B-CELL MATURATION ANTIGEN (BCMA)-DIRECTED NANOPARTICLES
20200384130 ยท 2020-12-10
Assignee
Inventors
- Alexandre Detappe (Boston, MA, US)
- Irene Ghobrial (Boston, MA, US)
- Mairead REIDY (Brookline, MA, US)
- Peter GHOROGHCHIAN (Boston, MA, US)
Cpc classification
A61K47/6931
HUMAN NECESSITIES
B82Y5/00
PERFORMING OPERATIONS; TRANSPORTING
A61K49/1881
HUMAN NECESSITIES
A61K49/0008
HUMAN NECESSITIES
International classification
A61K49/18
HUMAN NECESSITIES
A61K47/69
HUMAN NECESSITIES
Abstract
The present invention relates to compositions comprising B-cell maturation antigen-directed nanoparticles and methods for using the same.
Claims
1. A targeted nanoparticle conjugate comprising: a nanoparticle; a linker; and an anti-B-cell maturation (BCMA) antibody.
2. The targeted nanoparticle conjugate of claim 1, wherein the nanoparticle of the targeted nanoparticle conjugate is less than 10 nm in size.
3. The targeted nanoparticle conjugate of claim 1, wherein the nanoparticle is a gadolinium nanoparticle, optionally a silica-based gadolinium nanoparticle (SiGdNP).
4. The targeted nanoparticle conjugate of claim 1, wherein the nanoparticle of the targeted nanoparticle conjugate is 30 nm or more in size.
5. The targeted nanoparticle conjugate of claim 1, wherein the nanoparticle is a polymer brush nanoparticle or a nanoparticle comprising clustered regularly interspaced short palindromic repeats (CRISPR) machinery (i.e. sgRNA guides and/or Cas9 mRNA) agents.
6. The targeted nanoparticle conjugate of claim 1, wherein the nanoparticle is a polymer nanoparticle, optionally wherein the targeted nanoparticle conjugate further comprises a drug.
7. The targeted nanoparticle conjugate of claim 1, wherein the nanoparticle is an inorganic nanoparticle.
8. The targeted nanoparticle conjugate of claim 1, wherein the targeted nanoparticle conjugate is approximately 6-15 nm in size, optionally about 8-12 nm in size, optionally wherein the size of the targeted nanoparticle conjugate is stable over time, optionally wherein the size of the targeted nanoparticle conjugate is stable over a period of 15 min or longer, optionally wherein the size of the targeted nanoparticle conjugate is stable over a period of 30 min or longer.
9. The targeted nanoparticle conjugate of claim 1, wherein the targeted nanoparticle conjugate is approximately 15-60 nm in size, optionally about 30-50 nm in size, optionally wherein the size of the targeted nanoparticle conjugate is stable over time, optionally wherein the size of the targeted nanoparticle conjugate is stable over a period of 15 min or longer, optionally wherein the size of the targeted nanoparticle conjugate is stable over a period of 30 min or longer.
10. The targeted nanoparticle conjugate of claim 1, wherein the linker is selected from the group consisting of a N-hydroxysuccinimide (NHS)-to-NHS linker, a NHS-to-haloacetyl, a NHS-maleimide, and a NHS-pyridyldithiol linker.
11. The targeted nanoparticle conjugate of claim 1, wherein the anti-BCMA antibody is a monoclonal antibody or fragment thereof, optionally a human monoclonal antibody or fragment thereof.
12. The targeted nanoparticle conjugate of claim 1, wherein the anti-BCMA antibody is an anti-BCMA antibody fragment, optionally selected from the group consisting of a Fv, a Fab, a Fab, a Fab-SH, a F(ab).sub.2, a diabody, a linear antibody, a single-chain antibody molecule (e.g., scFv) and a multispecific antibody formed from antibody fragments.
13. The targeted nanoparticle conjugate of claim 1, wherein the anti-BCMA antibody is labeled, optionally wherein the anti-BCMA antibody is labeled with peridinin chlorophyll protein complex (PerCP)/Cy5.5.
14. The targeted nanoparticle conjugate of claim 1, wherein the targeted nanoparticle conjugate comprises a nanoparticle core decorated with free NHS groups, optionally wherein said NHS groups are conjugated on the surface of the anti-BCMA antibody via a bissulfosuccinimidyl suberate crosslinker.
15. The targeted nanoparticle conjugate of claim 1, further comprising a drug moiety, optionally wherein the drug moiety is an anti-CS1 agent or an anti-BCMA agent.
16. A formulation comprising the targeted nanoparticle conjugate of claim 1.
17. The formulation of claim 14, wherein the targeted nanoparticle conjugate is present at a dose equivalent of 0.1-1 mg/g of SiGdNP, optionally at about 0.25 mg/g of SiGdNP.
18. A pharmaceutical composition comprising the targeted nanoparticle conjugate of claim 1 and a pharmaceutically acceptable carrier.
19. A method for detecting the presence and/or localization of multiple myeloma (MM) and/or minimal residual disease (MRD) in a subject, the method comprising: administering the targeted nanoparticle conjugate of claim 1 to the subject; and detecting the presence and/or localization of the targeted nanoparticle conjugate in the subject, thereby detecting the presence and/or localization of MM and/or MRD in the subject.
20. The method of claim 19, wherein the step of administering is performed by injection, optionally by intravenous and/or intraperitoneal injection.
21. The method of claim 19, wherein the step of detecting comprises utilization of a magnetic resonance imaging (MRI) scan.
22. The method of claim 21, wherein the targeted nanoparticle conjugate acts as an imaging biomarker for the detection of MM cells and/or MRD in the subject.
23. The method of claim 22, wherein the targeted nanoparticle conjugate provides contrast that is improved by at least 5-fold, optionally by at least 10-fold, optionally about 12-fold or more as compared to an appropriate non-targeted NP control, optionally wherein a signal-to-noise ratio (SNR) and normalized SNR are calculated according to equations (1) and (2): (1) SNR=intensity/noise; (2) Normalized SNR(i)=SNR(i)/SNR.sub.baseline.
24. The method of claim 22, wherein the targeted nanoparticle conjugate possesses a MRI detection threshold for MRD of 100,000 or less plasma cells per subject, optionally 50,000 or less plasma cells per subject, optionally 30,000 or less plasma cells per subject, optionally 20,000 or less plasma cells per subject, optionally 10,000 or less plasma cells per subject, optionally 8,000 or less plasma cells per subject, optionally 6,000 or less plasma cells per subject, optionally 5,000 or less plasma cells per subject, optionally 4,000 or less plasma cells per subject, optionally 3,000 or less plasma cells per subject, optionally about 2,200 plasma cells per subject.
25. The method of claim 19, wherein the step of detecting is performed within approximately 1 hour of the step of administering the targeted nanoparticle conjugate, optionally within approximately 30 minutes of the step of administering the targeted nanoparticle conjugate.
26. The method of claim 19, wherein the targeted nanoparticle conjugate binds approximately 70% of MM cells at 30 minutes after the step of administering the targeted nanoparticle conjugate.
27. The method of claim 19, wherein the targeted nanoparticle conjugate is detected in spine, femur, other bone and/or in the spleen.
28. The method of claim 19, wherein tumor uptake of the targeted nanoparticle conjugate is enhanced relative to an appropriate control non-targeted nanoparticle.
