Carrier-PD-l1 binding agent composition and method for using the same for treating cancer

Abstract

To provide carrier-PD-L1 binding agent compositions and methods for using the same for treating cancers.SOLUTION: Described herein are compositions of binding agents and carrier proteins, and optionally at least one therapeutic agent, and methods of making and using the same, in particular, as a cancer therapeutic. Also described are lyophilized compositions of binding agents and carrier proteins, and optionally at least one therapeutic agent, and methods of making and using the same, in particular, as a cancer therapeutic. Still also described are methods for treating and / or increasing the therapeutic effectiveness of an immunotherapy of a patient suffering from a cancer which expresses PD-L1 or PD-L2 by administering to the patient a nanoparticle composition and a PD-1 immunotherapy.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application relates to novel compositions of binding agents and carrier proteins and methods of making and using them, particularly as cancer therapeutics. [Background technology]

[0002] Chemotherapy remains the mainstay of systemic treatment for many types of cancer, including melanoma. Most chemotherapeutic agents are only poorly selective for tumor cells and can be highly toxic to healthy proliferating cells (Allen TM. (2002) Cancer 2:750-763), often necessitating dose reductions and even cessation of treatment. In theory, chemotherapy can be used to treat a wide range of cancers, including melanoma, cancers, and cancers. One way to overcome the toxicity problems of the method and improve the efficacy is to target chemotherapy drugs to tumors using antibodies specific to proteins selectively expressed (or overexpressed) by tumor cells, attracting the targeted drugs to the tumor, thereby changing the biodistribution of chemotherapy and resulting in more drug reaching the tumor and less impact on healthy tissue.However, despite 30 years of research, specific targeting has rarely been successful in therapeutic situations.

[0003] Conventional antibody dependent chemotherapy (ADC) is designed with a toxic agent attached to a target antibody via a synthetic protease-cleavable linker. The efficacy of such ADC therapy depends on the ability of the target T cell to bind to the antibody, the linker to be cleaved, and the uptake of the toxic agent into the target T cell (Schrama, D. et al. (2006 Nature reviews.Drug discovery 5:147-159).

[0004] Antibody-targeted chemotherapy has promised advantages over conventional therapy because it offers a combination of targeting capability, multiple cytotoxic agents, and improved therapeutic potential with potentially low toxicity. Despite extensive research, clinically effective antibody-targeted chemotherapy remains elusive: the main obstacles include the instability of the linker between the antibody and the chemotherapy drug, the reduced tumor toxicity of the chemotherapeutic agent when bound to the antibody, and the inability of the conjugate to bind to and enter tumor cells. Moreover, these therapies have not allowed control over the size of the antibody-drug conjugate.

[0005] There remains a need in the art for antibody-based cancer therapeutics that retain cytotoxic effects for targeted drug delivery to provide reliable and improved anti-tumor efficacy over prior art therapies.

[0006] Programmed cell death protein-1 (PD-1, also known as CD279, hereafter referred to as "PD-1") receptor PD-1 ligands include programmed cell death ligand-1 (also known as PD-L1, B7-H1, CD274, hereafter referred to as "PD-L1") and programmed cell death ligand-2 (also known as PD-L2, B7-DC and CD273, hereafter referred to as "PD-L2"), and are commonly expressed on the surface of dendritic cells or macrophages. PD-L1 PD-L1 is expressed in many tumors, including cancers occurring in various organs, such as the head and neck, lung, stomach, colon, pancreas, breast, kidney, bladder, ovary, and cervix, as well as melanoma, glioblastoma, multiple myeloma, lymphoma, and various leukemias. PD-L1 is generally expressed by tumor cells, such as metastatic non-small cell lung cancer (NSCLC), as well as in cancers occurring in the breast, ovaries, and cervix. It is overexpressed on the surface of non-small cell lung carcinomas (NSLC).

[0007] Upon binding to the PD-1 receptor on activated T cells, tumor cells expressing PD-L1 can utilize inhibitory signaling of the PD-1 pathway, thereby limiting or even stopping the host's own anti-tumor immune response from T cells. On the other side of this inhibitory signaling, blocking or impeding the interaction of PD-1 with PD-L1 / PD-L2 would prevent the inhibition signaled by the pathway. Thus, immunotherapeutics based on antibodies against PD-1, PD-L1 or PD-L2 aim to overcome such immune response resistance of tumors and restore or restimulate the host's own immune mechanisms against tumors. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there is a need to increase the therapeutic effectiveness of immunotherapeutic treatment of patients suffering from cancers that express PD-L1 or PD-L2. Effect of the Invention

[0009] The therapeutic efficacy of immunotherapeutic treatment of patients suffering from cancer expressing PD-L1 or PD-L2 is demonstrated by (a) A therapeutically effective amount of a nanoparticle or nanoparticle composition as described herein, wherein the nanoparticle is capable of binding to PD-L1 or PD-L2, and (b) PD-1 is increased by administration of an immunotherapeutic agent. It is surprising and surprising that this can be achieved.

[0010] Both anti-PD-1 and anti-PD-L1 antibodies have been developed and approved to treat a variety of cancers. Anti-PD-1 antibodies include, but are not limited to, nivolumab (OPDIVO®), developed by Bristol-Myers Squibb US and used to treat metastatic melanoma and squamous NSCL. Approved in the US for the treatment of cancer; pembrolizumab (KEYTRUDA®), developed by Merck US and approved for the treatment of metastatic melanoma. Anti-PD-L1 antibodies include, but are not limited to: atezolizumab (Tecentriq®), developed by Roche, Switzerland (Genentech US) and approved for the treatment of the most common type of bladder cancer, i.e. urinary BMS-936559 / MDX-1105 (Bristol Myers Squibb), eDI4736 (MedImmune / AstraZeneca) and MSB00100718C (EMD Serono). See Atkins “Therapeutic uses of anti-PD-1 and anti-PD-L1 antibodies” International Immunology Vol.27(1) pp.39-46.

[0011] Atezolizumab is a humanized monoclonal antibody that targets the PD-1 pathway to block immune checkpoint inhibition signaled by it. As used herein, the PD-1 pathway signals inhibition of T cell immune responses upon interaction of PD-1 and PD-L1 / PD-L2. A variety of other types of cancer, including non-squamous NSCLC, renal cell carcinoma, and bladder cancer, are Therapies using other anti-PD-L1 antibodies for treatment (e.g., avelumab, durvalumab, BMS 936559) are also under research and development.

[0012] Like PD-L1, PD-L2 binds to PD-1. Human PD-L1 and PD-L2 bind to each other approximately 41 percent It has been reported that PD-L2 and PD-1 share amino acid sequence identity and have similar functional properties. Binding of PD-L2 to PD-1 also inhibits T cell proliferation and cytokine production, promoting a similar T cell immune response. For example, various cancers, including non-squamous NSCLC, renal cell carcinoma, and bladder cancer, have been shown to downregulate Therapies using anti-PD-L2 antibodies to treat other types of cancer are also under research and development. [Means for solving the problem]

[0013] In accordance with the present invention, the nanoparticles comprise (a) a carrier protein, (b) a first binding agent, and (c) optionally a therapeutic agent, where the nanoparticles are capable of binding to PD-L1 or PD-L2. In a preferred embodiment, the nanoparticles are prepared by combining the components of the nanoparticle (the carrier protein , binding agent and / or therapeutic agent).

[0014] In one aspect, a method is provided for treating a patient suffering from a cancer that expresses PD-L1 or PD-L2, the method comprising: (a) administering to a patient a plurality of nanoparticles (or a nanoparticle composition comprising nanoparticles), each of the nanoparticles comprising a carrier protein, a first nanoparticle having an antigen-binding moiety, and / or a second nanoparticle having an antigen-binding moiety. a binding agent, wherein the antigen is PD-L1 or PD-L2 and, optionally, at least one therapeutic and (b) administering to the patient a PD-1 immunotherapeutic agent, wherein the nanoparticles are capable of binding to PD-L1 or PD-L2. In one embodiment, the PD-1 immunotherapeutic agent comprises a second binding agent capable of binding to PD-1.

[0015] In another aspect, the invention provides a method for increasing the therapeutic efficacy of an immunotherapeutic treatment in a patient suffering from a cancer that expresses PD-L1 or PD-L2, comprising administering to the patient a therapeutically effective amount of any of the immunotherapeutic agents described herein. and (b) administering to the patient a PD-1 immunotherapeutic agent. In one embodiment, the PD-1 immunotherapy comprises administering a second binding agent capable of binding to PD-1.

[0016] In one aspect, the invention provides a method for treating a patient suffering from a cancer that expresses PD-L1 or PD-L2, comprising: (a) administering to the patient a plurality of nanoparticles (or a nanoparticle composition comprising nanoparticles); wherein each of the nanoparticles comprises albumin, an antibody having an antigen-binding moiety, where the antigen is PD-L1 or PD-L2, and paclitaxel, and the nanoparticles are capable of binding to PD-L1 or PD-L2; and (b) administering to the patient a PD-1 immunotherapeutic agent. In one embodiment, the PD-1 immunotherapeutic agent comprises a second antibody capable of binding to PD-1 (an anti-PD-1 antibody). In one embodiment, the antibody is an anti-PD-L1 antibody. In one embodiment, the antibody is an anti-PD-L2 antibody.

[0017] In some embodiments, a CTLA-4 immunotherapeutic agent is administered to a patient in combination with the nanoparticles capable of binding PD-L1 or PD-L2. In one embodiment, the CTLA-4 immunotherapeutic agent is administered in addition to the PD-1 immunotherapeutic agent. In one embodiment, the CTLA-4 immunotherapeutic agent is administered instead of the PD-1 immunotherapeutic agent. In one embodiment, the CTLA-4 immunotherapeutic agent is an anti-CTLA-4 antibody.

[0018] In one aspect, each of the nanoparticles of the nanoparticle composition comprises about 400 to about 1000 A first binder is included.

[0019] In some aspects, the first binding agent is an aptamer. The second binding agent of the PD-1 immunotherapeutic agent is an aptamer.

[0020] In some aspects, the first binding agent is an antibody (e.g., an anti-PD-L1 antibody or an anti-PD-L2 antibody). In some aspects, the second binding agent of the PD-1 immunotherapeutic is an antibody (e.g., In some aspects, the anti-PD-1 antibody is nivolumab, pembrolizumab, or izumab, pidilizumab, PDR001, or derivatives thereof. In some aspects, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, or BMS 936559 (MDX1105). In some aspects, the binding agent of the CTLA-4 immunotherapeutic agent is an anti-CTLA-4 antibody. In one embodiment, the anti-CTLA-4 antibody is ipilimumab.

[0021] In some aspects, the first binding agent and / or the second binding agent is a fusion protein. In one embodiment, the fusion protein is AMP-224 (PD-L2 IgG2a fusion protein; Amplimmune / GlaxoSmith Klein); AMP-514 (MEDI0680) (PD-L2 fusion protein; Amplimmune / GlaxoSmith Klein), or a derivative thereof.

[0022] In some aspects, the nanoparticles or nanoparticle compositions are lyophilized. In some aspects, the second binding agent of the PD1 immunotherapeutic agent is a free binding agent, wherein the free binding agent is not complexed to or incorporated on and / or within a nanoparticle composition.

[0023] In some aspects, the PD-1 immunotherapeutic agent is a second binding site conjugated to the nanoparticle composition. Immunotherapeutics Comprising a Second Binding Agent Incorporated onto and / or Within a Combination or Nanoparticle Composition A nanoparticle composition, wherein the immunotherapeutic nanoparticle composition comprises a carrier protein and the second binding agent.

