies (mabs) can be used to stimulate innate anti-tumor immunity (Topalian et al, 2011).
NEO-201 is a novel humanized IgG1mAb generated against the Hollinshead allogeneic colorectal cancer vaccine platform (Hollinshead et al, 1970; Hollinshead et al, 1972). The immunogenic component of this vaccine is a tumor-associated antigen (TAA) derived from a fraction of tumor cell membranes pooled from a surgically excised specimen from 79 colon cancer patients (Hollinshead et al, 1985). These cell membrane fractions were semi-purified, screened for delayed-type hypersensitivity (DTH) in colon cancer patients versus healthy volunteers, and evaluated in clinical trials with refractory colorectal cancer (Hollinshead et al, 1985; Hollinshead, US4810781,1989; Bristol and Kantor, U.S. Pat. No. 7829678,2010). These trials report clinical benefit as defined by a significant prolongation of anti-tumor response and overall survival in patients who develop a sustained IgG response in addition to the cell-mediated response to the vaccine, suggesting that the vaccine contains an immunogenic component capable of producing anti-tumor antibodies (Hollinshead, 1991). This original colorectal cancer vaccine was used to generate monoclonal antibodies in mice, thereby producing the previously described enriximab (NPC-1C/NEO-102) (Luka et al, 2011; Patel et al, 2013; Beg et al, 2016; Kim et al, 2017) and NEO-201. Preliminary investigations have shown that NEO-201 can bind to tumor-associated variants of CEACAM family members (Zeligs et al, 2017), and efforts are underway to further characterize one or more antigens recognized by NEO-201 and one or more specific epitopes.
The human carcinoembryonic antigen (CEA) family consists of 29 genes arranged in tandem on chromosome 19q 13.2. Based on nucleotide homology, these genes are classified into two major subfamilies, CEACAM and the pregnancy-specific glycoprotein subgroup. CEACAM encoded proteins including CEA (CEACAM5), CEA-related cell adhesion molecules (CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7 and CEACAM 8. the CEACAM family belongs to the Ig superfamily structurally, each of the human CEACAMs contains an N-terminal domain comprising 108-110 amino acids and being homologous to an Ig variable domain, followed by a different number (0 to 6) of constant-like domains of the Ig C2 type, the CEACAM proteins can interact homophilically and heterophilically with each other, CEACAM1 is a unique protein in this family because it contains in its cytoplasmic domain an ITIM (immunoreceptor tyrosine-based inhibitory motif) like PD1, this inhibition is triggered by phosphorylation of tyrosine residues with ITIM, which results in the homology-1 and-2 of the
Monoclonal antibodies (mabs) consist of a unique antigen-binding region (antigen-binding fragment, Fab) specific for a given mAb and a constant region (crystallizable fragment, Fc) common to all mabs of the same isotype. The Fc region is capable of modulating immune cells by engaging with Fc receptor (FcR) family members expressed on the surface of specific immune cell typesAnd (4) activity. In particular, human IgG1 mabs can interact with Fc γ receptor IIIa receptor (Fc γ RIIIa, CD16) expressed on macrophages and NK cells. This interaction can stimulate macrophages to phagocytose mAb-opsonized cancer cells, and can activate NK cells to degranulate and lyse cancer cells through a mechanism known as antibody-dependent cellular cytotoxicity (ADCC). ADCC has been demonstrated to be a key mediator of anti-tumor effects in vivo in many preclinical studies and plays an important role in the mechanism of action of several mabs for cancer therapy (Seidel et al, 2013). Examples of clinically approved mabs that can mediate ADCC include trastuzumab, which targets the HER2 receptor of breast cancer (Seidel et al, 2013;etc., 2013); rituximab, which targets the pan-B cell marker CD20 of 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 avilamumab, which targets the immunosuppressive ligand PD-L1 of merkel cell carcinoma and bladder cancer (boyerin et al, 2015). In addition, the Fc region can potentially interact with the C1 complex to activate Complement Dependent Cytotoxicity (CDC), where the proteolytic cascade peaks upon formation of a void in the plasma membrane of the cell, causing lysis of the cell targeted by the antibody. Even where anti-tumor CDC has been demonstrated in vitro, it remains controversial whether anti-tumor CDC is critical to the clinical efficacy of mAb therapy for cancer (Meyer et al, 2014).
Applicants' previous U.S. patent nos. 5,688,657, 7,314,622, 7,491,801, 7,763,720, 7,829,678, 8,470,326, 8,524,456, 8,535,667, 8,802,090, 9,034,588, 9,068,014, 9,371,375, 9,592,290, 9,718,866, and RE39,760 (each of which is hereby incorporated by reference in its entirety) disclose various anti-cancer antibodies, cancer antigens, and related technologies.
Disclosure of Invention
The studies described in the examples herein assess the in vitro binding characteristics as well as in vivo activity and localization of NEO-201 in preclinical models. NEO-201 showed broad reactivity against a range of human carcinoma (carcinoma) cell lines and tumor tissues, but no binding to most healthy tissues was observed. Furthermore, NEO-201 exhibits ADCC and CDC activity against human cancer tumor cells in vitro and attenuates the growth of human pancreatic xenograft tumors to a large extent in vivo, both alone and in combination with human Peripheral Blood Mononuclear Cells (PBMCs) as the effector cell source of ADCC. Finally, single dose toxicity studies in non-human primates demonstrated the safety and tolerability of NEO-201, as the only side effects observed were transient reductions in circulating neutrophils. These studies provide rationale for the potential clinical utility of NEO-201 as a new therapeutic for the treatment of a wide variety of solid tumors. Furthermore, the observed CDC activity of the subject antibodies opens the opportunity to treat immunocompromised patients where ADCC is not expected to be effective, for example in patients that are immunocompromised because of their disease or as a result of radiation, chemotherapy, and other disease treatments.
Preclinical antitumor activity (Patel et al, 2013) and clinical safety and efficacy (Beg et al, 2016; Kim et al, 2017) of mabs generated against the Hollinshead allogeneic colorectal cancer vaccine platform, known as enciximab (NPC-1C/NEO-102), were previously reported. This report describes the characterization of a second tumor antigen targeting mAb (designated NEO-201) derived from the same vaccine platform. NEO-201 is shown to positively stain a variety of human cancer tumor cell lines in vitro, including cells derived from a variety of tumor types, histological subtypes, and mutation profiles. NEO-201 positivity was more frequently observed in tumor cell lines derived from lung adenocarcinoma versus squamous cell carcinoma and HER2 positive breast cancer cell line versus triple negative cancer cell line. Staining of human tumor samples demonstrated that a wide variety of cancerous tumor tissues were positively stained for NEO-201, including colon, pancreatic, gastric, lung, breast and uterine tumors. Extensive research studies using larger sample sizes may reveal that NEO-201 can distinguish histological and molecular subtypes in various carcinomas. Interestingly, a higher proportion of tumor tissues reacted with NEO-201 compared to cultured cancer cell lines. This observation may indicate that the target recognized by NEO-201 is more readily expressed in vivo than in vitro, which would indicate that target expression depends at least in part on interaction with tumor cells from factors within the local microenvironment. Experiments are currently being conducted to further characterize one or more antigens and one or more epitopes recognized by NEO-201 and to identify known or various regulatory control mechanisms that control their expression in tumor tissues rather than normal tissues.