29. The method of claim 19, wherein detecting the presence and/or localization of MM and/or MRD in the subject is used to assess a MM therapy, optionally a therapy comprising administration of an anti-CS1 agent or an anti-BCMA agent, optionally wherein the targeted nanoparticle conjugate is administered in combination with the MM therapy.
30. The method of claim 19, wherein the subject is human.
31. The method of claim 19, wherein the subject is murine.
32. The method of claim 31, wherein the subject is a MRD model mouse, optionally wherein the MRD model mouse is induced by administration of Bortezomib and Melphalan.
33. The method of claim 31, wherein xenograft-derived MM is detected in SCID/beige mice.
34. The method of claim 19, wherein detecting the presence and/or localization of MM and/or MRD in the subject comprises detecting disease progression from MGUS to SMM and/or detecting early tumor and/or extramedullary MM disease.
35. The method of claim 19, wherein the detecting step comprises detecting gadolinium, optionally detecting Gd.sup.155 concentrations.
36. A targeted nanoparticle conjugate comprising: a nanoparticle comprising multiple sites of conjugation; and an anti-BCMA antibody.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0072] The present disclosure is directed, at least in part, to nanoparticle-antibody conjugates targeted to cell surface receptorsconjugates which, because of their targeted nature, possess enhanced ability as imaging agents for detection and localization of multiple myeloma and/or the presence of MRD in a cell line and/or subject. In certain embodiments, the nanoparticle moieties of the antibody-nanoparticle conjugates of the instant disclosure are gadolinium-based and optionally are of such small size (e.g., NPs of less than 5 nm) that such conjugate compositions, even when conjugated to targeting moieties (e.g., anti-BCMA monoclonal antibodies) via linkers (e.g., NHS linker moieties), are relatively rapidly cleared from the circulation of a subject via renal excretion, with no toxic impact. Thus, the nanoparticle-antibody conjugates described herein provide improved imaging contrast and allow for enhanced monitoring of MRD and/or therapeutic prediction of MM.
[0073] Many new therapeutic modalities for MM are currently in clinical trials, and recent rapid development of such compositions has been accompanied by an increased need for specific imaging biomarkers to monitor MRD, with the goal of improving the evaluation and efficacy of such treatments. Most MM patients are diagnosed as MRD positive, due to a rapid variation of M-spike/free light-chain (FLC) ratio level and/or end organ damage, as indicated by, e.g., elevated calcium rate, renal failure, anemia, and/or bone lesion (CRAB criteria; Kumar et al. Lancet Oncol 17: e328-346). Where the M-spike/FLC level ratio increases, patients are imaged by whole body-X ray to detect bone lesions. However, this imaging technique lacks sensitivity. Because new MM therapies have become available, early confirmation of MM can significantly improve patient outcomes.
[0074] Minimal residual disease (MRD) is directly linked to both shorter durations of treatment response as well as to inferior long-term survival outcomes in patients with multiple myeloma (MM; Kumar et al. Lancet Oncol 17: e328-346; Nishihori et al. Curr Hematol Malig Rep 11: 118-126; Anderson et al. Clin Cancer Res, doi:10.1158/1078-0432.CCR-16-2895). Current diagnostic methods that utilize serologic studies and/or bone marrow examinations do not take into account the spatial heterogeneity of the tumor microenvironment; they require serial invasive samplings to diagnose residual plasma cells. Available diagnostic imaging modalities are not sensitive nor specific for the detection of malignant plasma-cells (Lapa et al. Theranostics 6: 254-261) and often rely on ionizing radiation that precludes frequent testing (Fazel et al. N Engl J Med 361: 849-857).
[0075] It is predicted that establishment of imaging methods for the detection of MRD will have a transformative impact on the care of patients with MM, enabling noninvasive and repetitive testing to find residual plasma cells at earlier time points and when present even in focal distribution patterns that would otherwise preclude detection.
[0076] Magnetic resonance imaging (MRI) is known to provide a more reliable method for assessing disease burden, prognosis, and to monitor response to therapy, as compared to computed tomography (CT) scans and positron emission tomography (PET) (Spinnato P. et al, Eur J Radiol. 2012 81(12):4013-8). Techniques for magnetic resonance imaging (MRI) with conventional FDA-approved agents are being developed and have been shown to be more reliable at assessing disease burden (Pawlyn et al. Leukemia 30: 1446-1448), for enabling accurate disease prognostication (Dimopoulos et al. J Clin Oncol 33: 657-664), as well as for following therapeutic responses in MM patients when compared to computed tomography (CT), single-photon emission computed tomography (SPECT), or positron emission tomography (PET; Spinnato et al. Eur J Radiol 81: 4013-4018). MRI has the advantage of distinguishing between benign and malignant osteolytic regions, in addition to detecting early marrow infiltration (Shortt et al. AJR Am J Roentgenol 192: 980-986); however, the current protocols used to perform MRIi.e., fat-water imaging, diffusion weighted imaging, contrast enhancementare time-consuming, expensive, and rely on passive accumulation of non-targeted constrast agents within the tumor microenvironment (Matsumura and Maeda. Cancer Res 46: 6387-6392), which has hitherto limited both their detection specificity and sensitivity. CT scans can only detect bone destruction but not myeloma activity, and PET imaging is reliant upon imaged cells exhibiting active metabolism yet PET imaging does not possess sufficient sensitivity to visualize residual MM cells displaying slow proliferative activity (Freedenberg M I et al. Phys Med 2014 30(1):104-10). While SPECT and fluorodeoxyglucose-based (.sup.18F-FDG-) PET are able to accurately identify plasma cell populations (Cavo et al. Lancet Oncol 18: e206-e217), they utilize ionizing radiation that prevents repetitive testing in short intervals. .sup.18F-FDG-PET also displays poor detection sensitivity for malignant plasma cells in the MRD state, which are more slowly proliferative.
[0077] Prior to the invention described herein, there was a pressing need for an imaging biomarker for MM based on MRI acquisition, which would present clear advantages over existing imaging techniques. In MM, and more specifically for MRD diagnostics, the current MRI contrast agents (gadolinium chelates) rely upon the passive targeting pathway (Zhou et al. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2013 5(1):1-18) (EPR-effect), which does not allow for production of a contrast signal that is sufficiently specific to be detected. In nanomedicine, use of antibodies has been proposed for targeting of cell-surface receptors. However, recent proof-of-concept studies performed upon such targeting agents have demonstrated suboptimal results. Indeed, NP circulation half-time time was observed to have dropped dramatically in such studies, resulting in a low in vivo binding affinity due to the large size of these complexes, and imaging was further prevented by the inability of the NP to escape the vasculature in order to target the cell surface receptors to which the antibody was targeted. As a result, no difference was observed between passive and actively targeted forms of NP, limiting their application as effective and specific imaging agents. As described in detail below, to develop an improved imaging biomarker and noting that a primary issue to address was the final size of the complex nanoparticle-full antibody, an ultrafine sub-5 nm NP having high MRI properties was selected, as were relatively small monoclonal antibodies. The instant disclosure focuses upon generating a novel MM-targeted contrast agent capable of using short MRI sequences to identify minute tumor cell populations with high spatial localization. A primary goal of the current disclosure was to generate gadolinium (Gd)-based nanoparticles (GdNPs) that could be specifically targeted to plasma cells to enhance early detection of MRD. The use of antibodies has long been proposed for targeting of nanoparticles to tumors by binding receptors that are overexpressed on their cell surfaces (Ulbrich et al. Chem Rev 116: 5338-5431; Mulvey et al. Nat Nanotechnol 8: 763-771). As the sizes of these typical nanoparticle-antibody complexes (50-200 nm in diameter; Arruebo et al. J Nanomater, doi:10.1155/2009/439389 (2009)) are much larger than those of full monoclonal antibodies or of their molecular-conjugates (10-15 nm in length and 3-5 nm in diameter; Reth, M. Nat Immunol 14: 765-767), their pharmacology has been largely dictated by the nanoparticle rather than by the antibody. Moreover, most preclinical studies performed with such agents have been conducted in subcutaneous xenograft models (Smith et al. Nat Nanotechnol, doi:10.1038/nnano.2017.57 (2017); Qian et al. Nat Biotechnol 26: 83-90) that do not recapitulate the vascular patterns found in the natural tumor microenvironment (Mack and Marshall. Nat Biotechnol 28: 214-229). Many of these reported constructs have, thus, exhibited no differences with respect to their untargeted counterparts in achieving tumor localization (Kunjachan et al. Nano Lett 14: 972-981), which have stymied their further translational development.