[0024] In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is an antibody. In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is an anti-PD-1 antibody. In some aspects, the antibody of the immunotherapeutic nanoparticle composition comprises atezolizumab, nivolumab, pembrolizumab, avelumab or durvalumab, pidilizumab, BMS936559, PDR001, or post-emulsion formulations thereof.

[0025] In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is a fusion protein. In one embodiment, the fusion protein is AMP-224 (PD-L2 IgG2a fusion protein; Amplimmune / GlaxoSmith Klein); AMP-514 (MEDI0680) (PD-L2 fusion protein Amplimmune / GlaxoSmith Klein), or their derivatives.

[0026] In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is an aptamer. In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is a PD-1 apeptide. It's Tamar.

[0027] In some aspects, the immunotherapeutic nanoparticles and / or nanoparticle compositions are lyophilized.

[0028] In some aspects, the nanoparticle composition and the PD-1 immunotherapeutic agent are administered sequentially. In some aspects, the nanoparticle composition is administered prior to administration of the PD-1 immunotherapeutic agent. In some aspects, the PD-1 immunotherapeutic agent is administered prior to administration of the nanoparticle composition. In some aspects, the nanoparticle composition and the PD-1 immunotherapeutic agent are administered simultaneously.

[0029] In some embodiments, the present invention relates to a method for increasing the therapeutic efficacy of an immunotherapeutic treatment in a patient suffering from a cancer that expresses PD-L1 or PD-L2. In one embodiment, the method comprises administering to the patient a therapeutically effective amount of a nanoparticle or nanoparticle composition described herein, and a PD-1 or CTLA-4 immunotherapeutic agent comprising a second binding agent. In an embodiment, the second binding agent is capable of binding to PD-1 or CTLA-4. In embodiments, the PD-1 or CTLA-4 immunotherapeutic agent is coupled to a carrier protein (e.g., albumin). The nanoparticles comprise a first binding agent, a second binding agent, and, optionally, a therapeutic agent (e.g., paclitaxel).

[0030] In some embodiments, the present invention relates to a method for treating a patient suffering from a cancer that expresses PD-L1 or PD-L2. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a nanoparticle composition described herein and administering to the patient a second wherein the binding agent of the nanoparticle composition is capable of binding to PD-L1, PD-L2 or PD-1, and the second binding agent of the immunotherapeutic agent is capable of binding to PD-L1, PD-L2 or PD-1.

[0031] Without being bound by theory, it is believed that the binding agent is bound by the carrier protein through hydrophobic interactions, which are weak in nature. Nevertheless, the activity of the individual components and their relative relationship in the nanoparticles are maintained despite lyophilization and reconstitution of the composition. It is further believed that the binding to the carrier protein, such as the complexation of the binding agent to the carrier protein, occurs via an albumin binding motif on the binding agent and / or an antibody binding motif on the carrier protein. Albumin binding motifs and antibody binding motifs are described in International Patent Application PCT / US17 / 45643, filed on August 4, 2017, and are incorporated herein by reference in their entirety. In some embodiments, the binding agent is a non-therapeutic and non-endogenous human antibody, fusion protein, or aptamer.

[0032] For the nanoparticles to be used therapeutically, additional challenges are posed.

[0033] Although rearrangement of hydrophobic components in the nanoparticles can be mitigated through covalent bonds between the components, such covalent bonds pose challenges to the therapeutic use of nanoparticles in cancer treatment. The binding agent, carrier protein, and additional therapeutic agent typically act at different locations and through different mechanisms within the tumor. Non-covalent bonds allow the nanoparticle components to dissociate in the tumor. Thus, although covalent bonds may be advantageous for lyophilization, they may be disadvantageous for therapeutic applications.

[0034] The size and size distribution of nanoparticles is also important. Nanoparticles may behave differently according to their size. At larger sizes, nanoparticles or agglomerations of the particles may occlude blood vessels, both of which can affect the performance and safety of the composition.

[0035] When administered intravenously, large particles (e.g., greater than 1 μm) are typically not preferred because they may become lodged in the pulmonary microvasculature. At the same time, larger particles may accumulate in tumors or specific organs. For example, THERASPHERE®, a 20-60 micron glass particle injected into the coronary arteries that feed tumors in the liver for delivery of radioactive elements, also known as radioembolization, is used clinically for liver cancer.

[0036] Therefore, for intravenous administration, particles of 1 μm or less are used. Particles larger than 1 μm are more typically administered directly into a tumor (direct injection) or into an artery that supplies the site of a tumor.

[0037] Finally, cryoprotectants and agents that aid in the lyophilization process must be safe and tolerable for therapeutic use.

[0038] Without being bound by theory, it is believed that the binding agent is bound by the carrier protein through hydrophobic interactions, which are weak in nature. Nevertheless, the activity of the individual components, and their relative relationships in the nanoparticles, is still achieved despite lyophilization and reconstitution of the composition.

[0039] In one aspect, provided herein is a nanoparticle composition comprising nanoparticles, each of the nanoparticles comprising a carrier protein, a binding agent, and optionally at least one therapeutic agent, the binding agent disposed outwardly from a surface of the nanoparticle; and and the nanoparticles are capable of binding to PD-L1, PD-L2 or PD-1 in vivo.

[0040] In another aspect, provided herein is a nanoparticle composition comprising nanoparticles, each of the nanoparticles comprising a carrier protein that is not albumin and, optionally, at least one therapeutic agent, wherein the binding agent is disposed on an exterior surface of the nanoparticle; and the nanoparticles are capable of binding to PD-L1, PD-L2 or PD-1 in vivo. In one embodiment, the nanoparticles are bound to about 100 to about 1000 binding agents, preferably about 400 to about 800 binding agents. As the nanoparticles polymerize, the number of binders increases proportionally. For example, if a 160 nm nanoparticle contains 400 binders, a 320 nm dimer contains about 800 binders.

[0041] In another aspect, provided herein is a nanoparticle composition comprising nanoparticles, each of the nanoparticles comprising a carrier protein, a binding agent, and optionally at least one therapeutic agent that is not paclitaxel, the nanoparticles capable of binding to PD-L1, PD-L2, or PD-1 in vivo. In one embodiment, the nanoparticles further comprise: In one embodiment, the binding agent is disposed on the surface of the nanoparticle such that the binding portion of the binding agent (eg, a variable region of an antibody) faces away from the surface.

[0042] In other embodiments, the nanoparticles multimerize, e.g., dimerize. Multimerization is observed as a multiple of the weight or size of a single molecule, e.g., a 160 nm particle multiplies to about 320 nm, about 480 nm, about 640 nm, etc. In some embodiments, less than 20% of the nanoparticles in the population are dimerized. The particles are multimeric. In some embodiments, greater than 80% of the nanoparticles in the population are multimeric. be.

[0043] In one embodiment, the weight ratio of carrier-bound drug to binder (e.g., The ratio of the antibody to the PD-L1 or anti-PD-L2 antibody bound to the cytoplasmic lipase is about 5:1 to about 1:1. In one embodiment, the weight ratio of carrier-bound drug to binder is about 10:4. In one embodiment, the binder is substantially a monolayer on all or part of the surface of the nanoparticle.In one embodiment, less than 0.01% of the nanoparticles in the composition have a size selected from greater than 200nm, greater than 300nm, greater than 400nm, greater than 500nm, greater than 600nm, greater than 700nm and greater than 800nm.The larger size is believed to be the result of the multimerization of several nanoparticles, each of which comprises a core and a binder that coats all or part of the surface of each nanoparticle.

[0044] The present invention further includes lyophilized compositions and lyophilized compositions that do not substantially differ from or are the same as the properties of freshly prepared nanoparticles. In particular, the lyophilized compositions are similar or identical to the fresh compositions in terms of particle size, particle size distribution, toxicity to cancer cells, binder affinity, and binder specificity upon resuspension in an aqueous solution. Surprisingly, the lyophilized nanoparticles are also characterized by the presence of two different protein components in these particles. Nevertheless, after resuspension, the nanoparticles retain their properties as freshly produced nanoparticles.

[0045] In one aspect, the invention relates to a lyophilized nanoparticle composition comprising a plurality of nanoparticles, each of the plurality of nanoparticles comprising a carrier-bound drug core and an amount of a binding agent that binds PD-L1, PD-L2 or PD-1. In one embodiment, the binding agent is disposed on the surface of the core such that the binding moieties of the binding agent are directed outward from the surface, and the binding agents maintain their association with the outer surface of the nanoparticle upon reconstitution with an aqueous solution. In one embodiment, the lyophilized composition remains stable at room temperature for at least about 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more. In one embodiment, the lyophilized composition is stable at room temperature for at least 3 months. In one embodiment, the reconstituted nanoparticles retain the activity of the therapeutic agent and are capable of binding to targets in vivo. In another embodiment, the composition is stable at about 20° C. to about 25° C. for up to about 12 months or more.

[0046] In one embodiment, the average size of the reconstituted nanoparticles is about 90 nm to about 1 μm. In a preferred embodiment, the average size of the reconstituted nanoparticles is about 100 nm to about 200 nm, more preferably about 100 nm to about 160 nm. In one embodiment, the average size of the reconstituted nanoparticles is greater than 800 nm to about 3.5 μm, which is smaller than the average size of the smaller nanoparticles. In one embodiment, the core The weight ratio of to binder is greater than 1: 1 to about 1: 3. In one embodiment, the average size of the reconstituted nanoparticles is from about 90 nm to about 225 nm.

[0047] In one aspect, the present disclosure relates to a lyophilized nanoparticle composition comprising a plurality of lyophilized nanoparticles or a plurality of nanoparticles, each of the nanoparticles comprising: (a) a (b) an albumin-bound paclitaxel core, and (b) binding moieties of the binding agent facing outward from its surface. the lyophilized composition comprises binding agents that bind PD-L1, PD-L2 or PD-1 disposed on the surface of an albumin-bound paclitaxel core such that the binding agents maintain their association with the outer surface of the nanoparticles upon reconstitution with an aqueous solution, the lyophilized composition is stable at about 20°C to about 25°C for at least three months, and the reconstituted nanoparticles are capable of binding PD-L1, PD-L2 or PD-1 in vivo.

[0048] In one embodiment, the average size of the reconstituted nanoparticles is not substantially different from the particle size of freshly prepared nanoparticles. In some embodiments, the average particle size is 90 nm to 800 nm, including, for example, 90, 100, 110, 130, 150, 160, 200, 300, 400, 500, 600, 700 or 800 nm. In other embodiments, the average particle is, for example, greater than 800 nm to about 3.5 μm. In some embodiments, the particle is a multimer of nanoparticles. In some embodiments, the nanoparticles have an average particle size of about 90 nm to about 225 nm, either immediately after nanoparticle production or after lyophilization and resuspension in an aqueous solution suitable for injection.

[0049] In some embodiments, the weight ratio of albumin-bound paclitaxel to binder is about 5:1 to about 1:1. In other embodiments, the weight ratio of albumin-bound paclitaxel to binder is about 10:4. In further embodiments, the weight ratio of albumin-bound paclitaxel to binder is about 10:4. The weight ratio of taxel to binder is greater than 1:1 to about 1:3.