This investigation also revealed that NEO-201 was significantly tumor specific in its stain spectrum, as the vast majority of healthy normal tissues and normal tissues adjacent to the tumor tissue were found to be negative for NEO-201. Although NEO-201 positivity was observed in normal tongue and exocervical tissue, staining intensity was weak, and the microarray represented only a minimum sample size (n ═ 2). A further extensive analysis of NEO-201 staining in normal tissue samples will be performed to confirm these observations. Furthermore, NEO-201 administration did not induce any observable severe toxicity in mice and was well tolerated when administered to non-human primates. The observed depletion of neutrophils in non-human primates indicates that one or more antigens reactive with NEO-201 are expressed on these immune cells and an assessment of the reactivity of NEO-201 with hematopoietic cell types is made. These encouraging results indicate that 1) NEO-201 may have diagnostic utility in differentiating cancerous from benign tissue from a patient biopsy; and 2) NEO-201 can effectively target tumors without causing significant toxicity or off-target effects other than neutropenia. Efforts are currently underway to further evaluate the safety and tolerability of NEO-201, and clinical trials are planned for the treatment of carcinomas using NEO-201.
Innate immune effector mechanisms have been shown to play a major role in promoting and enhancing host anti-tumor immunity. It is well known that the Fc portion of the human IgG1mAb activates innate immunity against opsonized targets, thereby potentially mediating ADCC and/or CDC (Strome et al, 2007; Hayes J, et al, 2017). In particular, the ability to mediate ADCC is considered to be a key component of the therapeutic efficacy of the various human IgG1 mabs approved for the treatment of cancer (Boyerinas et al 2015; Seidel et al 2013; petriceic et al 2013; Dall' Ozzo et al 2004; Levy et al 2009; Kawaguchi et al 2007; Lopez-Albaitero et al 2009). Importantly, the V158F polymorphism of the FCGR3A gene (encoding Fc γ RIIIa) is associated with differential affinity for human IgG1mAb (Koene et al, 1997; Wu et al, 1997), where immune cells from donors with high affinity V/V genotypes show greater trastuzumab-mediated ADCC activity in vitro (Musolino et al, 2008). The V/V genotype also showed significant correlation with objective response rate and progression-free survival in breast cancer patients treated with trastuzumab (Musolino et al, 2008), providing indirect clinical evidence of the role of ADCC in mAb-based therapy. NEO-201 can mediate ADCC in vitro because treatment of tumor cells with NEO-201 enhances the cytotoxic activity of NK cells by 2-5 fold and ADCC activity is retained at even low concentrations of antibody (0.1. mu.g/mL). These data raise the following possibilities: patients with the V/V genotype may receive increased benefit from NEO-201 treatment. Another prospect is the potential to enhance ADCC activity and potentially the potential clinical benefit of NEO-201 by enhancing NK cell function and cytokine stimulation. It is well known that IL-2 is a potent activator of NK cells (Hank et al, 1990) and that IL-21 has been shown to enhance ADCC activity mediated by trastuzumab and cetuximab (Watanabe et al, 2010). Recent preclinical studies of novel fusion protein superagonists of IL-15 signaling (referred to as ALT-803) showed greatly enhanced proliferation, activation and lytic capacity of NK cells (and CD8+ T cells) leading to significant antitumor activity in various animal models of cancer (Han et al, 2011; Gomes-Giacoia et al, 2014; Mathies et al, 2016; Rhode et al, 2016; Kim et al, 2016; Felice et al, 2017). Interestingly, ALT-803 was found to significantly enhance NK cell degranulation, IFN- γ production, and rituximab-mediated ADCC against B-cell lymphoma cell lines and primary follicular lymphoma cells in vitro, and combined treatment with ALT-803 and rituximab in two B-cell lymphoma models resulted in significantly reduced tumor cell burden and improved survival in vivo (Rosario et al, 2016).
Another innate immune effector mechanism potentially conjugated by mabs is activation of the complement system to promote CDC, and NEO-201 was found to have the ability to mediate CDC to kill tumor cells. The contribution of CDC to the therapeutic efficacy of mabs is controversial, but has been proposed to be beneficial for cancer therapy, at least in some specific cases (Meyer et al, 2014). In addition, several different Complement Regulatory Proteins (CRP) act to inhibit complement activation, and it has been reported that certain membrane-bound CRPs such as CD46, CD55, CD59 are abnormally expressed in various cancers (Seya et al, 1994; Niehans et al, 1996; Donin et al, 2003), which may confer resistance to CDC. Future investigations will determine whether strategies to block CRP can enhance CDC of NEO-201 mediated resistant tumor cells.
Evaluation of NEO-201 in vivo revealed a great antitumor effect when administered in combination with activated human immune effector cells. This combination even resulted in a complete regression of some mice (5/20, 25%) from both combination groups. Furthermore, it was found that NEO-201 preferentially localizes to xenograft tumor tissue, but not to various healthy tissues. These data demonstrate that the mechanism of action of NEO-201 against tumors is ADCC-dependent lysis of tumor cells by innate immune cells. However, it should be noted that antitumor activity was also observed in the case of NEO-201 alone without adding human immune cells to immunodeficient mice. This phenomenon may be specific to the conditions encountered in vivo, as treatment of CFPAC-1 tumor cells with NEO-201 did not induce significant toxicity in an in vitro ADCC assay. One hypothesis of NEO-201 activity in the absence of immune effector cells may be the induction of CDC. The CDC activity of NEO-201 was directly demonstrated in a further experiment described in example 3.
Taken together, this research study indicates that NEO-201 is a significant tumor-specific antibody capable of engaging innate immune effector mechanisms (including both ADCC and CDC) to kill tumor cells. Furthermore, NEO-201 demonstrated safety and anti-tumor efficacy in an in vivo xenograft model of pancreatic cancer, as well as tolerance in non-human primates. These findings provide supporting rationale for the clinical development of NEO-201 as a diagnostic and therapeutic agent for patients with a wide variety of carcinomas. The results also support the use of NEO-201 in immunocompromised patients (with low NK cell levels) because anti-tumor effects can be produced by CDC even in the absence of robust ADCC activity.
Drawings
FIGS. 1A-1D: flow cytometry of NEO-201 bound to human carcinoma cell line. Representative human cancer tumor cell lines with various levels of NEO-201 antigen expression, (FIG. 1A) pancreatic CFPAC-1 (high), (FIG. 1B) NSCLC H441 (medium), (FIG. 1C) mammary HCC1937 (low), and (FIG. 1D) colon SW1116 (negative). For each cell line, the results are expressed as% NEO-201 positive and Mean Fluorescence Intensity (MFI). Red, NEO-201 stained cells; black, unstained cells. NEO-201 positivity was defined as% positivity ≧ 10%.