[0078] The targeting efficiencies of monoclonal antibodies directed to two specific antigensthe B-cell maturation antigen (BCMA) and the signaling lymphocytic activation molecule-F7 (SLAMF7) receptorwere initially compared. Both targets are well-established antigens almost exclusively present on the cell surface of non-malignant B-cells (Lonial et al. N Engl J Med 373:621-631; Novak et al. Blood 103:689-694). BCMA, as distinguished from SLAMF7, is a highly specific plasma cell antigen having an important role in the maturation and differentiation of the B-cell into a plasma cell (Carpenter R O et al. Clin Cancer Res 201319(18):2048-60). The high prevalence and expression level of BCMA increases with the advancement of the MM progression (
[0079] Herein, magnetic resonance imaging (MRI) of ultra-small gadolinium-based nanoparticles that were conjugated to monoclonal antibodies has been utilized to enable rapid detection of clonal plasma cells in the bone marrow microenvironment. It is believed that the instant disclosure represents the first example of utilizing a non-invasive and safe imaging agent to improve early detection of MRD after therapeutic administration.
[0080] Certain targeted nanoparticle conjugates of the disclosure are capable of enhancing the sensitivity of detecting MM cells in a subject (e.g., in a mammalian subject). Targeted nanoparticles of the disclosure can, for example, improve sensitivity by at least 1.5-fold relative to untargeted NPs. Optionally, sensitivity is improved by at least two-fold relative to untargeted NPs. Optionally, sensitivity is improved by at least three-fold relative to untargeted NPs. Optionally, sensitivity is improved by at least five-fold relative to untargeted NPs. Optionally, sensitivity is improved by at least ten-fold relative to untargeted NPs.
[0081] Certain targeted nanoparticle conjugates of the disclosure can additionally and/or alternatively enhance the specificity of detecting MM cells in a subject (e.g., in a mammalian subject). Targeted nanoparticles of the disclosure can, for example, improve specificity by at least 1.5-fold relative to untargeted NPs. Optionally, specificity is improved by at least two-fold relative to untargeted NPs. Optionally, specificity is improved by at least three-fold relative to untargeted NPs. Optionally, specificity is improved by at least five-fold relative to untargeted NPs. Optionally, specificity is improved by at least ten-fold relative to untargeted NPs.
[0082] In certain embodiments, a targeted nanoparticle conjugate of the disclosure can possess a lower MRI detection threshold for MRD than a non-targeted nanoparticle. For example, the MRI detection threshold for MRD in a subject for certain targeted nanoparticles of the disclosure can be 100,000 or less plasma cells per subject, optionally 50,000 or less plasma cells per subject, optionally 30,000 or less plasma cells per subject, optionally 20,000 or less plasma cells per subject, optionally 10,000 or less plasma cells per subject, optionally 8,000 or less plasma cells per subject, optionally 6,000 or less plasma cells per subject, optionally 5,000 or less plasma cells per subject, optionally 4,000 or less plasma cells per subject, optionally 3,000 or less plasma cells per subject, optionally about 2,200 plasma cells per subjecte.g., optionally 2,200450 plasma cells per subject (optionally, where the subject is a mouse).
Anti-BCMA Monoclonal Antibodies
[0083] B cell maturation antigen (BCMA) is member 17 of the tumor necrosis factor receptor superfamily (TNFRSF). Its native ligands are the B cell activating factor (BAFF; also called BLS or TALL-1, TNFSF13B) and a proliferation-inducing ligand (APRIL, TNFSF13, CD256) (Mackay et al. (2003) Annu Rev Immunol 21:231-264) which are ultimately involved (through interaction with further ligands) in regulating various aspects of humoral immunity, B cell development, and homeostasis. The affinity for BAFF lies in the low micromolar range whereas APRIL binds nearly 100 fold tighter to BCMA (Bossen et al. (2006) Semin Immunol 18:263-275). Expression of BCMA is restricted to the B cell lineage where it is predominantly expressed on plasma blasts and plasma cells but is absent from naive B cells, germinal center B cells and memory B cells (Darce et al. (2007) J Immunol 179:7276-7286; Benson et al. (2008) J Immunol 180:3655-3659; Good et al. (2009) J Immunol 182:890-901).
[0084] BCMA expression is important for the survival of long-lived, sessile plasma cells in the bone marrow (O'Connor et al. (2004) J Exp Med 199:91-98). Consequently, BCMA-deficient mice show reduced plasma cell numbers in the bone marrow whereas the level of plasma cells in the spleen in unaffected (Peperzak et al. (2013) Nat Immunol [Epub 2013 Feb. 3, 10.1038/ni.2527]). The differentiation of mature B cells into plasma cells is normal in BCMA knockout mice (Schiemann et al. (2001) Science 293:21 1 1-21 14; Xu et al. (2001) Mol Cell Biol 21:4067-4074). The binding of BAFF or APRIL to BCMA triggers NF-B activation (Hatzoglou et al. (2000) J Immunol 165:1322-1330), which induces upregulation of anti-apoptotic Bcl-2 members such as Bcl-xL or Bcl-2 and Mcl-1 (Peperzak et al. (2013) Nat Immunol [Epub 2013 Feb. 3, 10.1038/ni.2527]).