[0050] In some embodiments, the core comprises albumin-bound paclitaxel (e.g., albumin-bound paclitaxel). and the binding agent selectively recognizes PD-L1 or PD-L2. In some embodiments, the core is albumin-bound paclitaxel (e.g., Abraxane®) and the binding agent selectively recognizes PD-1. In some embodiments, the core is albumin-bound paclitaxel (e.g., Abraxane®), and the binding agent selectively recognizes CTLA-4.

[0051] In some embodiments, the at least one therapeutic agent is located within the interior of the nanoparticle. In other embodiments, the at least one therapeutic agent is located on the exterior surface of the nanoparticle. In yet other embodiments, the at least one therapeutic agent is present within the nanoparticle and and located on the outer surface of the nanoparticle.

[0052] In some embodiments, the nanoparticles include more than one type of therapeutic agent. Sun and platinum drugs, such as paclitaxel and cisplatin.

[0053] In some embodiments, the binding agent is atezolizumab, nivolumab, pembrolizumab, avelumab or durvalumab, pidilizumab, BMS936559, or a derivative thereof. In some embodiments, the binding agent is substantially a monolayer of binding agent on all or a portion of the surface of the nanoparticle.

[0054] In further embodiments, the antibody is less glycosylated than typically found in naturally occurring human antibodies. Such glycosylation can be influenced, for example, by the expression system or the presence of glycosylation inhibitors during expression. In some embodiments, the glycosylation state of an antibody or other binding agent is altered through enzymatic or chemical action.

[0055] In some embodiments, the at least one therapeutic agent is selected from the group consisting of abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leukotriene ... It is selected from prorelin, melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib, oxaliplatin, paclitaxel, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine and cyclophosphamide.

[0056] In some embodiments, the binding agent, carrier protein, and, if present, therapeutic agent are attached via non-covalent bonds.

[0057] In some embodiments, the carrier protein is selected from gelatin, elastin, gliadin, legumin, zein, soy protein, milk protein, and whey protein. In other embodiments, the carrier protein is albumin, such as human serum albumin. In some embodiments, the carrier protein is a recombinant protein, such as recombinant human serum albumin.

[0058] In some embodiments, the nanoparticle composition is formulated for intravenous delivery, hi other embodiments, the nanoparticle composition is formulated for direct injection or perfusion into a tumor.

[0059] In some embodiments, the second binding agent of the immunotherapeutic agent is formulated for intravenous delivery. In other embodiments, the second binding agent of the immunotherapeutic agent is administered by direct injection into a tumor. or formulated for perfusion.

[0060] In some embodiments, the average nanoparticle size in the nanoparticle composition is less than or equal to 800 nm Ultra-3.5 μm.

[0061] In some embodiments, the nanoparticles have a dissociation constant of about 1×10 -11 M ~ approx. 1x10 -9 Having M .

[0062] In another aspect, provided herein is a method for making a nanoparticle composition, the method comprising: incorporating the carrier protein and, optionally, at least one therapeutic agent in a nanoparticle composition comprising: Under conditions and ratios of components that will allow the formation of the desired nanoparticles, a pH of 5.0 to 7.5 and a pH of about 5 The method comprises contacting the antibody in a solution having a temperature of about 23° C. to about 60° C., about 23° C. to about 60° C., or about 55° C. to about 60° C. In one embodiment, the nanoparticles are prepared at 55-60° C. and pH 7.0. In another aspect, a nanoparticle composition is provided herein, wherein the method includes (a) contacting the carrier protein and, optionally, the at least one therapeutic agent to form a core, and (b) exposing the core to conditions and compositions that will permit the formation of a desired nanoparticle. The method includes contacting the antibody in a solution having a pH of about 5.0 to about 7.5 and a temperature of about 5°C to about 60°C, about 23°C to about 60°C, or about 55°C to about 60°C under conditions of a ratio of about 1:1.

[0063] The amounts of components (e.g., carrier protein, antibody, therapeutic agent, or combinations thereof) are controlled to provide for the formation of the desired nanoparticles. Compositions in which the amounts of components are too dilute will not form the nanoparticles described herein. In a preferred embodiment, the weight ratio of carrier protein to binder is 10:4. In some embodiments, In some embodiments, the amount of carrier protein is about 1 mg / mL to about 100 mg / mL. In some embodiments, the amount of binder is about 1 mg / mL to about 30 mg / mL. For example, in some embodiments, For this purpose, the ratio of carrier protein:binder:solution is approximately 9 mg of carrier protein (e.g., albumin) to 4 mg of binder in 1 mL of solution (e.g., saline). A therapeutic agent (eg, paclitaxel) can also be added to the carrier protein.

[0064] The nanoparticles described herein are preformed and contain the carrier protein ( For example, albumin), a therapeutic agent (for example, paclitaxel) and a binding agent (for example, an antibody) By preformed nanoparticles is meant premixed in vitro under conditions that permit the formation of the nanoparticles prior to administration to a patient (and / or prior to lyophilization of the nanoparticles). In some embodiments, the preformed nanoparticles are diluted in an aqueous solution prior to administration to a patient. As non-limiting examples, the preformed nanoparticles are diluted in an aqueous solution within 5, 10, 20, 30, 45, or 60 minutes of administration to a patient. May be diluted 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 24 hours prior to use. .

[0065] In a further embodiment, the nanoparticles are prepared as described above and then lyophilized.

[0066] In another aspect, provided herein is a method for treating a cancer cell, the method comprising contacting the cell with an effective amount of a nanoparticle composition disclosed herein and an immunotherapeutic agent to treat the cancer cell.

[0067] In another aspect, provided herein are methods for treating a tumor in a patient in need thereof, comprising contacting the tumor with an effective amount of a nanoparticle composition disclosed herein and an immunotherapeutic agent to treat the tumor. In some embodiments, the tumor is reduced in size.

[0068] Generally, the immunotherapeutics (PD-1 immunotherapeutics and / or CTLA-4 immunotherapeutics) will be administered in a manner consistent with standard clinical protocols, e.g., consistent with FDA- (or other regulatory agency) approved labeling.

[0069] In some embodiments, the methods provided herein include a) administering the nanoparticle composition and the immunotherapeutic agent once a week for three weeks; b) discontinuing administration of the nanoparticle composition and the immunotherapeutic agent for one week; and c) repeating steps a) and b) as necessary to treat a cancer or tumor. The process includes repeating the steps of:

[0070] In a related embodiment, the treatment comprises administering the nanoparticle composition prior to administration of the immunotherapeutic agent. In one embodiment, the nanoparticle composition is administered about 6 to 48 or 12 to 48 hours prior to administration of the immunotherapeutic agent. In another embodiment, the nanoparticle composition is administered about 6 to 48 hours prior to administration of the immunotherapeutic agent. In yet another embodiment, the composition is administered 6 to 12 hours prior to administration of the immunotherapeutic agent. The nanoparticle composition is administered 2-8 hours prior to administration of the immunotherapeutic agent. In yet other embodiments, the nanoparticle composition is administered 1 week prior to administration of the immunotherapeutic agent.

[0071] In a related embodiment, the treatment comprises administering the immunotherapeutic agent prior to administration of the nanoparticle composition. In one embodiment, the immunotherapeutic agent is administered about 6 to 48 or 12 to 48 hours prior to administration of the nanoparticle composition. In another embodiment, the immunotherapeutic agent is In still other embodiments, the immune system is administered 6 to 12 hours prior to administration of the nanoparticle composition. The immunotherapeutic agent is administered 2-8 hours prior to administration of the nanoparticle composition. In yet other embodiments, the immunotherapeutic agent is administered 1 week prior to administration of the nanoparticle composition.

[0072] In some embodiments, the therapeutically effective amount of the nanoparticle composition is about 75 mg / m 2 ~about 175mg / m 2 of the carrier protein (i.e., 1 ml of the patient 2 In other embodiments, the therapeutically effective amount is about 75 mg / m of therapeutic agent (e.g., paclitaxel). 2 ~about 175mg / m 2 In other embodiments, the therapeutically effective amount is about 30 mg / m of the binding agent. 2 ~about 70mg / m 2 In yet other embodiments, the therapeutically effective amount is about 30 mg / m of bevacizumab. 2 ~about 70mg / m 2 Includes.

[0073] In one embodiment, the lyophilized composition has a concentration of about 75 mg / m 2 ~about 175mg / m 2 Career - protein (preferably albumin); about 30 mg / m 2 ~about 70mg / m 2 of binder; and about 75 mg / m 2 ~about 175mg / m 2 Contains paclitaxel.

[0074] In some embodiments, the present invention provides a method for preparing a nanoparticle composition comprising the steps of: (a) administering to a subject a subject the nanoparticle composition as defined herein; (b) an amount of an immunotherapeutic agent capable of binding to PD-1, and, optionally, (c) instructions for use.

[0075] The following figures are merely representative of the present invention and are not intended as limitations. For consistency, nanoparticles using Abraxane® and Rituximab are meant to use "AR" and the number following AR, e.g. AR160, gives the average particle size of these nanoparticles (in nanometers, based on Mastersizer 2000 analysis). Similarly, when the binding agent is Atezolizumab, the initial letter is "AA" and the number following is the average particle size of the nanoparticles (in nanometers, based on Malvern Nanosight analysis). [Brief description of the drawings]