FIGS. 2A-2C: IHC staining of human tumor samples with NEO-201. (FIG. 2A) staining of adjacent normal and malignant tissues from colon, pancreas, stomach and lung samples for representative NEO-201. All images were obtained at 100X. (FIG. 2B) quantification of NEO-201 positive staining of human tumor microarray samples from various cancer tumor tissues. (FIG. 2C) quantification of NEO-201 positive staining of human tumor microarray samples from normal tissue adjacent to tumor tissue. n is the number of samples.
FIGS. 3A-3C: NEO-201 mediates ADCC and CDC against human tumor cell lines. (FIG. 3A) ADCC Activity Using CFPAC-1 or ASPC-1 cells as target cells. Cells were treated with 10. mu.g/mL NEO-201 or human IgG1 (negative control). Purified NK cells from two healthy donors were used as effector cells at the indicated E: T ratio. Statistically significant by T-test (p < 0.05). (FIG. 3B) ADCC assay of CFPAC-1 cells treated with increasing doses of NEO-201 NK cells isolated from healthy donors were used as effector cells at an E: T ratio of 12.5: 1. The graph depicts the fold increase (%) in specific lysis for tumor cells treated with NEO-201 versus control cells treated with 10. mu.g/mL
FIGS. 4A-4D: antitumor efficacy of NEO-201 in CFPAC-1 tumor xenografts. (fig. 4A) tumor volume measurements of CFPAC-1 xenografts from each treatment group at different time points. Mice (
FIGS. 5A-5B: NEO-201 biodistribution in mice carrying CFPAC-1 xenografts. Measurement of normalized radioactivity of indicated tissues from female (fig. 5A) and male (fig. 5B) mice bearing CFPAC-1 tumors given intravenously radiolabeled NEO-201. n-4 animals/time point.
FIGS. 6A-6C: body weight and neutrophil counts from cynomolgus monkeys treated with NEO-201. (figure 6A) the percentage change in body weight of monkeys from baseline (day-1) was measured 7 and 14 days after receiving a single dose of NEO-201 at the indicated dose level. n-4 animals/group (2 females, 2 males). (figure 6B) percentage change in neutrophil levels from baseline (day-7) in blood from monkeys treated with a single dose of NEO-201 at the indicated dose levels. n-4 animals/group (2 females, 2 males). (fig. 6C) neutrophil levels versus p-value for the 0mg/kg control for each dose and time point. Statistically significant by T-test (p < 0.05).
FIGS. 7A-7C: haNK ADCC assay using NEO-201 (4 hours). Target cells were 3000 cells/well. (FIGS. 6A-6B) percent specific lysis as a function of effector to target (E: T) ratio for H520 lung carcinoma (FIG. 6A) or OV90 ovarian carcinoma (FIG. 6B) cells treated at 4 hours with NEO-201 (upper line, square symbols) or IgG1 negative control (lower line, circle symbols). The ratio of E to T is 6.25:1, 12.5:1, or 25: 1. The mAb concentration was 10. mu.g/mL. The values shown are the mean of 3 replicates +/-SD. Asterisks indicate statistical significance of control IgG negative controls (p <0.01, two-tailed t-test). (FIG. 6C) percent specific lysis of lung, (H520, HCC827), breast, (ZR-75-1) and ovarian (OV90) carcinoma cells treated with NEO-201 (right, light gray bar) or negative control IgG (left, solid black bar) at a constant E: T ratio of 25:1 for four hours. The mAb concentration was 10. mu.g/mL. The values shown are the mean of 3 replicates +/-SD. Asterisks indicate statistical significance of control IgG negative controls (p <0.01, two-tailed t-test).
FIG. 8: treatment with ALT-803 enhanced ADCC activity mediated by NEO-201. NK cells isolated from two normal donors were treated with ALT-803(25ng/ml) or media control for 48 hours and used as effector cells in a 4 hour nonradioactive ADCC assay using a Celigo imaging cytometer. CF-PAC1 (human pancreatic cancer cell line) cells were stained with calcein AM and used as targets at 3,000 cells/well. The results are expressed as specific lysis% (SE).
FIG. 9: treatment with ALT-803 enhanced the expression of TIM-3 and NKG2D on human NK cells. Purified human NK cells from normal donors were cultured for 48 hours with or without ALT-803(25 ng/ml). Results are expressed as% of positive cells (MFI).
FIG. 10: treatment with ALT-803 enhanced the expression of TIM-3 and NKG2D on human NK cells. Purified human NK cells from another normal donor were cultured for 48 hours with or without ALT-803(25 ng/ml). Results are expressed as% of positive cells (MFI).
FIG. 11: treatment with ALT-803 enhanced ADCC activity mediated by low concentrations of NEO-201. NK cells isolated from normal donors (ND #6) were treated with ALT-803(25ng/ml) or media control for 48 hours and used as effector cells in a 4 hour non-radioactive ADCC assay using a Celigo imaging cytometer. NEO-201 was used at three different concentrations (10. mu.g/ml, 1. mu.g/ml and 0.1. mu.g/ml). CF-PAC1 (human pancreatic cancer cell line) cells were stained with calcein AM and used as targets at 3,000 cells/well. E: T25: 1. The results are expressed as specific lysis% (SE). Statistically significant (p < 0.01).
FIG. 12: treatment with ALT-803 enhanced ADCC activity of the normal donor (ND #8) with the lowest ADCC activity mediated by NEO-201, and the activity could be blocked by anti-CD 16 and anti-TIM-3 antibodies. NK cells isolated from normal donors with minimal ADCC activity were treated with ALT-803(25ng/ml) or media control for 48 hours and used as effector cells in a 4 hour nonradioactive ADCC assay using a Celigo imaging cytometer. anti-CD 16 and anti-TIM-3 were used at concentrations of 30. mu.g/ml and 15. mu.g/ml. NK cells were pre-treated with anti-CD 16 or anti-TIM-3 for 2 hours prior to addition of NEO-201 and effector cells. CF-PAC1 (human pancreatic cancer cell line) cells were stained with calcein AM and used as targets at 3,000 cells/well. NEO-201 was used at a concentration of 10. mu.g/ml. E: T25: 1. The results are expressed as specific lysis% (SE). Statistically significant (p <0.01) compared to treatment without ALT-803. # was statistically significant (p <0.01) compared to treatment without anti-CD 16 and anti-TIM-3.