[0085] BCMA is also highly expressed on malignant plasma cells, for example in multiple myeloma, (MM), which is a B cell non-Hodgkin lymphoma of the bone marrow, and plasma cell leukemia (PCL), which is more aggressive than MM and constitutes around 4% of all cases of plasma cell disorders. In addition to MM and PCL, BCMA has also been detected on Hodgkin and Reed-Sternberg cells in patients suffering from Hodgkin's lymphoma (Chiu et al. (2007) Blood 109:729-739). Similar to its function on plasma cells, ligand binding to BCMA has been shown to modulate the growth and survival of multiple myeloma cells expressing BCMA (Novak et al. (2004) Blood 103:689-694). Signaling of BAFF and APRIL via BCMA are considered as pro-survival factors for malignant plasma cells; hence, the depletion of BCMA-positive tumor cells and/or the disruption of ligand-receptor interaction should improve the therapeutic outcome for multiple myeloma and autoantibody-dependent autoimmune diseases. There are presently various approaches available for the treatment of multiple myeloma (Raab et al. (2009) Lancet 374:324-339). Chemotherapy leads in most subjects only to partial control of multiple myeloma; only rarely does chemotherapy lead to complete remission. Combination approaches are therefore often applied, commonly involving an additional administration of corticosteroids, such as dexamethasone or prednisone. Corticosteroids are, however, plagued by side effects, such as reduced bone density. Stem cell transplantation has also been proposed, using one's own stem cells (autologous) or using cells from a close relative or matched unrelated donor (allogeneic). In multiple myeloma, most transplants performed are of the autologous kind. Such transplants, although not curative, have been shown to prolong life in selected patients (Suzuki (2013) Jpn J Clin Oncol 43:1 16-124). Alternatively, thalidomide and derivatives thereof have recently been applied in treatment but are also associated with sub-optimal success rates and high costs. More recently, the proteasome inhibitor bortezomib (PS-341) has been approved for the treatment of relapsed and refractory MM and was used in numerous clinical trials alone or in combination with established drugs resulting in an encouraging clinical outcome (Richardson et al. (2003) New Engl J Med 348:2609-2617; Kapoor et al. (2012) Semin Hematol 49:228-242). Therapeutic approaches are often combined. The costs for such combined treatments are correspondingly high and success rates still leave significant room for improvement. The combination of treatment options is also not ideal due to an accumulation of side effects if multiple medicaments are used simultaneously.
[0086] The ability to specifically target plasma cells is also of great benefit for the treatment of autoimmune diseases. Conventional therapy for autoimmune conditions such as systemic lupus erythematosus (SLE) and rheumatic arthritis (RA), in which autoreactive antibodies are crucial to disease pathology, depend on the severity of the symptoms and the circumstances of the patient (Scott et al. (2010) Lancet 376:1094-1 108, D'Cruz et al. (2007) Lancet 369, 587-596). In general, mild forms of disease are first treated with nonsteroidal antiinflammatory drugs (NSAID) or disease-modifying anti-rheumatic drugs (DMARD). More severe forms of SLE, involving organ dysfunction due to active disease, usually are treated with steroids in conjunction with strong immunosuppressive agents such as cyclophosphamide, a cytotoxic agent that targets cycling cells. Only recently Belimumab, an antibody targeting the cytokine BAFF, which is found at elevated levels in serum of patients with autoimmune diseases, received approval by the Food and Drug Administration (FDA) for its use in SLE. However, only newly formed B cells rely on BAFF for survival in humans, whereas memory B cells and plasma cells are less susceptible to selective BAFF inhibition (Jacobi et al. (2010) Arthritis Rheum 62:201-210). For rheumatoid arthritis, TNF inhibitors were the first licensed biological agents, followed by abatacept, rituximab, and tocilizumab and others: they suppress key inflammatory pathways involved in joint inflammation and destruction, which, however, comes at the price of an elevated infection risk due to relative immunosuppression (Chan et al. (2010) Nat Rev Immunol 10:301-316, Keyser (201 1) Curr Rheumatol Rev 7:77-87). Despite the approval of these biologicals, patients suffering from RA and SLE often show a persistence of autoimmune markers, which is most likely related to the presence of long-lived, sessile plasma cells in bone marrow that resist e.g. CD20-mediated ablation by rituximab and high dosage glucocorticoid and cyclophosphamid therapy. Current strategies in SLE include a reset of the immune system by immunoablation and autologous stem cell transplantation, though the risk for transplant-related mortality remains a serious concern (Farge et al. (2010) Haematologica 95:284-292). The use of proteasome inhibitors such as Bortezomib might be an alternative strategy for plasma cell depletion: owing to the high rate of protein synthesis and the limited proteolytic capacity, plasma cells are hypersensitive to proteasome inhibitors. Bortezomib has recently been approved for the treatment of relapsed multiple myeloma and a recent study in mice with lupus-like disease showed that bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis (Neubert et al. (2008) Nat Med 14:748-755). However, proteasome inhibitors do not specifically act on plasma cells and the incidence of adverse effects such as peripheral neuropathy is high (Arastu-Kapur et al. (2011) Clin Cancer Res 17:2734-2743).
[0087] Therapeutic antibodies can act through several mechanisms upon binding to their target. The binding itself can trigger signal transduction, which can lead to programmed cell death (Chavez-Galan et al. (2009) Cell Mol Immunol 6:15-25). It can also block the interaction of a receptor with its ligand by either binding to the receptor or the ligand. This interruption can cause apoptosis if signals important for survival are affected (Chiu et al. (2007) Blood 109:729-739). With regard to cell-depletion there are two major effector mechanisms known. The first is the complement-dependent cytotoxicity (CDC) towards the target cell. There are three different pathways known. However, in the case of antibodies the important pathway for CDC is the classical pathway which is initiated through the binding of C1 q to the constant region of IgG or IgM (Wang and Weiner (2008) Expert Opin Biol Ther 8:759-768).
[0088] The second mechanism is called antibody-dependent cellular cytotoxicity (ADCC). This effector function is characterized by the recruitment of immune cells which express Fc-receptors for the respective isotype of the antibody. ADCC is largely mediated by activating Fc-gamma receptors (FcyR) which are able to bind to IgG molecules either alone or as immune complexes. Mice exhibit three (FcyRI, FcyRIII and FcyRIV) and humans five (FcyRI, FcyRIIA, FcyRIIC, FcyRIIIA and FcyRIIIB) activating Fcy-receptors. These receptors are expressed on innate immune cells like granulocytes, monocytes, macrophages, dendritic cells and natural killer cells and therefore link the innate with the adaptive immune system.
[0089] Depending on the cell type, there are several modes of action of FcgR-bearing cells upon recognition of an antibody-marked target cell. Granulocytes generally release vasoactive and cytotoxic substances or chemoattractants but are also capable of phagocytosis. Monocytes and macrophages respond with phagocytosis, oxidative burst, cytotoxicity, or the release of pro-inflammatory cytokines, whereas Natural killer cells release granzymes and perforin and can also trigger cell death through the interaction with FAS on the target cell and their Fas ligand (Nimmerjahn and Ravetch (2008) Nat Rev Immunol 8:34-47; Wang and Weiner (2008) Expert Opin Biol Ther 8:759-768; Chavez-Galan et al. (2009) Cell Mol Immunol 6:15-25).
[0090] Antibodies which bind CD269 (BCMA) and their use in the treatment of various B-cell related medical disorders are described in the art. Ryan et al (Molecular Cancer Therapeutics, 2007 6 (11), 3009) describe an anti-BCMA antibody obtained via vaccination in rats using a peptide of amino acids 5 to 54 of the BCMA protein. The antibody described therein binds BCMA, blocks APRIL-dependent NF-KB activation and induces ADCC. No details are provided on the specific epitope of the antibody. WO 2012/163805 describes BCMA binding proteins, such as chimeric and humanized antibodies, their use to block BAFF and/or APRIL interaction with BCMA, and their potential use in treating plasma cell malignancies such as multiple myeloma. The antibody disclosed therein was obtained via vaccination in mouse using a recombinant peptide of amino acids 4 to 53 of the BCMA protein. WO 2010/104949 also describes various antibodies that bind preferably the extracellular domain of BCMA and their use in treating B cell mediated medical conditions and disorders. No details are provided on the specific epitope of the antibodies.