[0076] [Figure 1]Figure 1 shows the results of an experiment in which CD20-positive Daudi lymphoma cells were labeled with fluorescently tagged anti-human CD20 or an isotype-matched control in panels F and A, respectively, and analyzed by flow cytometry. In other panels, the Daudi cells were pretreated with Abraxane® (ABX), ABX-rituximab nanoparticles (AR160), lyophilized and resuspended AR160 (AR160L), or Rituxan prior to CD20 labeling. CD20 binding was specifically blocked by AR160 nanoparticles and Rituxan, but not by ABX alone, indicating that AR160 and AR160 bind CD20 on these cells and block the binding of fluorescent anti-CD20 antibodies. [Diagram 2] FIG. 2 is a histogram overlay of the scatter plot of FIG. [Figure 3A] FIG. 3A shows a comparison of ABX alone with ABX / rituximab nanoparticles (AR; FIG. 3A), both freshly made and lyophilized / resuspended. [Figure 3B] Figures 3A-B show a comparison of ABX alone with ABX / trastuzumab nanoparticles (AT; Figure 3B), both freshly made and lyophilized / resuspended. [Figure 4] FIG. 4 compares the toxicity of ABX and AR particles in a Daudi cell proliferation assay. [Figure 5A]5A-5C show results obtained in mice treated with either labeled Abraxane®, labeled Abraxane® coated with a nonspecific (bevacizumab) antibody (AB IgG), or labeled Abraxane® coated with rituximab (AR160). FIG. 5A shows fluorescence accumulation in regions of interest (ROIs 2, 3, and 4) within each tumor as well as in background regions (ROIs 1, 5, and 6). ROIs 1, 5, and 6 serve as background references. The data demonstrate that administration of AR160 nanoparticles results in increased fluorescence compared to Abraxane® alone or Abraxane® coated with a nonspecific antibody. [Figure 5B] Figures 5A-5C show results obtained in mice treated with either labeled Abraxane®, labeled Abraxane® coated with a nonspecific (bevacizumab) antibody (AB IgG), or labeled Abraxane® coated with Rituximab (AR160). Figure 5B is a bar graph of the mean fluorescence per unit tumor area for mice in all three treatment groups and shows total tumor delivery. The data demonstrate that administration of AR160 nanoparticles results in increased fluorescence compared to Abraxane® alone or Abraxane® coated with a nonspecific antibody. [Figure 5C]Figures 5A-5C show results obtained in mice treated with either labeled Abraxane®, labeled Abraxane® coated with a nonspecific (bevacizumab) antibody (AB IgG), or labeled Abraxane® coated with rituximab (AR160). Figure 5C is a bar graph of the mean fluorescence per unit of tumor area normalized by background ROI to give the percentage of drug-to-body delivered to the tumor. The data demonstrate that administration of AR160 nanoparticles results in increased fluorescence compared to Abraxane® alone or Abraxane® coated with a nonspecific antibody. [Figure 6] Figure 6 shows the survival rate of mice treated with a single dose of saline, BEV24 (bevacizumab at 24 mg / kg), ABX30 (ABX at 30 mg / kg), AB160 (BEV at 12 mg / kg and ABX at 30 mg / kg) and AB225 (BEV at 24 mg / kg and ABX at 30 mg / kg). Thirty days after dosing, the survival rate of mice treated with AB225 and AB160 far exceeds that of mice treated with BEV alone or Abraxane® alone. [Figure 7] Figure 7 shows the binding affinity between Atezolizumab and ABX. The Kd was determined to be 1.462x10-9. Biolayer interferometry (BLItz) (Forte Bioscience) was performed using a streptavidin probe. [Figure 8A] FIG. 8A shows the particle size distribution of ABX alone (90 nm average size) and ABX-atezolizumab nanoparticles (AA; 129 nm average size) as determined by Mastersizer NS300. [Figure 8B] FIG. 8B is a photograph of ABX-atezolizumab nanoparticles from FIG. 8A. [Figure 9A]Figure 9A shows flow cytometry of ABX-atezolizumab nanoparticles (AA130) competing with labeled anti-PD-L1 antibody for binding to the PD-L1 positive human melanoma cell line, C8161. C8161 cells were pretreated with an isotype control antibody (Figure 9A) and then labeled with fluorescently labeled anti-PD-L1 antibody. [Figure 9B] Figure 9B shows flow cytometry of ABX-atezolizumab nanoparticles (AA130) competing with labeled anti-PD-L1 antibody for binding to the PD-L1 positive human melanoma cell line, C8161. C8161 cells were pretreated with no treatment (Figure 9B) and then labeled with fluorescently labeled anti-PD-L1 antibody. [Figure 9C] Figure 9C shows flow cytometry of ABX-atezolizumab nanoparticles (AA130) competing with labeled anti-PD-L1 antibody for binding to the PD-L1 positive human melanoma cell line, C8161. C8161 cells were pretreated with Abraxane® (Figure 9C) and then labeled with fluorescently labeled anti-PD-L1 antibody. [Figure 9D] Figure 9D shows flow cytometry of ABX-atezolizumab nanoparticles (AA130) competing with labeled anti-PD-L1 antibody for binding to the PD-L1 positive human melanoma cell line, C8161. C8161 cells were pretreated with atezolizumab (Figure 9D) and then labeled with fluorescently labeled anti-PD-L1 antibody. [Figure 9E] Figure 9E shows flow cytometry of ABX-atezolizumab nanoparticles (AA130) competing with labeled anti-PD-L1 antibody for binding to the PD-L1 positive human melanoma cell line, C8161. C8161 cells were pretreated with AA130 (Figure 9E) and then labeled with fluorescently labeled anti-PD-L1 antibody. [Figure 10] FIG. 10 shows the dose-dependent toxicity of ABX (solid line) and AA130 (dashed line) against C8161 cells. [Figure 11A]Figure 11A shows the time course of tumor volume in mice injected with 2x106 PD-L1 positive C8161 melanoma tumor cells and then treated once with 100ul IV tail vein injection of saline (Figure 11A). Tumor growth was monitored three times a week. Tumor size was calculated by the formula: (length x width2) / 2. [Figure 11B] Figure 11B shows the time course of tumor volume in mice injected with 2x106 PD-L1 positive C8161 melanoma tumor cells and then treated once with atezolizumab alone (18 mg / kg; Figure 11B). Tumor growth was monitored three times a week. Tumor size was calculated by the following formula: (length x width2) / 2. [Figure 11C] Figure 11C shows the time course of tumor volume in mice injected with 2x106 PD-L1 positive C8161 melanoma tumor cells and then treated once with ABX alone (45mg / kg; Figure 11C). Tumor growth was monitored three times a week. Tumor size was calculated by the formula: (length x width2) / 2. [Figure 11D] Figure 11D shows the time course of tumor volume in mice injected with 2x106 PD-L1 positive C8161 melanoma tumor cells and then treated once with AA130 (18 mg / kg atezolizumab and 45 mg / kg ABX; Figure 11D). Tumor growth was monitored three times a week. Tumor size was calculated by the following formula: (length x width2) / 2. [Figure 12] Figure 12 shows the survival of mice from the experiment shown in Figures 11A-11D. Kaplan Meier curves were generated using Graph Pad software. Median survival for each group was 14, 13, 16, and 21.5 days for saline, atezolizumab, Abraxane, and AA130, respectively. The difference in survival between AA130 and all other groups was significant with p values ​​of 0.0008 for saline, 0.0015 for atezolizumab, and 0.0113 for ABX. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] After reading this specification, it will be apparent to those skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, not all of the various embodiments of the present invention will be described herein. It will be understood that the embodiments presented herein are presented by way of example only, and not by way of limitation. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present invention described below.

[0078] Before the present invention is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or their own uses, which may, of course, vary. It is also to be understood that the terms used herein are only to describe specific embodiments and are not intended to be limiting.

[0079] The detailed description of the present invention has been divided into various sections for the convenience of the reader only, and disclosure appearing in any section may be combined with sections appearing in other sections. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of the invention.

[0080] definition

[0081] 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. In this specification and in the claims that follow, reference will be made to certain terms that shall be defined to have the following meanings.

[0082] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0083] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur.

[0084] When used before numerical designations including ranges, e.g., temperature, time, amounts, concentrations, and the like, the word "about" means to denote (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. It indicates an approximation that can vary. Preferably, when used in relation to a dosage, the term "about" means that the dosage can vary by + / - 10%. For example, "about 400 to about 800 binding agent" means that the dosage can vary by + / - 10%. This indicates that the outer surface of the nanoparticles contains an amount of binder of 360 to 880 particles.

[0085] "Comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. When used to define compositions and methods, "consisting essentially of" is intended to mean excluding other elements that are of essential importance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect one or more of the basic and novel characteristics of the claimed invention. "Consisting of" means excluding more than trace amounts of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0086] As used herein, the term "nanoparticle" or "nanoparticle complex" refers to a nanoparticle having a diameter of 5 mm or less. The term "particles" refers to particles having at least one dimension that is less than 1 mm. In a preferred embodiment, For administration, e.g., intravenous, the nanoparticles have at least one dimension that is less than 1 micron. For direct administration, the nanoparticles can be larger. Even larger particles are expressly contemplated by the present invention.

[0087] In a population of particles, the size of each particle is distributed evenly. Therefore, the particle size for the population can be expressed by the average value and also by percentile. D50 is the particle size below which 50% of particles fall. 10% of particles are smaller than the D10 value, and 90% of particles are smaller than D90. In case of uncertainty, the "average" size is equivalent to D50. Thus, for example, AB160 and AR160 refer to nanoparticles with an average size of 160 nanometers (nm).

[0088] The term "nanoparticle" may also include discrete multimers of smaller unit nanoparticles. For example, a 320 nm particle contains a dimer of a nanoparticle of unit 160 nm. Therefore, for a 160 nm nanoparticle, For example, multimers may have wavelengths of approximately 320 nm, 480 nm, 640 nm, 800 nm, 960 nm, 1120 nm, etc.

[0089] As used herein, the term "carrier protein" refers to a protein that functions to transport a binding agent and / or a therapeutic agent. The binding agents of the present disclosure can reversibly bind to a carrier protein. Examples of carrier proteins are described in more detail below.

[0090] As used herein, the term "core" refers to the central or interior portion of a nanoparticle, which may consist of a carrier protein, a carrier protein and a therapeutic agent, or other agent or combination of agents. In some embodiments, the binding agent portion may be associated with the core (e.g., may be non-covalently bound to the core).

[0091] As used herein, the term "therapeutic agent" refers to an agent that is therapeutically useful, e.g., for the treatment, amelioration, or attenuation of a disease state, physiological condition, symptom, or etiological factor, or for the assessment or diagnosis thereof. The therapeutic agent can be a chemotherapeutic agent, e.g., a mitotic inhibitor, a topoisomerase inhibitor, a steroid, an antitumor antibiotic, an antimetabolite, an alkylating agent, an enzyme, a proteasome inhibitor, or any combination thereof.

[0092] As used herein, the terms "binding agent," "specific binding agent," or "binding agent that specifically binds to" refer to an agent that binds to a target antigen and does not significantly bind to unrelated compounds. Examples of binding agents that can be effectively used in the disclosed methods include lectins, proteins, and antibodies, such as monoclonal antibodies, such as humanized monoclonal antibodies, chimeric antibodies, or polyclonal antibodies, or antigen-binding fragments thereof, as well as antibodies that bind to a target antigen. Examples of suitable binding agents include, but are not limited to, aptamers, fusion proteins, and aptamers. In one embodiment, the binding agent is an exogenous antibody. An exogenous antibody is an antibody that is not naturally produced in a particular mammal, such as a human, by the mammalian immune system.

[0093] As used herein, the terms "antibody" or "antibodies" refer to an antibody or antibodies that are Immunoglobulin molecules and immunologically active parts of immunoglobulin molecules (i.e., those that respond to antigens) The term also refers to antibodies that are composed of two heavy immunoglobulin chains and two light immunoglobulin chains, as well as full-length antibodies and portions thereof. Various forms include, for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv. , scFv, single domain antibodies (dAbs), diabodies, multispecific antibodies, dual specific antibodies, anti-idiotypic antibodies, bispecific antibodies, their functionally active epitope-binding fragments, bifunctional hybrids Lid antibodies, e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)), and single chain (e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988)), which are incorporated herein by reference. (See generally, Hood et al., Immunology, Benjamin, NY, 2nd ed.). (1984); Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference). The antibody can be of any type (e.g., IgG, IgA, IgM, IgE, or IgD). Preferably, the antibody is an IgG. The antibody is The antibody may be human, non-human (e.g., mouse, goat, or any other animal), fully human, humanized, or chimeric. An "antibody" or "antibodies" refers to any of the antibodies disclosed herein. As used herein, a biosimilar includes one or more of any of the following biosimilars: A biopharmaceutical product that is deemed equivalent in quality, safety, and effectiveness to a reference product marketed by the innovator company (Section 351(i) of the Public Health Service Act (42 U.S.C. 262(i)).

[0094] The term "dissociation constant," also referred to as "Kd," refers to the degree to which a particular substance dissociates between its individual components (e.g., t The term "protein carrier, antibody, and optional therapeutic agent" refers to the amount that represents the degree to which the antibody is separated into a single molecule.

[0095] As used herein, the terms "lyophilized", "freeze-drying" and the like refer to a method in which the material to be dried (e.g., nanoparticles) is first frozen, and then the ice or frozen solvent is removed by sublimation in a vacuum environment. Additives are optionally included in the pre-lyophilized formulation to enhance the stability of the lyophilized product during storage. In some embodiments, the nanoparticles are pre-freezed by freezing the lyophilized components (e.g., nanoparticles) prior to use as a therapeutic agent. The nucleic acid sequence may be formed from a nucleic acid sequence comprising a nucleic acid sequence of a nucleic acid molecule, ... In the method, the carrier protein, binding agent, such as an antibody, and any therapeutic agent are first combined with nanoparticles and then lyophilized. The lyophilized sample may further comprise additional additives.