FIG. 13: NK-92 killing assay using NEO-201 (16 hours). Target tumor cells (ASPC-1, BxPC-3, CFPAC-1 or LS174T) were seeded at 3000 cells/well. Cells were then treated with 10 μ g/mL of human IgG1 isotype control antibody or NEO-201, and Natural Killer (NK) cell line NK-92 was then added at effector to target (E: T) ratios of 1.5625:1, 3.125:1, 6.25:1, and 12.5: 1. After 16 h incubation at 37 ℃, cell viability was quantified using a Celigo imaging hemocytometer and GraphPad Prism 7 software. Viable target cells (calcein AM +/PI-) were counted per well and specific lysis was calculated. The results are tabulated graphically and below for each tumor cell type. Statistically significant (p < 0.05).
Detailed Description
In one aspect, the present disclosure provides a method of killing cancer cells comprising administering to a patient in need thereof an effective amount of an antibody to NEO-201.
In one aspect, the present disclosure provides a method of treating cancer comprising administering to a patient in need thereof an effective amount of an antibody to NEO-201.
In one aspect, the present disclosure provides a method of preventing cancer recurrence comprising administering to a patient in need thereof an effective amount of an antibody to NEO-201.
In one aspect, the present disclosure provides a method of reducing tumor burden in a patient having a carcinoma, the method comprising administering to a patient in need thereof an effective amount of an antibody to NEO-201.
The antibody may mediate complement-mediated cytotoxicity (CDC), thereby killing cancerous cells in the patient.
Prior to or at the time of the administration, the patient may be natural killer cell ("NK") depleted. Prior to or at the time of the administration, the patient may be severely NK-attenuating. The patient may have an NK cell deficiency (NKD), such as CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD 1). The patient may be NK-depleted or severely NK-depleted as a result of another therapy, for example a cancer therapy (such as 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., radicicolin), 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel), and/or immunomodulatory drugs (e.g., lenalidomide).
The method can comprise determining whether the patient is NK-attenuating prior to or at the time of the administering.
The method can comprise determining whether the patient is severe NK-attenuating prior to or at the time of the administration.
In the method, prior to or at the time of the administering, NK cells may comprise less than 5% of Peripheral Blood Mononuclear Cells (PBMCs) in the individual.
In the method, prior to or at the time of the administering, NK cells may comprise less than 3% of Peripheral Blood Mononuclear Cells (PBMCs) in the individual.
In the method, less than 70% of PBMC NK cells in the patient prior to or at the time of said administering may be CD56dimCD16+ NK cells.
In the method, less than 50% of PBMC NK cells in the patient prior to or at the time of said administering may be CD56dimCD16+ NK cells.
The NEO-201 antibody can comprise at least one, two, three, four, five, or all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO: 29.
The NEO-201 antibody can comprise a variable heavy chain sequence having at least 90% identity to SEQ ID NO 38.
The NEO-201 antibody can comprise a variable light chain sequence having at least 90% identity to SEQ ID NO: 39.
The NEO-201 antibody can comprise a variable heavy chain sequence having at least 90% identity to SEQ ID NO. 38 and a variable light chain sequence having at least 90% identity to SEQ ID NO. 39.
The NEO-201 antibody can comprise a heavy chain sequence having at least 90% identity to amino acids 20-470 of SEQ ID NO. 28 and a light chain sequence having at least 90% identity to amino acids 20-233 of SEQ ID NO. 29.
The NEO-201 antibody may comprise all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO: 29.
The NEO-201 antibody may comprise a human IgG1 constant domain.
The NEO-201 antibody can be a humanized antibody.
The NEO-201 antibody can be conjugated to another moiety.
The NEO-201 antibody can be conjugated to another cytotoxic moiety, label, radioactive moiety, or affinity tag.
The method can further comprise administering to the patient an effective amount of a cytokine agonist to enhance or stimulate killing of the cells of the carcinoma. The cytokine agonist may comprise interleukin 2(IL-2), interleukin 21(IL-21), ALT-803, an IL-15 inhibitor, a checkpoint inhibitor,
The method can further comprise administering to the patient an effective amount of a Complement Regulatory Protein (CRP) antagonist to enhance or stimulate killing of the cells of the carcinoma. The CRP antagonist can antagonize one or more of CD46, CD55, or CD 59. The CRP antagonist can comprise an antibody or antigen-binding fragment thereof.
The cytokine agonist may include an IL-15 agonist or an IL-15 superagonist.
The cytokine agonist may include a complex consisting of an IL-15 mutant (IL-15N72D) that binds to an IL-15 receptor alpha/
The effective dose of the NEO-201 antibody is reduced compared to treatment with the NEO-201 antibody alone in the absence of the cytokine agonist.
The cancer may include colon cancer. The carcinoma may comprise a pancreatic carcinoma. The carcinoma may comprise ovarian cancer. The carcinoma may comprise gastric carcinoma. The carcinoma may comprise lung cancer. The carcinoma may comprise breast cancer. The carcinoma may comprise a uterine cancer.
In another embodiment, the present disclosure provides a method of killing cancer tumor cells, comprising administering to a patient in need thereof an effective amount of an antibody to NEO-201, wherein prior to or at the time of said administering, said patient is natural killer ("NK") depleted. The NK depletion may be comprised in a sample, e.g. a blood sample, derived from a patient whose less than 5% or less than 3% of Peripheral Blood Mononuclear Cells (PBMCs) are NK cells. Alternatively or additionally, less than 70% (or optionally less than 50%) of PBMC NK cells in said patient may be CD56dimCD16+ NK cells prior to or at the time of said administering.
In another embodiment, the present disclosure provides a method of treating cancer comprising administering to a patient in need thereof an effective amount of an antibody to NEO-201, wherein prior to or at the time of said administering, said patient is natural killer cell ("NK") depleted.
In another embodiment, the present disclosure provides a method of preventing the recurrence of a cancer tumor, comprising administering to a patient in need thereof an effective amount of an antibody to NEO-201, wherein prior to or at the time of said administering, said patient is natural killer cell ("NK") depleted.
In another embodiment, the present disclosure provides a method of reducing tumor burden in a patient having a cancer tumor, the method comprising administering to a patient in need thereof an effective amount of a NEO-201 antibody, wherein prior to or at the time of said administering, the patient is natural killer cell ("NK") depleted.
In the foregoing methods, the antibody may mediate CDC, thereby killing cancer cells in the patient, e.g., despite the absence of effective ADCC due to the patient being NK-depleted. Upon said administering, said patient may be severe NK-attenuating. Optionally, the method further comprises determining whether the patient is NK-depleting or severe NK-depleting, e.g., at or within a time period prior to said administering, such as within 1 or 2 weeks prior. NK-depleted or severe NK-depleted states can also be inferred from the patient's history, such as prior to or concurrent with another therapy that depletes NK cells. For example, the patient has undergone or is concurrently undergoing cancer therapy, such as radiation therapy or chemotherapy. The cancer therapy can include administration of one or more proteasome inhibitors (e.g., bortezomib, MG132), histone deacetylase inhibitors (e.g., valproic acid, trichostatin a, suberoylanilide-hydroxamic acid (SAH), sodium butyrate), genotoxic agents (e.g., doxorubicin, melphalan, cisplatin, Ara-C, aphidicolin, mitomycin, methotrexate, etoposide), GSK inhibitors (e.g., LiCl, BIO, SB21), BET inhibitors (e.g., JQ1), HSP90 inhibitors (e.g., radicicolin (radicicola)), 17-AAG), microtubule assembly inhibitors (e.g., vincristine, cytochalasin D, nocodazole, docetaxel), and/or immunomodulatory drugs (e.g., lenalidomide).