[0091] WO 2002/066516 describes bivalent antibodies that bind both BCMA and TACI and their potential use in the treatment of autoimmune diseases and B cell cancers. An undefined extracellular domain of BCMA is used to generate the anti-BCMA portion of the antibodies described therein. WO 2012/066058 discloses bivalent antibodies that bind both BCMA and CD3 and their potential use in the treatment of B cell related medical disorders. Details regarding the binding properties and specific epitopes of the antibodies are not provided in either publication.
[0092] WO 2012/143498 describes methods for the stratification of multiple myeloma patients involving the use of anti-BCMA antibodies. Preferred antibodies are those known as Vicky-1 (lgG1 subtype from GeneTex) and Mab 193 (lgG2a subtype from R&D Systems). Details regarding the binding properties and specific epitopes of the antibodies are not provided.
[0093] WO 2014/068079 describes an anti-BCMA antibody evaluated as suitable for use in the treatment of plasma cell diseases such as multiple myeloma (MM) and autoimmune diseases. WO 2014/068079 provides an isolated antibody or antibody fragment that binds CD269 (BCMA), in particular an epitope of the extracellular domain of CD269 (BCMA). An isolated antibody or antibody fragment that binds CD269 (BCMA) was therefore provided, wherein the antibody binds an epitope comprising one or more amino acids of residues 13 to 32 of CD269 (BCMA).
[0094] To raise an anti-BCMA antibody, antigen comprising the extracellular domain of CD269 was used in vaccination in order to generate the binding specificity of the anti-BCMA antibody. Use of the entire CD269 protein, or fragments thereof comprising either a membrane-bound or intracellular domain, as an antigen during antibody generation could produce antibodies that bind concealed or intracellular domains of CD269, thereby rendering such agents unsuitable or disadvantageous for therapeutic application. The antibodies described in WO 2014/068079 were therefore defined by their binding to the extracellular portion of CD269. The specific epitope within the extracellular domain also represented a preferred novel and unexpected characterising feature of the WO 2014/068079 publication.
[0095] Fab fragments prepared from one embodiment of the WO 2014/068079 were crystallized in complex with the purified BCMA extracellular domain and the complex structure solved. The structural analysis revealed detailed information of the epitope of the anti-BCMA antibody of the WO 2014/068079 publication and its biological relevance. The binding of an epitope comprising one or more amino acids of residues 13 to 32 of CD269 (BCMA) of the extracellular domain by the antibody of the WO 2014/068079 publication was identified as an advantageous property, as this region showed a significant overlap with the binding sites of BAFF and APRIL, the two natural ligands of CD269. No anti-CD269 antibody described in the art previously had shown such comprehensive overlap with the BAFF and APRIL binding sites.
[0096] Certain anti-BCMA antibodies or antibody fragments described herein can bind an epitope comprising one or more of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27 or 32 of CD269 (BCMA). Optionally, an isolated anti-BCMA antibody or antibody fragment can be characterized in that the antibody binds an epitope consisting of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27 and 32 of CD269 (BCMA). These residues represent the amino acids that interact directly with the anti-BCMA antibody, as identified by the crystal structure data shown in WO 2014/068079. The numbering of these residues was carried out with respect to the N-terminal sequence of CD269.
[0097] In certain embodiments, the anti-BCMA antibody binds CD269 (BCMA) and disrupts the BAFF-CD269 and/or APRIL-CD269 interaction. BAFF/APRIL-CD269 interactions are thought to trigger anti-apoptotic and growth signals in the cell, respectively (Mackay, Schneider et al. (2003) Annu Rev Immunol 21:231-264; Bossen and Schneider (2006) Semin Immunol 18:263-275).
[0098] Exemplary humanization of anti-BCMA antibody J22.9-xi: J22.9-xi antibody was humanized based on sequence alignment and data obtained from a crystal structure. The sequences of the variable regions were aligned to their respective human homologs using IgBLAST (NCBI). Each proposed mutation was evaluated by visual inspection of the structure before alteration. Binding of the mutants to BCMA can be tested using flow cytometry. The affinity was measured using surface plasmon resonance (ProteOn XPR36; Bio-Rad). Preliminary assessment of the binding properties of the humanized sequences showed promising results with respect to their specificity and affinity to the same epitope as described for J22.9-xi binding.
[0099] To obtain a BCMA-binding antibody, standard hybridoma technique can be used. E.g., for production of initial anti-BCMA antibody, four (4) BL/6 wild type mice were immunized 6 times with incomplete Freund's adjuvant and 30 g of the extracellular domain of human BCMA C-terminally fused to Glutathione S-transferase (GST). After cell fusion followed by a screening period, the J22.9 hybridoma was shown to secrete an anti-BCMA antibody.
Linkers
[0100] Any number of art-recognized linker moieties can be used to join anti-BCMA antibodies with nanoparticles possessing enhanced imaging characteristics, thereby forming anti-BCMA antibody-nanoparticle compositions within the scope of the conjugates described herein. In exemplary embodiments, the reactive amine groups on the surface of compositions present heterobifunctional linker molecules, (e.g., anchoring points) via an N-hydroxysuccinimide ester (e.g., NHS) reaction with amine groups. In some embodiments, the heterobifunctional anchoring linker (e.g., a bifunctional PEG macromer) may include the amine-reactive NHS ester on one end, a short (e.g., approximately 2 kilo daltons (kDa)) PEG chain, and an acrylate group on the other end. In certain embodiments, the heterobifunctional linker (e.g., a bifunctional PEG macromer) may include the amine-reactive NHS ester on one end, the short PEG chain, and a thiol group on the other end. The short PEG linker also provides additional degrees of freedom to the acrylate group or the thiol group at the end, making it easier to link to the hydrogel coating in the second reaction. In specific embodiments of the instant disclosure, a bissulfosuccinimidylsuberate (BS3) linker is used for conjugation of NP to anti-BCMA antibody. Alternative linkerse.g., ones possessing more directed functionality than certain NHS-NHS homobifunctional linkers described hereinare also expressly contemplated.
[0101] It is contemplated that conjugation of NP to antibody can be performed in a number of ways, including use of an external linker to conjugate to the NP to create a link to the antibody (where two different functionalities can be selected and mixed together in the same linker, e.g., NHS linker (reactive towards amines) or maleimide (reactive towards thiols)). A number of other linkers can also be used, including alkyne-azide linkers (reacted via copper-catalyzed click chemistry), cyclooctyne-azide (copper-less click chemistry), TCO-tetrazine, etc. Since NPs of the disclosure possess amines, one end of the linker will tend to be NHS, but the composition of the other end of the linker can vary depending upon the antibody handle. Thiol-decorated/functionalized antibodies create scenarios where NHS-maleimide linkers and/or NHS-maleimidocaproyi linkers can be employed with good effect. Additionally and/or alternatively, extra arms can be created upon the polymer itself, thereby creating free amines with a thiol group, which can directly conjugate the NP to the antibody without requiring a linker to bridge the two moieties (NP and antibody).