[0096] The term "bulking agent" includes agents that provide structure to the lyophilized product. Common examples used for bulking agents include mannitol, glycine, lactose and sucrose. In addition to providing a pharmacopoeial cake, bulking agents can also provide useful qualities in terms of modifying collapse temperature, providing freeze-thaw protection, and enhancing protein stability over long-term storage. These agents can also serve as tonicity adjusters. In some embodiments, the lyophilized composition described herein includes a bulking agent. In some embodiments, the lyophilized composition does not include a bulking agent.

[0097] The term "buffer" includes agents that maintain the pH of the solution within an acceptable range prior to lyophilization, and includes, but is not limited to, succinate (sodium or potassium), histidine, phosphate (sodium or potassium), tris(hydroxymethyl)aminomethane, diethanolamine, citrate (sodium or potassium), and the like. The buffer of the present invention may contain, for example, 0.1 to 1.5% ammonium nitrate ... and 0.1 to 1.5% ammonium nitrate. has a pH of about 6.0. Examples of buffers that will control the pH in this range are succinate (e.g., sodium succinate), gluconate, histidine, citrate, and other organic acids. Contains a buffer.

[0098] The term "cryoprotectant" generally includes agents that provide stability to proteins against freeze-induced stress, presumably by being preferentially excluded from the protein surface. They may also provide protection during primary and secondary drying and long-term product storage. Examples are polymers, such as dextran and polyethylene glycol; sugars, such as sucrose, glucose, trehalose, and lactose; surfactants, such as polysorbates; and amino acids, such as glycine, arginine, and serine.

[0099] The term "cryoprotectant" includes agents that provide stability to proteins during the drying or "dehydration" steps (primary and secondary drying cycles), presumably by providing an amorphous glassy matrix and by binding to the protein through hydrogen bonds, replacing water molecules that are removed during the drying process. This helps to maintain protein conformation, minimize protein degradation during the lyophilization cycle, and improve long-term product. Examples include polyols, or sugars, such as sucrose and trehalose.

[0100] The term "pharmaceutical formulation" refers to a formulation that is in a form that allows the active ingredient to become effective and that does not contain additional ingredients that are toxic to the subject to which it will be administered.

[0101] A "pharmacologically acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a subject mammal to provide an effective amount of the active ingredient employed.

[0102] "Reconstitution time" is the time required to rehydrate the lyophilized formulation into solution.

[0103] A "stable" formulation is one in which the protein therein essentially retains its physical and / or chemical stability and / or biological activity upon storage. For example, various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability is measured by the degree of stability at a selected temperature. , can be measured over a selected period of time.

[0104] As used herein, the term "epitope" refers to the portion of an antigen that is recognized by a binding agent, such as an antibody. Epitopes include, but are not limited to, short amino acid sequences or peptides (optionally glycosylated or otherwise modified) that allow for specific interaction with a protein (e.g., an antibody) or ligand. For example, an epitope can be the portion of a molecule to which the antigen-binding site of a binding agent is attached.

[0105] The terms "treat" or "treatment" refer to the treatment of a disease or disorder (e.g., (ii) ameliorating the disease or disorder, i.e., causing the disease or disorder to regress; (iii) slowing the progression of the disease or disorder; and / or (iv) alleviating one or more symptoms of the disease or disorder. This includes inhibiting, alleviating or slowing the progression. In some embodiments, "treating" or "treatment" refers to killing cancer cells.

[0106] The term "kill" in the context of cancer treatment is intended to include any type of manipulation that will result in the death of at least a portion of the cancer cell or population of cancer cells.

[0107] The term "aptamer" refers to a nucleic acid molecule capable of binding to a target molecule, such as a polypeptide. For example, the aptamer of the present invention can specifically bind to PD-L1, PD-L2, PD-1, or CTLA-4. The generation of antibodies with specific binding specificities and the therapeutic use of aptamers are well established in the art, see, for example, U.S. Pat. Nos. 5,475,096, 5,270,163, 5,582,981, 5,840,867, 6,011,020, 6,051,698, 6,147,204, 6,180,348, and 6,699,843. , as well as the therapeutic effectiveness of Macugen® (Eyetech, New York) for treating age-related macular degeneration, each of which is incorporated herein by reference in its entirety.

[0108] The terms "oligomer" or "oligomeric" or "oligomerized" as used herein refer to an oligomer made up of two or more monomers.

[0109] Fusion proteins are bioengineered polypeptides that combine one peptide (e.g., the fragment crystallizable (Fc) domain of an antibody) with another biologically active agent, such as a protein domain, peptide, or nucleic acid or peptide aptamer, to generate a molecule with desirable structure-function characteristics and important therapeutic potential. The gamma immunoglobulin (IgG) isotype has been shown to possess favorable characteristics, such as recruitment and augmentation of effector functions. Due to their limited plasma half-lives, they are often used as the basis for generating Fc-fusion proteins. Given the range of aptamers (both peptide and nucleic acid) that can be used as fusion partners, fusion proteins have numerous biological and pharmaceutical applications.

[0110] As used herein, the term "sequentially" refers to the administration of two or more actions in any order. In some embodiments, the treatments are administered more than 48 hours apart. In some embodiments, the treatments are administered within about 48 hours of each other, within about 36 hours of each other, within about 24 hours of each other, within about 12 hours of each other, within about 10 hours of each other, within about 8 hours of each other, or within about 48 hours of each other. within about 6 hours of each other, within about 4 hours of each other, within about 2 hours of each other, or within about 1 hour of each other.

[0111] As used herein, the term "concurrently" refers to two or more treatments being administered at substantially the same time in any order.

[0112] As used herein, the term "PD-1" is also known as CD279. Referred to as programmed cell death protein-1, it is expressed on the surface of activated T cells, B cells, and myeloid cells.

[0113] As used herein, the term "PD-L1" refers to a polypeptide that is also known as B7-H1 or CD274. The term "programmed death ligand 1," which is commonly used, is a ligand for PD-1 that is typically expressed on the surface of dendritic cells or macrophages.

[0114] As used herein, the term "PD-L2" refers to a polypeptide also known as B7-DC or CD273. The term "programmed cell death ligand-2" is a ligand for PD-1 that is typically expressed on the surface of dendritic cells or macrophages.

[0115] As used herein, the term "biosimilar" or "biosimilar" can also mean "follow-on biologic" or "subsequent biologic." Also known as a "sequential entry biologic," it is designated by regulatory authorities as A biologic product is a substantially identical copy of a product approved for use in a clinical trial.

[0116] As used herein, the terms "synergy" or "synergistic effect" or "synergistically effective amount" or "synergistic effect" refer to the synergistic effect produced by the administration of at least two agents. It refers to a therapeutic effect that is greater than additive, which is the therapeutic effect of administering one of the agents without administering the other agent. For example, the therapeutic effect of the nanoparticle composition is increased when administered sequentially or simultaneously with a binding agent to produce a synergistic effect, provided that the increase is greater than the additive effect of the binding agent and the nanoparticle composition when administered alone. The term "synergistic therapeutic amount" typically refers to a substandard therapeutic amount of one or both therapeutic agents, meaning that a lower amount is required for the desired therapeutic efficacy than if the therapeutic agents were used alone. A synergistic therapeutic amount also includes cases where one therapeutic agent is given at a standard therapeutic dose and another therapeutic agent is administered at a substandard therapeutic dose.

[0117] As used herein, the term "therapeutically effective amount" or "therapeutic efficacy" of a nanoparticle composition or binding agent refers to a level of the nanoparticle composition or binding agent at which at least the physiological effects of a disease or disorder are ameliorated. A therapeutically effective amount is a dose of one or more tablets, capsules, or other medicinal agents. It may be given in one or more doses using a pharmaceutical unit. The amount of the nanoparticle composition or binding agent to be administered will vary depending on the nanoparticle composition or binding agent, the disorder and its severity, and the general health, age, sex, weight, and tolerance to drugs of the subject to be treated, but can be routinely determined by one of skill in the art. In some embodiments, the term "therapeutically effective amount" refers to a synergistically effective amount or a synergistically therapeutic amount.

[0118] Additionally, some terms used herein are more specifically defined below.

[0119] overview

[0120] The present invention is based in part on the surprising discovery that nanoparticles comprising a carrier protein, a binding agent, such as an antibody, aptamer or fusion protein, having a PD-L1 or PD-L2 binding domain, and a therapeutic agent, optionally lyophilized, provide targeted therapy to tumors while minimizing toxicity to the patient. Thus, the nanoparticles described herein are a significant improvement over conventional ADCs.

[0121] The present invention is further based, in part, on the synergism of immune checkpoint inhibitor immunotherapeutics (e.g., PD-1 immunotherapeutics and / or CTLA-4 immunotherapeutics) with nanoparticles. Without being bound by theory, binding of PD-L1 or PD-L2 by the binding agents (e.g., antibodies) described herein enhances T-cell proliferation and activation. The nanoparticles appear to deplete or reduce the amount of PD-L1 or PD-L2 available to bind PD-1 on T cells, thereby enhancing the therapeutic efficacy of PD-1-based immunotherapeutics. Administration of the nanoparticles alone or in combination with PD-1 immunotherapeutics reduces the number of T cells that are not subject to PD-1 pathway-mediated inhibition. and may restore a patient's immune response to cancers that express PD-L1 or PD-L2.

[0122] For conventional ADCs to be effective, the linker must not dissociate in the systemic circulation but must remain active at the tumor site. It is important that the carboxylate is stable enough to allow sufficient drug release from the carboxylate (Alley, SC, et al. (2008) Bioconjug Chem 19:759-765). This is an important consideration in developing effective drug conjugates. (Julien, DC, et al. (2011) MAbs 3:467-478; Alley, SC, et al. (2008) Bioconjug Chem 19:759-765); An attractive feature of the nanoparticles described herein is that no biochemical linkers are required.

[0123] Another drawback of current ADCs is that their ability to achieve higher drug penetration into tumors has not been demonstrated in human tumors. Early testing of ADCs in mouse models has demonstrated that they can be used to treat tumors with antibodies. It has been suggested that tumor targeting results in high concentrations of active agent in the tumor (Deguchi, T. et al. (1986) Cancer Res 46:3751-3755); however, this does not correlate to treatment of human disease, likely because human tumors are much more heterogeneous in permeability than mouse tumors (Jain, RK, et al. (2010) Nat Rev Clin Oncol 7 :653-664). Nanoparticle size is also important for extravasation from the vasculature into tumors. In a mouse study using a human colon adenocarcinoma xenograft model, vascular pores were permeable to liposomes up to 400 nm (Yuan, F., et al. (1995) Cancer Res 55:3752-3756). Another study of tumor pore size and permeability showed that regressing and cranial tumors were more sensitive to particles less than 200 nm. demonstrated that the nanoimmunoconjugates were permeable to IgG and IgG antibodies, both characteristics dependent on the location and growth state of the tumor (Hobbs, SK, et al. (1998) Proc Natl Acad Sci USA 95:4607-4612). The nanoimmunoconjugates (nanoparticles) described herein are large intact nanoparticles less than 200 nm in size. This problem is overcome by the fact that the large conjugates are partially dissociated into smaller functional units that can easily penetrate tumor tissue in the systemic circulation. Moreover, a smaller toxic payload is released once the conjugate reaches the tumor site. and only the toxic moiety, and not the entire conjugate, needs to be taken up by the tumor cells.