The patient may have an NK cell deficiency (NKD), such as CNKD (e.g., CNKD1, CNKD2) or FNKD (e.g., FNKD 1).
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise at least one, two, three, four, five or all six of the CDR sequences contained in SEQ ID NO 28 and SEQ ID NO 29.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise a variable heavy chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to SEQ ID NO 38. The variable heavy chain having the percentage of sequence identity may comprise all 3 of the CDR sequences contained in SEQ id No. 38.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise a variable light chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to SEQ ID NO 39. The variable light chain may comprise all 3 of the CDR sequences contained in SEQ ID NO 39.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise a variable heavy chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to SEQ ID No. 38 and a variable light chain sequence having at least 80%, at least 85%, at least 90%, or most preferably at least 95% identity to SEQ ID No. 39. The variable light chain can comprise all 3 of the CDR sequences contained in SEQ ID No. 39, and the variable heavy chain having the sequence identity percentage can comprise all 3 of the CDR sequences contained in SEQ ID No. 38.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise a heavy chain sequence having at least 80%, at least 85%, at least 90% or most preferably at least 95% identity to amino acids 20-470 of SEQ ID NO. 28 and a light chain sequence having at least 80%, at least 85%, at least 90% or most preferably at least 95% identity to amino acids 20-233 of SEQ ID NO. 29. The light chain may comprise all 3 of the CDR sequences contained in SEQ ID NO. 29 and the heavy chain having the stated percentage of sequence identity may comprise all 3 of the CDR sequences contained in SEQ ID NO. 28.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise the heavy chain variable region sequence contained in SEQ ID NO:28 and the light chain variable region sequence contained in SEQ ID NO: 29.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the NEO-201 antibody may comprise a heavy chain sequence comprising amino acids 20-470 of SEQ ID NO 28 and a light chain sequence comprising amino acids 20-233 of SEQ ID NO 29.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the NEO-201 antibody comprises a human IgG1 constant domain.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the NEO-201 antibody may be a humanized antibody.
In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the NEO-201 antibody may be conjugated to another moiety, such as another cytotoxic moiety, a label, a radioactive moiety or an affinity tag.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the method may further comprise administering to the patient an effective amount of a cytokine agonist to enhance or stimulate killing of the cells of the carcinoma. The cytokine agonist may comprise interleukin 2(IL-2), interleukin 21(IL-21), ALT-803, an IL-15 inhibitor, a checkpoint inhibitor,
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the method may further comprise administering to the patient an effective amount of a Complement Regulatory Protein (CRP) antagonist to enhance or stimulate killing of the cancer cells. The CRP antagonist can antagonize one or more of CD46, CD55, or CD 59. The CRP antagonist can comprise an antibody or antigen-binding fragment thereof. The cytokine agonist may include an IL-15 agonist or an IL-15 superagonist. The cytokine agonist may comprise a complex consisting of a mutant of IL-15 (IL-15N72D) bound to an IL-15 receptor alpha/
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the effective dose of the NEO-201 antibody is reduced compared to treatment with the NEO-201 antibody alone in the absence of the cytokine agonist.
In a preferred embodiment of the invention that may be used with any of the preceding or following embodiments, the cancer may express the NEO-201 antigen. Said expression of NEO-201 antigen can be determined by detecting NEO-201 antigen in a sample of said cancer. The detection may be performed by techniques including, but not limited to: histological staining, flow cytometry, RT-PCR, dot blotting, western blotting, northern blotting, and other techniques known in the art. In the case of recurrent or metastatic cancer, expression of the NEO-201 antigen can also be inferred by expression of NEO-201 in the primary cancer or by responsiveness of the primary cancer to NEO-201 antibody therapy.
In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the cancer may comprise colon cancer.
In a preferred embodiment of the invention, which can be used with any of the preceding or following embodiments, the cancer can comprise pancreatic cancer.
In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the cancer may comprise ovarian cancer.
In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the cancer may comprise gastric cancer.
In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the cancer may comprise lung cancer.
In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the cancer may comprise breast cancer.
In a preferred embodiment of the invention, which may be used with any of the preceding or following embodiments, the cancer may comprise uterine cancer.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the present invention or the testing of the present invention, suitable methods and materials are described herein. The materials, methods, and examples are illustrative only and not intended to be limiting.
The meaning of "a/an" and "the" as used in the description herein and throughout the claims that follow, unless the context clearly dictates otherwise, includes plural referents.
As used herein, "amino acid" refers in a broad sense to both 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 the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, e.g., hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon, carboxyl, amino, and R groups bound to a hydrogen, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
The term "NK depletion" or "natural killer cell depletion" as used herein means that the patient has a low level of Natural Killer (NK) cells relative to the normal range. NK cells are cytotoxic innate immune lymphocytes. Typically, NK cells account for 5% -20% of Peripheral Blood Mononuclear Cells (PBMCs) in healthy individuals. Patients with NK cells that account for less than 5% of PMBC are termed NK-depleted. In addition, if NK cells comprise less than 3% of PMBC, the patient is said to be severely NK cell-depleted. In addition, in normal individuals, up to 90% of PBMC NK cells are CD56dimCD16+NK cells, and these are considered to be the most toxic subpopulations. If less than 70% of the PBMC NK cells are CD56dimCD16+NK cells, and the patient is said to be NK-depleted. In addition, if less than 50% of the PBMCNK cells are CD56dimCD16+NK cells, then the patient is said to be severely NK-depleted. A given patient may be referred to as NK-diminished or severe NK-diminished based on one or both of these individual criteria being met. Generally, the status of a patient is determined as NK-diminished or severe NK-diminished by testing a sample, e.g., a blood sample, taken from the patient, e.g., a sample obtained and tested within one or two weeks ago. The patient's status can also be inferred as NK-diminished or severely NK-diminished from disease diagnosis and/or the course of treatment associated with such depletion of NK cells.
NK-depleted also includes subjects with NK cell deficiency (NKD). Exemplary NKD conditions include classical NKD (cnkd), which is characterized by the absence of NK cells and their function in peripheral blood lymphocytes; functional nkd (fnkd), characterized by NK cells present in peripheral blood lymphocytes, with defective NK cell activity. In both CNKD and FNKD, NK cell abnormalities are major immune defects that result in an inadequate ADCC response. CNKD and FNKD can be further subdivided based on patient characteristics, such as the identity of one or more causative genes and other patient characteristics. CNKD includes CNKD subtype 1(CNKD1), which is autosomal dominant and associated with a defect in the GATA2 gene; and CNKD subtype 2(CNKD2), which is autosomal recessive and associated with a defect in the MCM4 gene. FNKD includes FNKD1, which is autosomal recessive and is associated with a defect in the FCCR3A gene.