Nanoparticles
[0102] Nanoparticles of uniform size and shape (e.g., 3-5 nm diameter) have been proven an effective tool for bioimaging. Nanoparticles have a high area-to-volume ratio; they are very reactive, good catalysts and adhere to biological molecules. One nanoparticle material is silicon as it is inert, non-toxic, abundant and economic. The silicon surface can be functionalized. Silicon nanoparticles show efficient photoluminescence in the visible part of the electromagnetic spectrum and are bioinert and chemically stable. One material which has similar biocompatibility is porous silicon. Particles smaller than 100 nm show an enhanced permeability and retaining effect (EPR effect) in tumours, an important nonspecific targeting effect. Silicon nanoparticles, also known as silicon quantum dots, can be used in imaging technologies but also for LED, photovoltaics, lithium ion batteries, transistors, polymers or two-photon absorption.
[0103] A number of nanoparticles can be used in the conjugate compositions of the current disclosures, including the exemplified silica-based gadolinium NPs as described herein and, e.g., polymer NPs such as those disclosed in U.S. Pat. No. 9,381,253 (polymer brush nanoparticle for organic MRI contrast) and an exemplary polymer nanoparticle for in vivo CRISPR modification (as described in WO 2017/004509).
Magnetic Resonance Imaging (MRI)
[0104] MRI is one of the most used techniques for medical diagnostics, combining the advantages of being non-invasive, quick and without danger for the patient. It is based on observation of the relaxation of the protons of water, which is directly dependent on magnetic fields (the important magnetic field BO and radio-frequency fields), pulse sequence, the environment of the water in the organism, etc. Interpretation of the MRI images then gives access to identification of most tissues. The contrast can be increased by two types of agents: positive T1 and negative T2 contrast agents. Positive contrast agents, i.e. T1, which permit lightening of the image as contact of water with the contrast agent makes it possible to reduce the longitudinal relaxation time: T1. Gd(III)DTPA or Gd(III)DOTA are examples of T1 contrast agents used in clinical practice and contemplated/employed within the instant disclosure.
[0105] Certain nanoparticles known in the field and as employed herein are useful in particular as contrast agents in imaging (e.g., MRI) and/or in other diagnostic techniques and/or as therapeutic agents, which give better performance than known nanoparticles of the same type and which combine both a small size (for example less than 20 nm) and a high loading with metals (e.g., rare earths), in particular so as to have, in imaging (e.g., MRI), strong intensification and a correct response (increased relaxivity) at high frequencies.
[0106] Exemplary nanoparticles according to the disclosure, possessing a diameter dl between 1 and 20 nm, can each comprise a polyorganosiloxane (POS) matrix including gadolinium cations optionally associated with doping cations; a chelating graft C1 DTPABA (diethylenetriaminepentaacetic acid bisanhydride) bound to the POS matrix by an SiC covalent bond, and present in sufficient quantity to be able to complex all the gadolinium cations; and optionally another functionalizing graft Gf* bound to the POS matrix by an SiC covalent bond (where Gf* can be derived from a hydrophilic compound (PEG); from a compound having an active ingredient PA1; from a targeting compound; and/or from a luminescent compound (fluorescein)).
Administration
[0107] A nanoparticle-anti-BCMA antibody conjugate of the instant disclosure may be administered via a number of routes of administration, including but not limited to: subcutaneous, intravenous, intrathecal, intramuscular, intranasal, oral, transepidermal, parenteral, by inhalation, or intracerebroventricular.
[0108] The term injection or injectable as used herein refers to a bolus injection (administration of a discrete amount of an agent for raising its concentration in a bodily fluid), slow bolus injection over several minutes, or prolonged infusion, or several consecutive injections/infusions that are given at spaced apart intervals.
[0109] In some embodiments of the present disclosure, a formulation as herein defined is administered to the subject by bolus administration.
[0110] The nanoparticle conjugate is administered to the subject in an amount sufficient to achieve concentrations at the desired site of imaging (and/or treatment, e.g., where a drug or other agent is administered) determined by a skilled clinician to be effective, for example in an amount sufficient to achieve concentrations in the vicinity of from about 110.sup.8 to about 110.sup.1 moles/liter. In some embodiments of the invention, the nanoparticle conjugate is administered at least once a year. In other embodiments of the invention, the nanoparticle conjugate is administered at least once a day. In other embodiments of the invention, the nanoparticle conjugate is administered at least once a week. In some embodiments of the invention, the nanoparticle conjugate is administered at least once a month.
[0111] Exemplary doses for administration of a nanoparticle conjugate of the disclosure to a subject include, but are not limited to, the following: 1-20 mg/kg/day, 2-15 mg/kg/day, 5-12 mg/kg/day, 10 mg/kg/day, 1-500 mg/kg/day, 2-250 mg/kg/day, 5-150 mg/kg/day, 20-125 mg/kg/day, 50-120 mg/kg/day, 100 mg/kg/day, at least 10 ug/kg/day, at least 100 ug/kg/day, at least 250 ug/kg/day, at least 500 ug/kg/day, at least 1 mg/kg/day, at least 2 mg/kg/day, at least 5 mg/kg/day, at least 10 mg/kg/day, at least 20 mg/kg/day, at least 50 mg/kg/day, at least 75 mg/kg/day, at least 100 mg/kg/day, at least 200 mg/kg/day, at least 500 mg/kg/day, at least 1 g/kg/day, and an imaging and/or therapeutically effective dose that is less than 500 mg/kg/day, less than 200 mg/kg/day, less than 100 mg/kg/day, less than 50 mg/kg/day, less than 20 mg/kg/day, less than 10 mg/kg/day, less than 5 mg/kg/day, less than 2 mg/kg/day, less than 1 mg/kg/day, less than 500 ug/kg/day, and less than 500 ug/kg/day.
[0112] In some embodiments of the invention, a therapeutic agent distinct from the nanoparticle conjugate is administered prior to, in combination with, at the same time, or after administration of the imaging and/or therapeutically effective amount of a nanoparticle conjugate of the disclosure. In some embodiments, the second therapeutic agent is selected from the group consisting of a chemotherapeutic, an antioxidant, an antiinflammatory agent, an antimicrobial, a steroid, etc.
[0113] The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988. Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rero), (4th Ed., Univ. of Oregon Press, Eugene, 2000).
[0114] 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, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
[0115] The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.
Example 1: Materials and Methods
Cell Lines
[0116] The human MM cell line MM.1S was purchased from ATCC (Manassas, Va., USA). The MM.1S GFP.sup.+ Luc.sup.+ cell line was generated by retroviral transduction, using the pGC-GFP/Luc vector. Cells were authenticated by short tandem repeat DNA profiling. MM.1S, OPM2 and KMS11 cells were cultured in RPMI media (e.g., RPMI-1640 media; Sigma, USA) supplemented with 10% fetal bovine serum (Sigma, USA), 1% penicillin-streptomycin (Invitrogen, USA) and 1% glutamine (Invitrogen, USA). Optimal conditions of 37 C. and 5% CO.sub.2 were maintained in a humidified incubator.