[0124] The advent of antibody (i.e., AVASTIN®) coated albumin nanoparticles containing a therapeutic agent (i.e., Abraxane®) provides a new paradigm for the directed delivery of two or more therapeutic agents to a predetermined site in vivo. See International Publication Nos. WO 2012 / 154861 ​​and WO 2014 / 055415, each of which is incorporated herein by reference in its entirety.

[0125] When compositions of albumin and a binding agent, such as an antibody, are mixed together in an aqueous solution at specific concentrations and ratios, the binding agents useful in the present invention spontaneously self-assemble within and onto the albumin to form nanoparticles with multiple copies (up to 500 or more) of the binding agent. Without being limited to any theory, it is believed that a binding agent (e.g., an antibody) is capable of binding to one or more of the amino acids of the binding agent. via a ubiquitin binding motif and one or more antibody binding motifs on the carrier protein It is believed that the motif non-covalently binds to a carrier protein (e.g., albumin) via a nucleotide sequence. Examples of such motifs can be found in International Patent Application No. PCT / US17 / 45643, which is incorporated herein by reference in its entirety.

[0126] Although protein compositions containing a single source protein are generally stored in lyophilized form where they exhibit significant shelf life, such lyophilized compositions do not contain self-assembled nanoparticles of two different proteins held together by hydrophobic-hydrophobic interactions. Moreover, nanoparticle configurations in which the majority of the binding moieties of the binding agent are exposed on the surface of the nanoparticle are apt to be susceptible to migration or reorganization by conditions that would otherwise be considered benign. For example, during lyophilization, the ionic charges on the protein are dehydrated, thereby exposing the underlying charges. The exposed charges allow charge-charge interactions between the two proteins, which can change the binding affinity of each protein for the other. In addition, the concentration of the nanoparticles increases significantly as the solvent (e.g., water) is removed. Such increased concentration of nanoparticles can lead to irreversible oligomerization. Oligomerization is a known property of proteins that reduces the biological properties of the oligomers compared to the monomeric form and sometimes increases the size of the particles to more than 1 micron.

[0127] On the other hand, a stable nanoparticle composition is required to have a shelf life of at least 3 months, and a shelf life of more than 6 or 9 months is required for preferred clinical and / or commercial use. Such a stable composition must be readily available for intravenous injection, must retain its self-assembled morphology upon intravenous injection in order to target the nanoparticles to the desired site in vivo, must have a maximum size of less than 1 micron to avoid any ischemic events when delivered into the bloodstream, and finally, must be compatible with the aqueous solutions used for injection. It must be compatible with the composition.

[0128] compound

[0129] As will be apparent to one of skill in the art upon reading this disclosure, the present disclosure provides a composition of nanoparticles comprising a carrier protein, a binding agent, and, optionally, at least one therapeutic agent, the composition comprising The composition is optionally lyophilized.

[0130] In some embodiments, the carrier protein is albumin, gelatin, elastin (including topoelastin) or elastin-derived polypeptides (e.g., alpha-elastin and elastin-like polypeptides (ELPs)), gliadin, leucine, erythrocyte selenite, erythrocyte sarcoma, and the like). Glycine max, zein, soy protein (e.g., soy protein isolate (SPI) isolates), milk proteins (e.g., β-lactoglobulin (BLG) and casein), or whey proteins (e.g., whey protein concentrates (WPC) and whey protein isolates (WPI)). In a preferred embodiment, the carrier protein is albumin. In a preferred embodiment, the albumin is egg white (ovalbumin), bovine serum albumin (BSA), or the like. In an even more preferred embodiment, In some embodiments, the carrier protein is human serum albumin (HAS). In some embodiments, the carrier protein is a recombinant protein, such as recombinant human serum albumin. In some embodiments, the carrier protein is a generally regarded as safe (GRAS) excipient approved by the U.S. Food and Drug Administration (FDA).

[0131] In some embodiments, the binding agent is an antibody.

[0132] In some embodiments, the anti-PD-1 antibody comprises nivolumab, pembrolizumab, pidilizumab, PDR001, or a derivative thereof. The antibody is atezolizumab, avelumab, durvalumab or BMS936559 (MDX1105). In some aspects, the binding agent of the CTLA-4 immunotherapeutic agent is an anti-CTLA-4 antibody. In one embodiment, the anti-CTLA-4 antibody is ipilimumab.

[0133] In some aspects, the first binding agent and / or the second binding agent is a fusion protein. In one embodiment, the fusion protein is AMP-224 (PD-L2 IgG2a fusion protein; Amplimmune / GlaxoSmith Klein); AMP-514 (MEDI0680) (PD-L2 fusion protein; Amplimmune / GlaxoSmith Klein), or a derivative thereof. AMP-224 and AMP-514 target PD-1.

[0134] In some embodiments, the antibody is substantially a monolayer of antibody on all or a portion of the surface of the nanoparticle.

[0135] Table 1 provides a non-limiting list of antibodies.

[0136] [Table 1]

[0137] In some embodiments, the at least one therapeutic agent is abiraterone, bendamus The therapeutic agent is selected from the group consisting of rifabutin, bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib, oxaliplatin, paclitaxel, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine and cyclophosphamide. Preferably, the therapeutic agent is paclitaxel. Additional therapeutic agents are known, for example, those listed in International Publication WO2017 / 031368, the entire contents of which are incorporated herein by reference.

[0138] It is to be understood that the therapeutic agent may be located inside the nanoparticle, on the outer surface of the nanoparticle, or both. The nanoparticles may contain more than one therapeutic agent, e.g., two therapeutic agents, three therapeutic agents, or more than one therapeutic agent. , four therapeutic agents, five therapeutic agents, or more. Additionally, the nanoparticles can contain the same or different therapeutic agents on the inside and outside of the nanoparticle.

[0139] In one aspect, the nanoparticle comprises at least 100 binding agents non-covalently attached to the surface of the nanoparticle. In one aspect, the nanoparticle comprises at least 200 binding agents attached thereto. In one aspect, the nanoparticle comprises at least 300 binding agents non-covalently attached to the surface of the nanoparticle. The nanoparticles comprise at least 400 binding agents non-covalently attached to the surface of the nanoparticles. In one aspect, the nanoparticle comprises at least 500 binding agents non-covalently attached to the surface of the nanoparticle. It comprises at least 600 binding agents bound thereto.

[0140] In one aspect, the nanoparticle comprises about 100 to about 1000 binding agents non-covalently attached to the surface of the nanoparticle. In one aspect, the nanoparticles include about 200 to about 1000 binders. The particle comprises about 300 to about 1000 binding agents non-covalently attached to the surface of the particle. The nanoparticles comprise about 400 to about 1000 binding agents non-covalently attached to the surface of the nanoparticles. In one aspect, the nanoparticle comprises about 500 to about 1000 binding agents non-covalently attached to the surface of the nanoparticle. In one aspect, the nanoparticles include about 600 to about 1000 binders. The nanoparticle comprises about 200 to about 800 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises about 300 to about 800 binding agents non-covalently bound to the surface of the nanoparticle. In a preferred embodiment, the nanoparticle comprises about 400 to about 800 binding agents non-covalently bound to the surface of the nanoparticle. Approximately 800 binders are included. Possible values ​​are any value within any of the recited ranges, including the endpoints. Any value or subrange may be included.

[0141] In one aspect, the average particle size in the nanoparticle composition is less than about 1 μm. In one aspect, the average particle size in the nanoparticle composition is from about 90 nm to about 1 μm. In one aspect, the average particle size in the nanoparticle composition is from about 90 nm to about 900 nm. In one aspect, the average particle size in the nanoparticle composition is less than about 90 nm. In one aspect, the average particle size in the nanoparticle composition is about In one aspect, the average particle size in the nanoparticle composition is about 90 nm to about 700 nm. In one aspect, the average particle size of the nanoparticle composition is about 90 nm to about 600 nm. The particle size is about 90 nm to about 500 nm. The average particle size is about 90 nm to about 400 nm. In one aspect, the nanoparticle composition has an average particle size of about 90 nm to about 300 nm. The average particle size in the composition is about 90 nm to about 200 nm. The average particle size in the nanoparticle composition is about 100 nm to about 180 nm. In particularly preferred embodiments, the average particle size in the nanoparticle composition is about 130 nm or about 160 nm. Contemplated values ​​include any value, subrange or range within any of the recited ranges, including the endpoints. In one embodiment, the nanoparticle size is measured using a Mastersizer 2000. In one embodiment, the nanoparticle size is determined using a Malvern Nanosight.

[0142] In one aspect, the nanoparticle composition is formulated for intravenous injection. To avoid ischemic events, the nanoparticle composition formulated for intravenous injection comprises nanoparticles having an average particle size of less than about 1 μm.

[0143] In one aspect, the average particle size in the nanoparticle composition is greater than about 1 μm. In one aspect, the average particle size in the nanoparticle composition is about 1 μm to about 5 μm. In one aspect, the average particle size in the nanoparticle composition is about 1 μm to about 4 μm. In one aspect, the average particle size in the nanoparticle composition is about 1 μm to about 3 μm. In one aspect, the average particle size in the nanoparticle composition is about 1 μm to about 2 μm. In one aspect, the average particle size in the nanoparticle composition is about 1 μm to about 1.5 μm. Contemplated values ​​include any value, subrange or range within any of the recited ranges, including the endpoints.

[0144] In one aspect, the nanoparticle composition is formulated for direct injection into a tumor. Direct injection includes injection into or near the tumor site, perfusion into the tumor, and the like. When formulated for direct injection into a tumor, the nanoparticles can comprise any average particle size. Without being bound by theory, it is believed that larger particles (e.g., greater than 500 nm, greater than 1 μm, etc.) are more effective at injecting into tumors. It is believed that larger particles are more likely to become immobilized within the tumor, thereby providing a beneficial effect. Larger particles can accumulate in tumors or specific organs. For example, a drug called "TheraSphere" (in clinical use in liver cancer) is used to nourish tumors in the liver. See the 20-60 micron glass particles used for injection into the hepatic artery that supplies the tumor. Thus, for intravenous administration, particles smaller than 1 μm are typically used. Particles larger than 1 μm are more typically placed directly into a tumor ("direct injection") or administered into an artery that supplies the tumor site.

[0145] In one aspect, less than about 0.01% of the nanoparticles in the composition have a particle size greater than 200 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm, greater than 600 nm, greater than 700 nm, or greater than 800 nm. In one aspect, less than about 0.001% of the nanoparticles in the composition have a particle size greater than 200 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm, greater than 600 nm, greater than 700 nm, or greater than 800 nm. In a preferred embodiment, In a more preferred embodiment, less than about 0.01% of the nanoparticles in the composition have a particle size greater than 800 nm. In a more preferred embodiment, less than about 0.001% of the nanoparticles in the composition have a particle size greater than 800 nm. do.

[0146] In a preferred aspect, the sizes and size ranges recited herein relate to particle sizes of reconstituted lyophilized nanoparticle compositions, i.e., lyophilized nanoparticles that have a particle size or average particle size within the ranges recited herein after being resuspended in an aqueous solution (e.g., water, other pharma- ceutically acceptable excipients, buffers, etc.).