The term "antibody" as used herein refers broadly to any polypeptide chain-containing molecular structure having a specific shape that coordinates to and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. A typical antibody molecule is an immunoglobulin and all types of immunoglobulins IgG, IgM, IgA, IgE, IgD from all sources (e.g. human, rodent, rabbit, cow, sheep, pig, dog, chicken) are considered "antibodies". Antibodies include, but are not limited to, chimeric, human and other non-human mammalian antibodies, humanized antibodies, single chain antibodies (scFv), camelid antibodies, nanobodies, IgNAR (single chain antibodies obtained from sharks), Small Molecule Immunopharmaceuticals (SMIP), and antibody fragments (e.g., Fab ', F (ab')2). A number of antibody coding sequences have been described; and other antibody coding sequences can be generated by methods well known in the art. See Streltsov, et al (2005)Protein Sci.14(11) 2901-9; greenberg, et al (1995)Nature374(6518) 168-; nuttall, et al (2001)Mol Immunol.38(4) 313-26; Hamers-Casterman, et al (1993)Nature363(6428) 446-8; gill, et al (2006)Curr Opin Biotechnol.17(6):653–8。
"NEO-201 antibody" refers to heavy and light chains comprising SEQ ID NOS: 28 and 29 or the variable regions contained therein, optionally along with constant regions, as well as fragments and variants thereof. Such variants include sequences comprising one, two, three, four, five or preferably all six of the CDR sequences contained in SEQ ID NO 28 and SEQ ID NO 29, namely the heavy chain CDR1 of SEQ ID NO 32, the heavy chain CDR2 of SEQ ID NO 33, the heavy chain CDR3 of SEQ ID NO 34, the light chain CDR1 of SEQ ID NO 35, the light chain CDR2 of SEQ ID NO 36 and the light chain CDR3 of SEQ ID NO 37. The antibody may be a humanized antibody. The antibody may be expressed to contain one or more leader sequences that may be removed during expression and/or processing and secretion of the antibody. The antibodies can be presented in monovalent, bivalent, or higher multivalent forms, including but not limited to bispecific or multispecific antibodies comprising the NEO-201 antibody sequence and binding fragments of different antibodies. Typically, the antibody specifically binds to a cancer cell and competes for binding to the cancer cell with an antibody comprising the variable heavy chain of SEQ ID NO:38 and the variable light chain of SEQ ID NO:39 or the heavy chain of SEQ ID NO:28 and the light chain of SEQ ID NO: 29. One or more of those CDR sequences contained in SEQ ID NO 28 and/or SEQ ID NO 29 may be substituted by a variant sequence such as the light chain CDR1 of
As used herein, "antigen" refers broadly to a molecule or portion of a molecule capable of being bound by an antibody, which antigen is otherwise capable of inducing an animal to produce an antibody capable of binding to an epitope of the antigen. An antigen may have one epitope, or more than one epitope. Specific reactions as referred to herein indicate that an antigen will react in a highly selective manner with its corresponding antibody and not with a variety of other antibodies that may be elicited by other antigens. The antigen may be tumor specific (e.g., expressed by neoplastic cells of pancreatic and colon carcinomas.)
As used herein, "cancer" refers broadly to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division, resulting in malignant growth or tumors.
As used herein, "chimeric antibody" refers broadly to antibody molecules in which the constant regions, or a portion thereof, are altered, replaced, or exchanged such that the antigen binding site (variable region) is linked to the constant region of a different or altered class, effector function and/or species, or an entirely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that confers new properties to the chimeric antibody; or the variable region or a portion thereof is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
As used herein, "conservatively modified variants" applies to both amino acid sequences and nucleic acid sequences, and with respect to a particular nucleic acid sequence, refers in a broad sense to conservatively modified variants, to those nucleic acids that encode identical or substantially identical amino acid sequences, or to substantially identical sequences when the nucleic acids do not encode an amino acid sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Each nucleic acid sequence herein encoding a polypeptide also illustrates each possible silent variation of the nucleic acid. The skilled artisan will recognize that each codon in a nucleic acid (except AUG, which is codon only, typically methionine, and TGG, which is codon only, typically tryptophan) can be modified to produce a functionally identical molecule.
As used herein, "complementarity determining region," "hypervariable region," or "CDR" refers broadly to one or more hypervariable or Complementarity Determining Regions (CDRs) found in the variable region of a light chain or a heavy chain of an antibody. See Kabat, et al (1987) "Sequences of Proteins of Immunological Interest"National Institutes of health, Bethesda, Md. These expressions include, for example, those made by Kabat, et al (1983) "Sequences of Proteins of Immunological InterestThe hypervariable regions defined by "U.S. Dept. of Health and Human Services or hypervariable loops in the 3-dimensional structure of antibodies. Chothia and Lesk (1987)J Mol.Biol.196:901-917. The CDRs in each chain are held in close proximity by the framework regions and, together with the CDRs from the other chain, facilitate the formation of an antigen binding site. Within the CDRs, there are selected amino acids that have been described as Selectivity Determining Regions (SDRs), which represent key contact residues used by the CDRs in antibody-antigen interactions. Kashmiri (2005)Methods36:25–34。
As used herein, "control amount" refers broadly to any amount or series of amounts that a marker can be compared to a test amount of the marker. For example, a control amount of a marker can be the amount of the marker in a patient having a particular disease or condition or in a human not having such a disease or condition. The control amount can be an absolute amount (e.g., micrograms/ml) or a relative amount (e.g., relative intensity of a signal).
As used herein, "differentially present" refers in a broad sense to a difference in the amount or quality of a marker present in a sample taken from a patient having one of the diseases or conditions as compared to a comparable sample taken from a patient not having the disease or condition. For example, if the amount of nucleic acid fragments in one sample is significantly different from the amount of nucleic acid fragments in another sample, the nucleic acid fragments may optionally be present differentially between the two samples, e.g., as measured by hybridization and/or NAT-based assays. If the amount of polypeptide in one sample is significantly different from the amount of polypeptide in another sample, the polypeptide may optionally be present differentially between the two samples. It should be noted that a marker may be considered differentially present if it is detectable in one sample and undetectable in another sample. Optionally, a relatively low amount of upregulation can serve as a marker.
As used herein, "diagnosis" refers broadly to identifying the presence or nature of a pathological condition. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive ("percentage of true positives"). Diseased individuals not detected by the assay are "false negatives". Subjects who are not diseased and who test negative in the assay are referred to as "true negatives". The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those that test positive but do not have the disease. Although a particular diagnostic method may not provide a definitive diagnosis of the condition, it is acceptable if the method provides a positive indication that aids in diagnosis.