Silica-Based Gadolinium Nanoparticle Synthesis. Including Synthesis of Antibody-Conjugated Gadolinium-Based Nanoparticles (GdNPs)
[0117] Ultra-small, silica-comprised GdNPs were provided by NH Theraguix, Inc. (Villeurbanne, France) and were synthesized following previously reported procedures (Detappe et al. J Control Release 238: 103-113; Detappe et al. Sci Rep 6: 34040). Such nanoparticles are also described, e.g., in US 2013/0195766
[0118] The NP constructs were conjugated with mouse-anti-human SLAMF7 and BCMA monoclonal antibodies (Biolegend Inc., San Diego, Calif.), using a previously reported homobifunctional linker chemistry (Schmidt and Robinson. Nat Protoc 9: 2224-2236). Briefly, GdNPs were diluted in UltraPure water to a final concentration of 50 nM. A 1:10 molar ratio of bissulfosuccinimidylsuberate (BS3) linker was mixed with GdNPs for 30 min and at room temperature to promote the generation of linker-bound nanoparticles. These surface-modified GdNPs were then combined with the monoclonal antibodies at a 1:100 molar ratio; and, the suspensions were stirred for 1 h at room temperature. The nanoparticle-antibody complexes were purified by centrifugation filtration, using a filtration device equipped with a 50 kDa molecular weight cutoff membrane (Milipore) that was spun at 5,000 r.p.m.; centrifugation concentration was subsequently followed by resuspension of the nanoparticle-antibody complexes in 1M PBS. This process was conducted in triplicate to assure removal of all excess free antibodies into the filtrate and to concentrate the suspensions of pure NP-SLAMF7 and NP-BCMA. The final concentrations of the nanoparticle-antibody complexes were determined by ICP-MS, using an Agilent 7900 (Agilent Technologies, Inc., Santa Clara, Calif.).
In Vitro Assays Determining the Specificity of Nanoparticle-Antibody Complexes to Bind MM Cells
[0119] Flow cytometry analyses of MM cell lines treated with various nanoparticle-antibody complexes were performed. The cells were first mixed with suspensions of GdNP, NP-SLAMF7 or NP-BCMA (0.5 mM) for 30 min, washed with fresh media, and resuspended in solution (110.sup.6 cells/mL). The treated cells were then incubated with PerCP/Cy5.5-labeled anti-human BCMA antibodies at 37 C. and for one hour, which served as a competitive label to NP-BCMA (whose binding decreased fluorescent labeling with this reagent). Populations of Cy5.5-labeled cells were subsequently detected by flow cytometry. To cross-validate the results, ICP-MS was utilized to quantify the amounts of Gd bound per cell. To perform these later experiments, the treated MM cell lines were lysed with 0.3% Triton-X 100 solution prior to precise enumeration of the amounts of Gd in each sample, using ICP-MS. As a third method of validating the binding of nanoparticle-antibody complexes to MM cells, confocal microscopy was performed to visualize the colocalization of fluorescently-labeled antibodies with nanoparticle-antibody complexes, which had been labeled with a separate fluorophore, on the surfaces of MM cells. For these experiments, GdNPs were first conjugated with Cy5-NHS at 1:1000 molar ratio of fluorophore to nanoparticle, using EDC/NHS chemistry. The monoclonal antibodies were similarly labeled with AF488-NHS (1:1000 molar ratio of fluorophore to antibody) prior to nanoparticle conjugation as previously described (vide supra). MM cell lines were incubated with the dual-fluorophore conjugated nanoparticle-antibody complexes for 30 min, fixed in iced-cold methanol, and mounted on cover slips coated with Dapi Fluoromount-G (SouthernBiotech). Confocal microscopy (Olympus FV12000, Olympus) then proceeded to verify co-location of the two fluorophores in a punctate distribution on cellular surfaces.
Animal Model
[0120] GFP.sup.+/Luc.sup.+ MM1.S cells were administered to SCID/beige mice (510.sup.6 cells/mouse; n=5 mice per group) via IV dissemination, establishing an orthotopic murine xenograft model of MM. Tumor growth was monitored weekly by bioluminescence imaging (BLI), using an IVIS Spectrum-bioluminescent and fluorescent imaging system (Perkins Elmer). A murine model of MRD was further established by treating these mice with Bortezomib (0.5 mg/kg daily3 doses) followed by Melphalan (5.5 mg/kg1 dose).
Imaging Studies
[0121] MR image acquisition was conducted with a preclinical 7-Tesla BioSpec 70/20 MRI scanner (Bruker BioSpin, Billerica, Mass.). A dose equivalent of 0.25 mg/g of GdNPs conjugated to 80 g/mL of anti-BCMA antibodies were administered by IV injection into each mouse prior to imaging. A T1 GRE sequence employing a repetition time of 87 ms, echo time of 3.9 ms, and a flip angle of 60 was utilized for imaging. The acquisition matrix size and reconstructed matrix was 256256 pixels; the slice thickness was 5 mm. When comparing imaging parameters obtained with the different Gd-based contrast agents, MRI was performed at various time intervals after contrast administration; and, the results were compared to baseline images. For the early diagnostic and MRD quantification studies, MRI was performed 30 min post-IV injection. CT acquisitions were conducted on a preclinical Inveon CT scanner (Siemens) equipped with a 50 kVp source; the image resolution was 10.2 pixels/mm; and, a slice thickness of 0.1 mm was utilized. CT imaging was performed at various time intervals and before the injection of each MR contrast agent in order to compare changes in the SNR for different disease burdens detected via each imaging modality (vide infra).
Quantitative Comparisons of Imaging Modalities
[0122] Evaluation of the relative detection sensitivity for plasma cells at different time points and/or via different imaging modalities was performed by conducting a signal-noise-ratio (SNR) calculation on each acquired image. These SNR values were obtained after first performing a 3D segmentation of the spine and a femur of each animal, using Fiji freeware (https://fiji.sc/). Each image was normalized to the same intensity level and a region of interest (ROI), including the whole examined organ (i.e. spine or femur), was segmented; the signal intensity in the ROI was recorded and compared to the background level, which was measured on each scan. SNR and normalized SNR values were calculated according to equations (1) and (2): (1) SNR=intensity/noise; (2) Normalized SNR(i)=SNR(i)/SNR.sub.baseline. Absolute quantification of the uptake of various Gd-based contrast agents was determined, using ICP-MS (Agilent 7900) and by following previously described protocols. Briefly, animals were sacrificed at 30 min after contrast injection; their excised organs were dissolved in a 70% HCl solution; and, the Gd content of each organ was determined.
Lambda Light-Chain Quantification
[0123] Mice were bled once per week and immediately before imaging. Serum was separated from blood samples and frozen at 80 C. until the end of the study. Serum samples were diluted 1:10 v:v with PBS and a clinical-grade immunoassay, which is routinely performed in the pathology core of the Brigham and Women's Hospital (Boston, Mass.), was used to quantify the amounts of lambda light chains present in each sample.
Receiver Operator Characteristic Comparison
[0124] The ROC curve was used to represent the ability of the SNR to discriminate the presence or absence of tumor cells. The SNR at 5 weeks post-tumor cell implantation was enumerated for each of the various imaging modalities and served as a metric by which to compare their detection sensitivities. The class was defined for each time point using the following method: baseline measurements prior to tumor cell implantation served as the control (Ct.sub.0=0) and were compared against subsequent time points (C.sub.t=1) with the assumption that tumor cells were thereafter always present. To ensure that the prediction was not random, a two-sided Wilcoxon rank-sum test was employed. A p-value below 0.05 indicates that the SNR value for a given class was significantly different than that of another class.
Statistical Analyses
[0125] All in vitro statistical analyses were performed using GraphPad Prism software (V.7.1). The ability to discriminate the presence of MRD using each of the different medical imaging techniques was performed using R version 3.3.3.