[0147] In one aspect, at least about 50%, 60%, 70%, 80%, 90%, 95%, 96% of the nanoparticles , 97%, 98%, 99%, 99.5% or 99.9% as single nanoparticles in the reconstituted composition. That is, less than about 50%, less than about 40%, less than about 30%, etc. of the nanoparticles are dimerized or is polymerized (oligomerized).

[0148] In some embodiments, the nanoparticles in the composition have a dimerization number of less than 20%, a dimerization number of less than 10%, and preferably a dimerization number of less than 5%.

[0149] In some embodiments, the size of the nanoparticles can be controlled by adjusting the amount (e.g., ratio) of carrier protein to binder. The size and size distribution of the nanoparticles are also important. The nanoparticles of the present invention may behave differently according to their size. At large sizes, aggregation may block blood vessels. Thus, aggregation of nanoparticles may affect the performance and safety of the composition. On the other hand, larger particles may be more therapeutic under certain conditions (e.g., when not administered intravenously).

[0150] In one aspect, the nanoparticle composition comprises at least one additional therapeutic agent. In one embodiment, the at least one additional therapeutic agent is non-adhesive on the outer surface of the nanoparticle. In one embodiment, the at least one additional therapeutic agent is covalently attached to the nanoparticle. In one embodiment, the at least one additional The therapeutic agent is selected from abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine, and cyclophosphamide. In one embodiment, the at least one additional therapeutic agent is an anti-cancer binding agent, e.g. Anti-cancer antibodies. Additional anti-cancer antibodies are known, for example, those listed in International Publication WO2017 / 031368, the entirety of which is incorporated herein by reference.

[0151] Method for producing nanoparticles

[0152] In some aspects, the invention relates to methods of making the nanoparticle compositions described herein.

[0153] In one aspect, the nanoparticles of the nanoparticle composition are carrier protein or carrier protein therapeutic agent particles in a ratio of about 10:1 to about 10:30 of the binder and the carrier protein particle. The therapeutic agent is formed by contacting the carrier protein particles with a ratio of about 10:2 to about 10:25. In a preferred embodiment, the ratio is about 10:2 to about 1:1. In a preferred embodiment, the ratio is about 10:2 to about 10:6. In a particularly preferred embodiment, the ratio is about 10:4. Contemplated ratios include any value, subrange or range within any of the recited ranges, including the endpoints.

[0154] In one embodiment, the amount of solution or other liquid medium used to form the nanoparticles is particularly important. The nanoparticles are formed by binding the carrier protein (or In overly dilute solutions of the therapeutic agent and antibody, no chromatin is formed. In overly concentrated solutions, This will result in unstructured aggregates. In some embodiments, the amount of solution used (e.g., sterile water, saline, phosphate buffered saline) is about 0.5 mL of solution. In some embodiments, the amount of carrier protein is about 1 mg / mL to about 100 mg / mL. In some embodiments, the amount of binder is about 1 mg / mL to about 20 mL of solution. For example, in some embodiments, the ratio of carrier protein:binding agent:solution is approximately 9 mg of carrier protein (e.g., 1 mL) in 1 mL of solution (e.g., saline). For example, albumin) to 4 mg of binding agent, such as an antibody (e.g., BEV). The amount of therapeutic agent (e.g., taxol) can also be added to the carrier protein. For example, 1 mg of taxol can be added to 9 mg of carrier protein (10 mg of carrier protein) in 1 mL of solution. A standard IV bag can be used to add 4 mg of carrier protein / therapeutic agent (therapeutic agent) and 4 mg of binding agent, such as antibody, Fc fusion molecule or aptamer. For example, with approximately 1 liter of solution and a typical IV bag, it would be necessary to use 1000 times the amount of carrier protein / carrier protein therapeutic agent and antibody compared to the amount used in 1 mL. Therefore, the nanoparticles of the present invention cannot be formed in a standard IV bag. Moreover, when the components are added to a standard IV bag in the therapeutic amount of the present invention, the components do not self-assemble to form nanoparticles.

[0155] In one embodiment, the carrier protein or carrier protein therapeutic particle is contacted with the binder in a solution having a pH of about 4 to about 8. In one embodiment, the carrier protein or carrier protein therapeutic particle is contacted with the binder in a solution having a pH of about 4 to about 8. In one embodiment, the carrier protein or carrier protein-therapeutic agent particles are contacted with the binding agent in a solution having a pH of about 5. In an embodiment, the carrier protein or carrier protein therapeutic particle is in a solution having a pH of about 6. The protein therapeutic particles are contacted with the binder in a solution having a pH of about 7. The carrier protein or carrier protein therapeutic agent particles are maintained at a pH of about 8. In a preferred embodiment, the carrier protein or carrier protein therapeutic particle is contacted with the binder in a solution having a pH of about 5 to about 7.

[0156] In one embodiment, the carrier protein particles or carrier protein therapeutic particles are heated to a temperature of about 5° C. to about 60° C., or any range, subrange or endpoint therein. In a preferred embodiment, the carrier protein particles or carrier protein therapeutic agent particles are contacted with the binder at a temperature of about 23°C to about 60°C.

[0157] Without being bound by theory, the stability of the nanoparticles within the nanoparticle composition is determined, at least in part, by the temperature and / or pH at which the nanoparticles are formed, as well as the components in the solution (i.e., The binding activity is believed to depend on the concentration of the carrier protein, the binding agent, and, optionally, the therapeutic agent. In one embodiment, the K d is approximately 1x10 -11 M ~ approx. 2x10 -5 M. One In an embodiment, the K d is approximately 1x10 -11 M ~ approx. 2x10 -8 M. In one embodiment In the above, the K d is approximately 1x10 -11 M ~ approx. 7x10 -9 M. In a preferred embodiment The K of the nanoparticles d is approximately 1x10 -11 M ~ approx. 3x10 -8 M. Possible values ​​are the columns containing the end points. Including any value, subrange or range within any of the recited ranges.

[0158] Freeze drying

[0159] Lyophilization, or freeze-drying, removes water from a composition. In this method, the material to be dried is first frozen, and then the water or frozen solvent is removed by sublimation in a vacuum environment. Additives can be included in the pre-lyophilized formulation to enhance stability during the lyophilization process and / or to improve the stability of the lyophilized product during storage (Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawa et al., Pharm. Res. 8(3):285-291 (1991)).

[0160] While proteins can be lyophilized, the process of lyophilization and reconstitution can affect the properties of the protein. Because proteins are larger and more complex (i.e., have multiple functional groups in addition to complex three-dimensional structures) than traditional organic and inorganic drugs, the formulation of such proteins poses special problems. For a protein to remain biologically active, the formulation must protect the protein's multiple functional groups from degradation while at the same time maintaining the conformational integrity of at least the core sequence of the protein's amino acids. Protein degradation pathways can include chemical instability (i.e., any process that leads to the modification of the protein by bond formation or cleavage and results in a new chemical entity) or physical instability (i.e., changes in the higher-order structure of the protein). Chemical instability can result from deamidation, racemization, hydrolysis, oxidation, beta-elimination, or disulfide exchange. Physical instability can occur, for example, by denaturation, aggregation, precipitation, or adsorption. The three most common protein degradation pathways are protein aggregation, deamidation, and oxidation. (Cleland et al., Critical Reviews in Therapeutic Drug Carrier Systems 10(4) :307-377 (1993)).

[0161] The lyophilized compositions of the present invention are prepared by standard freeze-drying techniques in the presence or absence of stabilizers, buffers, etc. Surprisingly, these conditions do not alter the relatively fragile structure of the nanoparticles. Moreover, at best, these nanoparticles retain their size distribution upon freeze-drying, and more importantly, can be reconstituted for in vivo administration (e.g., intravenous delivery) in substantially the same form and proportions as if they were freshly made.

[0162] formulation

[0163] In one aspect, the nanoparticle composition is formulated for systemic delivery, eg, intravenous administration.

[0164] In one aspect, the nanoparticle composition is formulated for direct injection into a tumor. Direct injection includes injection into or near the tumor site, or perfusion into the tumor. Because the nanoparticle composition is not administered systemically, nanoparticle compositions formulated for direct injection into a tumor can include any average particle size. Without being bound by theory, it is believed that larger particles (e.g., greater than 500 nm, greater than 1 μm, etc.) are more likely to be immobilized within the tumor, thereby resulting in increased tumor mass. It is believed that this provides what are believed to be favorable beneficial effects.

[0165] In another aspect, a composition comprising a compound provided herein, and at least one Pharmaceutically acceptable excipients are provided herein.

[0166] In general, the compounds provided herein can be formulated for administration to a patient by any of the accepted modes of administration. A variety of formulations and drug delivery systems are available in the art. See, for example, Gennaro, AR, ed. (1995) Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co.

[0167] Generally, the nanoparticles provided herein will be administered as a pharmaceutical composition by any one of the following routes: orally, systemically (e.g., transdermally, intranasally, or via suppository). by intravenous) or parenteral (e.g., intramuscular, intravenous or subcutaneous) administration.

[0168] The compositions generally comprise the composition of the present invention in combination with at least one pharma- ceutically acceptable excipient. The composition is made up of nanoparticles of the claimed compound. Acceptable additives are non-toxic, aid in administration, and do not adversely affect the therapeutic efficacy of the claimed compounds. Such additives may be any solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous additives generally available to one skilled in the art.

[0169] Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, and the like. Liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol, and various oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols. Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th Edition, 1990).

[0170] The compositions of the present invention may, if desired, be packaged in a pack containing one or more unit dosage forms containing the active ingredient. The nanoparticles may be presented in a pack or dispenser device. Such a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass and rubber stopper, such as a rubber stopper in a vial. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising the nanoparticles of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0171] Treatment

[0172] The nanoparticle compositions described herein are useful for treating cancer cells and / or tumors in a mammal having a cancer or tumor that expresses PD-L1 and / or PD-L2. In a preferred embodiment, the mammal is a human (i.e., a human patient). Preferably, the lyophilized nanoparticle compositions are reconstituted (suspended in an aqueous excipient) prior to administration.

[0173] In one aspect, there is provided a method for treating a cancer cell, comprising contacting the cell with an effective amount of a nanoparticle described herein and an immunotherapeutic agent (e.g., PD-1 or CTLA-4) to treat the cancer cell, including, but not limited to, reducing proliferation, killing the cell, preventing metastasis of the cell, etc.

[0174] As used herein, "immune therapy" refers to a combination of The terms "immunotherapy," "single immunotherapy," or "multiple immunotherapies" generally refer to a combination of immunotherapy methods that induce, enhance, or suppress an immune response. In some cases, an immunotherapy or immunotherapies elicit or activate or amplify an immune response (also known as "activated immunity"). Cancer immunotherapy can either stimulate or suppress the immune response (also known as "suppressive immunotherapy"). For example, cancer immune therapy or cancer immunotherapy involves the suppression of the immune response against tumor or cancer cells. As will be appreciated by those skilled in the art, immune therapy or immunotherapy can utilize a variety of approaches or mechanisms, including, but not limited to: antibodies, antigens; immunoresponsive cells, such as lymphocytes, macrophages, dendritic cells, other antigen presenting cells, natural killer cells (NK cells, e.g., NK-92), T cells (e.g., helper T cells, cytotoxic T lymphocytes (CTLs)); and the like); therapies involving immunomodulators, including but not limited to interleukins (e.g., IL-2, IL-7, IL-12, etc.), cytokines (e.g., interferon, G-CSF, imiquimod, etc.), chemokines (e.g., CCL3, CCL26, CXCL7, etc.), immunomodulatory imid drugs, etc. Immunotherapy or immunotherapy can be administered through the use of one type of antibody or multiple types of antibodies. Immunotherapy or immunotherapy approaches can also be administered alone or in combination with other therapeutic agents or mechanisms, such as chemotherapeutic agents, for example to enhance the immune response against tumors.