As used herein, "diagnosis" refers broadly to grading a disease or symptom, determining the severity of a disease, monitoring disease progression, predicting the outcome of a disease and/or the prospect of recovery. The term "detecting" may also optionally encompass any of the foregoing. In some embodiments, diagnosis of a disease according to the invention may be affected by determining the level of a polynucleotide or polypeptide of the invention in a biological sample obtained from the subject, wherein the determined level may be related to a susceptibility to, or the presence or absence of, the disease. It should be noted that "a biological sample obtained from a subject" may also optionally include a sample that has not been removed from the subject.
As used herein, an "effective amount" refers broadly 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 the treatment of the disease. An effective amount may be an amount effective for prophylaxis (prophyxiases), and/or an amount effective for prophylaxis (prevention). An effective amount can be an amount effective to reduce, prevent, reduce the severity of, eliminate, slow the progression of, prevent and/or effectively prevent the occurrence of signs/symptoms. The "effective amount" may vary depending on the disease and its severity in the patient to be treated as well as the age, weight, medical history, susceptibility and preexisting condition. For the purposes of the present invention, the term "effective amount" is synonymous with "therapeutically effective amount".
As used herein, "expression vector" refers in a broad sense to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention, either constitutively or inducibly, in vitro or in vivo, in any cell, including prokaryotic cells, yeast cells, fungal cells, plant cells, insect cells, or mammalian cells. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the genome of the host cell. The expression system may have the ability to self-replicate or not (i.e., drive only transient expression in a cell). The term includes recombinant expression cassettes that contain only the minimal elements required for transcription of the recombinant nucleic acid.
As used herein, "framework region" or "FR" refers broadly to one or more framework regions within the variable region of a light or heavy chain of an antibody. See Kabat, et al (1987) "Sequences of Proteins of Immunological Interest"National Institutes of Health, Bethesda, Md. These expressions include those amino acid sequence regions inserted between the CDRs within the variable regions of the light and heavy chains of the antibody.
As used herein, "heterologous" refers broadly to a portion of a nucleic acid, indicating that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are often produced recombinantly such that two or more sequences from unrelated genes are arranged to make new functional nucleic acids, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that a protein comprises two or more subsequences that are not related in nature to each other (e.g., a fusion protein).
As used herein, "high affinity" refers in a broad sense to an antibody having at least 10 to a target antigen–8M, more preferably at least 10–9M and even more preferably at least 10–10KD of M. However, for other antibody isotypes, "high affinity" binding may vary. For example, for IgM isotypes, "high affinity" binding means having at least 10–7M, more preferably at least 10–8Antibody to KD of M.
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 may be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. A partially homologous nucleic acid or amino acid sequence is a nucleic acid or amino acid sequence that is not identical to a reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The term "sequence identity" is used interchangeably with "homology".
As used herein, "host cell" refers broadly to a cell that contains an expression vector and supports replication or expression of the expression vector. The host cell may be a prokaryotic cell, such as E.coli, or a eukaryotic cell, such as yeast, insect (e.g., SF9), amphibian, or mammalian cell, such as CHO, HeLa, HEK-293, e.g., cultured cells, explants, and in vivo cells.
As used herein, "hybridization" refers broadly to the physical interaction of complementary (including partially complementary) polynucleotide strands by forming hydrogen bonds between complementary nucleotides when the strands are arranged antiparallel to one another.
As used herein, the term "K-assoc" or "Ka" refers broadly to the binding rate of a particular antibody-antigen interaction; as used herein, the term "Kdiss" or "Kd" refers to the off-rate of a particular antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant, which is obtained from the ratio of KD to Ka (i.e., KD/Ka) and is expressed as molar concentration (M). The KD value of an antibody can be determined using well established methods in the art.
As used herein, "immunoassay" refers in a broad sense to an assay that uses an antibody that specifically binds an antigen. Immunoassays may be characterized by the use of the specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
As used herein, "isolated" refers in a broad sense to a material that is removed from its original environment in which it naturally occurs and is thus artificially altered from its natural environment. An isolated material may be, for example, an exogenous nucleic acid contained in a vector system, an exogenous nucleic acid contained within a host cell, or any material that has been removed from its original environment and thus artificially altered (e.g., an "isolated antibody").
As used herein, "label" or "detectable moiety" refers in a broad sense to a composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means.
As used herein, "low stringency," medium stringency, "high stringency," or "very high stringency conditions" refers broadly to conditions for nucleic acid hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel, et al (2002)Short Protocols in Molecular Biology(5thEd.)John Wiley&Sons, NY. Exemplary specific hybridization conditions include, but are not limited to: (1) low stringency hybridization conditions of washing 2 times in 6X sodium chloride/sodium citrate (SSC) at about 45 ℃ followed by at least 50 ℃ in 0.2XSSC, 0.1% SDS (for low stringency conditions, the temperature of the wash may be increased to 55 ℃); (2) moderate stringency hybridization conditions of one or more washes in 6XSSC at about 45 ℃ followed by 0.2XSSC, 0.1% SDS at 60 ℃; (3) high stringency hybridization conditions that are washed one or more times in 6XSSC at about 45 ℃ followed by 0.2XSSC, 0.1% SDS at 65 ℃; and (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65 ℃, followed by one or more washes in 0.2XSSC, 1% SDS at 65 ℃.
As used herein, "mammal" refers broadly to any and all warm-blooded vertebrate of the class mammalia, including humans, characterized by skin covered with hair, and in females that produce mammary glands for the rearing of litters. Examples of mammals include, but are not limited to, alpaca, armadillo, dolphin, cat, camel, chimpanzee, chinchilla, cow, dog, goat, gorilla, hamster, horse, human, lemur, llama, mouse, non-human primate, pig, mouse, sheep, shrew, squirrel, and tapir. Mammals include, but are not limited to, bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammals also include any and all mammals listed on the world species of mammals maintained by the national natural history museum, smith society, of washington, d.
As used herein, "nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single-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 indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
As used herein, the term "operably linked" refers in a broad sense to the joining of two DNA fragments such that the amino acid sequences encoded by the two DNA fragments remain in frame.
As used herein, a "paratope" refers broadly to a portion of an antibody that recognizes an antigen (e.g., the antigen binding site of an antibody.) the paratope may be a small region of the Fv region of an antibody (e.g., 15-22 amino acids), and may contain portions of the heavy and light chains of an antibody. See Goldsby, et alAntigens(Chapter 3)Immunology (5 th edition) New York: W.H.Freeman and Company, pages 57-75.
As used herein, "patient" refers broadly to any animal in need of treatment to alleviate a disease state or prevent the occurrence or recurrence of a disease state. Furthermore, as used herein, "patient" refers broadly to any animal that has previously been diagnosed with risk factors, medical history, susceptibility, symptoms, signs, or is at risk for or a member of a patient population for a disease. The patient may be a clinical patient, such as a human or veterinary patient, such as a companion animal, a domestic animal, a curious animal or a zoo animal. The term "subject" is used interchangeably with the term "patient".