Example 2: Development of Antibody-Conjugated, Ultra-Small, Gadolinum-Based Nanoparticles: NP-Anti-BCMA Conjugates Detected MM Presence and Progression in Cell Lines and in Mice
[0126] As shown in
[0127] The enhanced in vitro targeting efficiency of the NP-BCMA was subsequently verified by employing a human MM cell line (MM1.S). As shown in
Example 3: In Vivo Targeting of Plasma Cells Using Nanoparticle-Antibody Complexes
[0128] The targeting efficiency of the different NP compositions (NP-SLAMF7 and NP-BCMA, and their ability to detect plasma cells) was then evaluated in a murine model of MM that was established via IV dissemination of MM1.S cells followed by their bone marrow engraftment within immunocompromised SCID-beige mice. Tumor burden (tumor dissemination) was followed by bioluminescence imaging (BLI) at bi-weekly intervals starting on day 19 post-cell (MM1.S) xenotransplantation (injection;
[0129] The specificity of each of the administered contrast agents to target MM cells (confirmation of the presence of gadolinium atoms in the tumor region) was confirmed by animal sacrifice immediately after MRI. The femurs and vertebral tissues of each animal were harvested for histologic assessment after staining by H&E and by Prussian blue, which showed sheets of marrow-infiltrating plasma cells labeled with Gd (
[0130] The pharmacokinetic profiles of NP-SLAMF7 and NP-BCMA were similar (
Example 4: Comparisons of the Sensitivity and Specificity of the BCMA-Targeted Nanoparticle-Antibody Complex with Respect to Conventional Methods for Detecting Minimal Residual Disease Revealed that NP-Anti-BCMA Conjugates Detected MRD in Mammalian Subjects
[0131] Possessing a near-ideal targeting efficiency for solid tumors (i.e. as noted above, 4.20.4% ID/g in the spine, 2.010.1% ID/g in the femur) at 30 min post-injection (as quantified by ICP-MS, the NP-anti-BCMA conjugate was examined to determine if this agent, when combined with a MRI scan, could be employed as an imaging biomarker for the detection of MRD. Comparison of the sensitivity and specificity of NP-BCMA with respect to currently available methods for clinical MRD diagnosis was performed. Significant recent progress in the treatment of MM has been achieved, at least in part attributable to recent growth of in-depth understanding of the MM disease pathogenesis. In particular, therapeutic options for treatment of MM have expanded, e.g., with approval of Elotuzumab and Daratumumab having occurred in 2015. However, even while the survival of MM patients has doubled, it also has been demonstrated that an early treatment of MM patients may increase survival even more. In contrast, it was also demonstrated that at the end of a MM treatment, if a MRD negative status was achieved, the patient had a greater chance of non-relapse, as compared to MRD-positive patients. It was therefore evaluated whether the NP-BCMA conjugate might provide both an early predictor of MM and a MRD biomarker agent.
[0132] MRD was established as one of the most relevant biomarkers for MMindeed, most MM patient relapses have been identified as due to the presence of MRD positive signal. It was demonstrated that MRD can be employed to assess the direct therapeutic efficacy of MM therapeutic agents, while also empowering evaluation of future therapeutic decisions. However, detection of MRD is not straightforward. Current techniques to evaluate the presence of a MRD positive status, such as multiparameter flow cytometry and allele-specific oligonucleotide PCR are based on an invasive process, are qualitative, rely on a bone marrow sample, are destructive to samples, and/or are highly time-consuming to administer and evaluate. A common failure in the treatment and imaging of MM is the inability of traditional therapies to reach and combat the bone homing of tumorigenic B-cells. Targeted delivery of effective intracellular agent(s) to target cells has therefore been needed, yet targeted delivery has also presented difficult obstacles. While it has been demonstrated that it is possible to target the bone microenvironment by using bisphosphonate-based nanoparticles that do not possess specific affinities for malignant plasma cells, the current disclosure has identified a new approach to targeting of MM cells specifically. For at least the above reasons, the NP-BCMA conjugate was evaluated as an imaging biomarker for MRD. To do so, a murine model of MRD was established by intravascular dissemination of GFP and luciferase-expressing MM1.S cells (GFP.sup.+/Luc.sup.+ MM1.S) followed by therapeutic debulking after 21 days, using three doses of Bortezomib (0.5 mg/kg) and one dose of Melphalan (5.5 mg/kg). Tumor growth was monitored by weekly bioluminescence imaging (BLI), where cell dissemination (MM1.S.sub.GFP.sup.+.sub./LUC.sup.+) was followed via monitoring once a week by BLI as a gold standard for preclinical monitoring in the tumor cell dissemination method (
[0133] Results obtained by BLI, MRI, CT, and by the serum light-chain assay were compared at 1 week after therapeutic debulking (i.e. 5 weeks after initial tumor cell implantation). A receiver operator characteristic (ROC) curve was generated to assess the sensitivity and specificity of each of the 4 diagnostic modalities to detect the presence of MRD and confirmed the superiority of MRI using NP-BCMA (
[0134] The specificity and ease of use attributable to the T1 signal enhancement have allowed for a rapid a priori detection of MM disease (including MRD) and can be employed clinically as a predictor, before performing a more in-depth diagnostic of the patient, e.g., performing next generation sequencing. Indeed, results obtained from week 5 (i.e., 1 week post-therapy) (
[0135] In summary, demonstrated herein is what is believed to be the first proof-of-concept example in which changes in the SNR obtained by serial MRI of ultra-small, Gd-based nanoparticle-antibody complexes have been used as an imaging biomarker to detect MRD. Importantly, the newly disclosed agents described herein were able to circumvent the challenges seen with the first generation of antibody-targeted nanoparticles to achieve precise localization of malignant plasma cells in their natural microenvironment. While they may not be suitable for patients with advanced renal failure, given the well-established risks of all Gd-based contrast agents (Barrett and Parfrey. N Engl J Med 354: 379-386), the constructs disclosed herein may otherwise find utility in prompting early cessation of ineffective therapies and/or therapeutic re-initiation after prolonged periods of MM remission. With the increasing utilization of cell-surface targeted agents in MM therapy (e.g., elotuzumab (Lonial et al. N Engl J Med 373: 621-631), BCMA-targeted chimeric antigen receptor T-cells (Ali et al. Blood 128: 1688-1700; CAR-T), and daratumumab (Lokhorst et al. N Engl J Med 373: 1207-1219)), NP-SLAMF7, NP-BCMA and future formulations of GdNP-anti-CD38-antibody complexes may enable MRI to guide patient-specific therapeutic selection. Their successful application may further afford insights into the fabrication of other targeted constructs that may help to translate the full potential of nanomedicine to improve the care and survival of cancer patients.
[0136] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
[0137] One skilled in the art would readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the disclosure. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the disclosure, and are defined by the scope of the claims.
[0138] In addition, where features or aspects of the disclosure are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
[0139] The use of the terms a and an and the and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms comprising, having, including, and containing are to be construed as open-ended terms (i.e., meaning including, but not limited to,) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
[0140] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.
[0141] Embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosed invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.
[0142] The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms comprising, consisting essentially of, and consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present disclosure provides preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the description and the appended claims.
[0143] It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present disclosure and the following claims. The present disclosure teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating conjugates possessing improved contrast, diagnostic and/or imaging activity. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying conjugates possessing improved contrast, diagnostic and/or imaging activity.
[0144] The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.