"polypeptide," "peptide," and "protein" are used interchangeably herein and refer in a broad sense to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an analogue or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Polypeptides may be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide", "peptide" and "protein" include glycoproteins as well as non-glycoproteins.
As used herein, "promoter" refers broadly to a nucleic acid sequence that directs the transcription of a nucleic acid. As used herein, "promoter" includes essential nucleic acid sequences near the transcription start site, such as a TATA element in the case of a type II polymerase promoter. Promoters also optionally include distal enhancer or repressor elements, which can be located as far as several thousand base pairs from the transcription start site. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.
As used herein, a "prophylactically effective amount" refers in a broad sense to an amount of a compound that, when administered to a patient for the prevention of a disease or the prevention of the recurrence of a disease, is sufficient to achieve such prevention of the disease or recurrence. A prophylactically effective amount may be an amount effective to prevent the occurrence of signs and/or symptoms. The "prophylactically effective amount" may vary depending on the disease and its severity of the patient to be treated as well as the age, weight, medical history, susceptibility to the condition, preexisting condition.
As used herein, "prophylaxis" refers in a broad sense to a course of treatment in which the signs and/or symptoms are not present in the patient, are in remission, or are previously present in the patient. Prevention and treatment includes prevention of diseases that occur following treatment of a disease in a patient. Furthermore, prevention includes treatment of patients who may potentially develop a disease, particularly patients who are predisposed to the disease (e.g., members of a patient population, patients with risk factors, or patients at risk for developing a disease).
As used herein, with respect to a product, "recombinant" refers broadly to, for example, a cell or nucleic acid, protein or vector, indicating that the cell or nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or indicating that the cell originates from a cell so modified. Thus, for example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.
As used herein, "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with … …" or "specifically interacts or binds" refers broadly to a protein or peptide (or other epitope), and in some embodiments to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological agents. For example, under specified immunoassay conditions, specific antibodies bind to a particular protein at least two times greater than background (non-specific signal) and do not substantially bind in significant amounts to other proteins present in the sample. Typically, a specific or selective reaction will be at least twice background signal or background noise, and more typically greater than about 10-fold to 100-fold background.
As used herein, "specifically hybridizable" and "complementary" are used in a broad sense to mean that the nucleic acid can be detected by conventional Watson-Crick (Watson)-Crick) type or other unconventional type forms one or more hydrogen bonds with another nucleic acid sequence. The binding free energy of a nucleic acid molecule to its complementary sequence is sufficient to allow for the relevant function of the nucleic acid, e.g., RNAi activity. Determining the binding free energy of a nucleic acid molecule is well known in the art. See, e.g., Turner, et al (1987)CSH Symp.Quant.Biol.LII of 123-33; frier, et al (1986)PNAS9373-77; turner, et al (1987)J.Am.Chem.Soc.109:3783-85. Percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., watson-crick base pairing) with a second nucleic acid sequence (e.g., at least about 5,6, 7,8, 9, 10 out of 10 are at least about 50%, 60%, 70%, 80%, 90%, and 100% complementary). "completely complementary" or 100% complementarity refers, in a broad sense, to all consecutive residues of a nucleic acid sequence that are hydrogen-bonded to the same number of consecutive residues in a second nucleic acid sequence. By "substantial complementarity" is meant that the polynucleotide strands exhibit at least about 90% complementarity in regions of the polynucleotide strands selected so as to be non-complementary, except for overhangs. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired (i.e., under physiological conditions in the case of an in vivo assay or therapeutic treatment, or under conditions in which the assay is performed in the case of an in vitro assay). Non-target sequences may typically differ by at least 5 nucleotides.
As used herein, "signs" of disease broadly refer to any abnormality indicative of disease that may be found at the time of examination of a patient; is an objective indication of the disease as compared to the symptoms of subjective indications of the disease.
As used herein, "solid support," "support," and "substrate" refer broadly to any material that provides a solid or semi-solid structure to which another material may be attached, including but not limited to smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) and textured and porous materials.
As used herein, "subject" refers broadly to subjects suitable for treatment according to the present invention, including but not limited to avian and mammalian subjects, and preferably mammals. Mammals of the invention include, but are not limited to, dogs, cats, cows, goats, horses, sheep, pigs, rodents (e.g., rats and mice), lagomorphs, primates, humans. Any mammalian subject in need of treatment according to the present invention is suitable. Human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult) can be treated according to the invention. The invention may also be carried out on animal subjects, in particular mammalian subjects, such as mice, rats, dogs, cats, cows, goats, sheep and horses for veterinary purposes and for drug screening and drug development purposes. "subject" is used interchangeably with "patient".
As used herein, "symptoms" of a disease refers broadly to any pathological phenomena or deviation from normal structure, function, or sensation that a patient experiences and is indicative of the disease.
As used herein, "Therapy", "therapeutic", "treating" or "treatment" refers broadly to treating a disease, arresting or reducing the development of a disease or clinical symptoms thereof, and/or relieving a disease, whereby the disease or clinical symptoms thereof are resolved. Therapy encompasses prevention (prophyxiases), treatment, remediation, reduction, alleviation and/or alleviation of the disease, signs and/or symptoms of the disease. Therapy encompasses the reduction of signs and/or symptoms of a patient with developing signs and/or symptoms of a disease (e.g., tumor growth, metastasis). Therapy also encompasses "prevention. For therapeutic purposes, the term "reduce" refers broadly to a clinically significant reduction in signs and/or symptoms. Therapy includes treatment of relapse or relapse signs and/or symptoms (e.g., tumor growth, metastasis). Treatment encompasses, but is not limited to, excluding any occurrence of signs and/or symptoms and reducing and eliminating existing signs and/or symptoms. Therapies include the treatment of chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing relapse or relapse of signs and/or symptoms (e.g., tumor growth, metastasis).
As used herein, "canThe variable region "or" VR "refers broadly to a domain within each pair of light and heavy chains in an antibody that is directly involved in binding the antibody to an antigen. Each heavy chain has a variable domain at one end (V)H) Followed by a plurality of constant domains. Each light chain has a variable domain at one end (V)L) And has a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
As used herein, "vector" refers broadly to a plasmid, cosmid, phagemid, phage DNA, or other DNA molecule that is capable of autonomous replication in a host cell and is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of essential biological function of the vector and into which DNA may be inserted in order to bring about its replication and cloning. The vector may also contain a marker suitable for use in the identification of cells transformed with the vector.
The techniques and procedures are generally performed according to conventional methods well known in the art and described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook, et al (2001)Molec.Cloning:Lab.Manual[ 3 rd edition)]Cold Spring Harbor Laboratory Press. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture, as well as transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The nomenclature used and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well known and commonly employed in the art. Standard techniques are available for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.
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