[0175] In one aspect, a method for treating a tumor in a patient in need thereof is provided, comprising administering to the patient a therapeutically effective amount of a nanoparticle composition and an immunotherapeutic agent as described herein to treat the tumor.In one embodiment, the size of the tumor is reduced.In one embodiment, the size of the tumor does not increase (i.e., progress) for at least a period of time during and / or after treatment.

[0176] In one embodiment, the nanoparticle composition is administered intravenously. In one embodiment, the nanoparticle composition is administered directly to the tumor. In one embodiment, the nanoparticle composition is administered by direct injection or perfusion into the tumor.

[0177] In one embodiment, the immunotherapeutic agent is administered intravenously. In one embodiment, the immunotherapeutic agent is administered directly to the tumor. In one embodiment, the immunotherapeutic agent is administered by direct injection or perfusion into the tumor.

[0178] In one aspect, a method is provided for treating a patient suffering from a cancer that expresses PD-L1 or PD-L2, the method comprising administering to the patient a nanoparticle composition comprising nanoparticles, each of the nanoparticles comprising a carrier protein, a binding agent having a PD-L1 or PD-L2 binding moiety, and optionally at least one therapeutic agent, the nanoparticles capable of binding to PD-L1 or PD-L2. In some embodiments, the method further comprises administering to the patient a PD-1 immunotherapeutic agent. In one embodiment, the PD-1 immunotherapeutic agent comprises administering a second binding agent capable of binding to PD-1.

[0179] In another aspect, the invention provides a method for increasing the therapeutic efficacy of an immunotherapeutic treatment in a patient suffering from a cancer that expresses PD-L1 or PD-L2, comprising administering to the patient a therapeutically effective amount of a nanoparticle composition described herein. In some embodiments, the method further comprises administering to the patient a PD-1 immunotherapeutic agent. In one embodiment, the PD-1 immunotherapeutic agent comprises administering a second binding agent capable of binding to PD-1. .

[0180] In one embodiment, the method comprises: a) administering the nanoparticle composition once a week for three weeks; b) ceasing administration of the nanoparticle composition for one week; and c) optionally repeating steps a) and b) as necessary to treat the tumor. Includes.

[0181] In one aspect, the PD-1 immunotherapeutic agent is administered simultaneously with the nanoparticle composition. In one aspect, the PD-1 immunotherapeutic agent is administered before the nanoparticle composition. In one aspect, the PD-1 immunotherapeutic agent is administered after the nanoparticle composition. In one aspect, the PD-1 immunotherapeutic agent is administered according to a label approved by a regulatory body (e.g., the FDA). is administered.

[0182] In some aspects, each of the nanoparticles of the nanoparticle composition comprises about 400 to about 800 binding agents.

[0183] In some aspects, the first binding agent (the binding agent in the nanoparticle) is an aptamer. In some aspects, the second binding agent of the PD-1 immunotherapeutic is an aptamer.

[0184] In some aspects, the first binding agent (the binding agent in the nanoparticle) is an antibody. In an aspect, the second binding agent of the PD-1 immunotherapeutic agent is an antibody.

[0185] In some aspects, the anti-PD-1 antibody comprises nivolumab, pembrolizumab, pidilizumab, PDR001, or a derivative thereof. is atezolizumab, avelumab, durvalumab, or BMS936559 (MDX1105), or a derivative thereof. In some aspects, the binding agent of the CTLA-4 immunotherapeutic agent is an anti-CTLA-4 antibody. In one embodiment, the anti-CTLA-4 antibody is ipilimumab, or a derivative thereof.

[0186] In some aspects, the first binding agent and / or the second binding agent is a fusion protein. In one embodiment, the fusion protein is AMP-224 (PD-L2 IgG2a fusion protein; Amplimmune / GlaxoSmith Klein); AMP-514 (MEDI0680) (PD-L2 fusion protein; Amplimmune / GlaxoSmith Klein), or a derivative thereof. In some aspects, the The nanoparticle composition is lyophilized.

[0187] In some aspects, the second binding agent of the PD-1 immunotherapeutic agent is a free binding agent, and the A free binding agent is not complexed to or incorporated on and / or within the nanoparticle composition.

[0188] In some aspects, the PD-1 immunotherapeutic agent is a PD-1 immunotherapeutic nanoparticle composition that includes a second binding agent complexed to the nanoparticle composition or incorporated on and / or within the nanoparticle composition. wherein the immunotherapeutic nanoparticle composition comprises a carrier protein and the second binding agent. In some aspects, the immunotherapeutic nanoparticle composition is lyophilized.

[0189] In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is an antibody. In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is an anti-PD-1 antibody. In some aspects, the anti-PD-1 antibody comprises nivolumab, pembrolizumab, pidilizumab, PDR001, or a derivative thereof.

[0190] In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is an aptamer. In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is a PD-1 aptamer. It is.

[0191] In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition is a fusion protein. In some aspects, the second binding agent of the immunotherapeutic nanoparticle composition binds PD-1. In one embodiment, the fusion protein is AMP-224 (PD-L2 IgG2a fusion protein; Amplimmune / GlaxoSmith Klein); AMP-514 (MEDI0680) (PD-L2 fusion protein; Amplimmune / GlaxoSmith Klein), or a derivative thereof. It is.

[0192] In some aspects, the nanoparticle composition and the PD-1 immunotherapeutic agent are administered sequentially. In some aspects, the nanoparticle composition is administered prior to administration of the PD-1 immunotherapeutic agent. In some aspects, the PD-1 immunotherapeutic agent is administered prior to administration of the nanoparticle composition. In some aspects, the nanoparticle composition and the PD-1 immunotherapeutic agent are administered simultaneously.

[0193] In some embodiments, the present invention relates to a method for increasing the therapeutic efficacy of an immunotherapeutic treatment in a patient suffering from a cancer that expresses PD-L1 or PD-L2, the method comprising administering to the patient a therapeutically effective amount of a nanoparticle composition described herein and a PD-1 immunotherapeutic agent comprising a second binding agent. wherein when the binding agent of the nanoparticle composition is capable of binding to PD-L1 and / or PD-L2, the second binding agent of the immunotherapeutic agent is capable of binding to PD-1, and If the binding agent of the nanoparticle composition is capable of binding to PD-1, the second The binding agent may bind to PD-L1 and / or PD-L2.

[0194] In some embodiments, the invention relates to a method for treating a patient suffering from a cancer that expresses PD-L1 or PD-L2, comprising administering to the patient a therapeutically effective amount of a nanoparticle composition described herein and an immunotherapeutic agent comprising a second binding agent, wherein the nanoparticles The binding agent of the particle composition can bind to PD-L1, PD-L2 or PD-1, and the second binding agent of the immunotherapeutic agent can bind to PD-L1, PD-L2 or PD-1.

[0195] In one embodiment, a method for treating a patient suffering from a cancer that expresses PD-L1 or PD-L2 comprises administering to the patient a nanoparticle composition comprising nanoparticles and a PD-1 immunotherapeutic agent. Each of the nanoparticles of the nanoparticle composition comprises (a) a carrier protein, (b) a binding agent having a PD-L1 or PD-L2 binding moiety, and (c) optionally, at least one therapeutic agent. When reconstituted in an aqueous solution, the binding agent of the nanoparticles is capable of binding to PD-L1 or PD-L2.

[0196] In one embodiment, there is provided a method for increasing the therapeutic efficacy of an immunotherapeutic treatment in a patient suffering from a cancer that expresses PD-L1 or PD-L2, comprising administering to the patient (a) a naphthalene derivative as described herein; and (b) a therapeutically effective amount of a PD-1 immunotherapeutic agent. In one embodiment, the nanoparticle composition is lyophilized and, upon reconstitution with an aqueous solution, the binding agent of the nanoparticles is capable of binding to PD-L1 or PD-L2.

[0197] In some aspects, the amount of nanoparticles and the amount of the second binding agent are determined relative to one another. It is determined.

[0198] In some aspects, the synergistic binding of the nanoparticle composition and the second binding agent to the immunotherapeutic agent is The ratio of amounts effective to increase the therapeutic efficacy of the immunotherapeutic agent such that the efficacy of the immunotherapeutic agent is substantially greater than its administration alone. In one aspect, the ratio of amounts of the nanoparticle composition to the second binding agent is about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6 , 1:7, 1:8, 1:9 or 1:10 to about 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11 , 1:12, 1:15 or about 1:20.

[0199] In another aspect, a method for increasing the therapeutic efficacy of an immunotherapeutic agent comprises administering to a patient a nanoparticle as described herein, the ratio of the amount of the nanoparticle composition and the amount of the second binding agent being in the range of about 1:1 to about 1:10, and / or the administration of such a combination. The synergistic therapeutic effectiveness of the second binding agent is at least about 5% greater than the therapeutic effectiveness of a single dose of the second binding agent. or about 10% or about 15% or about 20% or about 25% or about 30% or about 35% or about 40% or about 45% or about 50% or about 55% or about 60% or about 65% or about 70% or about 80% or about 90% or about 100% larger In another aspect, the synergistic therapeutic efficacy of administration of such a combination can be achieved by administering a single dose of either the nanoparticle composition or the second binding agent. At least about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% greater than the therapeutic efficacy.

[0200] In one embodiment, the second binding agent of the immunotherapeutic agent comprises about 60 mg / mL for about 30 minutes to about 60 minutes (eg, atezolizumab).

[0201] In one embodiment, the second binding agent of the immunotherapeutic agent is administered over a period of about 60 minutes. For intravenous delivery, include about 1.0 mg / kg to about 3.0 mg / kg (e.g., nivolumab).

[0202] In one embodiment, the second binding agent of the immunotherapeutic agent is administered over a period of about 30 minutes. Includes about 2 mg / kg for intravenous delivery (e.g., pembrolizumab).

[0203] In some embodiments, the present invention relates to a method for increasing the therapeutic efficacy of an immunotherapeutic treatment in a patient suffering from a cancer that expresses PD-L1 or PD-L2, the method comprising administering to the patient a therapeutically effective amount of a nanoparticle composition as described herein above, and an immunotherapy comprising a second binding agent. and administering a therapeutic agent to the subject, wherein the binding agent of the nanoparticle is capable of binding to PD-L1 and / or PD-L2, and the second binding agent of the immunotherapeutic agent is capable of binding to PD-1. and if the binding agent of the nanoparticle is capable of binding to PD-1, the second binding agent of the immunotherapeutic agent is capable of binding to PD-L1 and / or PD-L2.

[0204] In some embodiments, the present invention relates to a method for treating a patient suffering from a cancer that expresses PD-L1 or PD-L2, comprising administering to the patient a therapeutically effective amount of a nanoparticle composition as described hereinabove, and an immunotherapy agent comprising a second binding agent, wherein the binding agent of the nanoparticle is capable of binding to PD-L1, PD-L2, and PD-1, and the second binding agent of the immunotherapeutic agent binds to the same PD-L1, PD-L2, and PD-1 as the binding agent of the nanoparticle. can be done.

[0205] In some aspects, the amount of nanoparticles is an effective amount of the nanoparticle composition. In some aspects, the amount of nanoparticles is an amount that is less than the effective amount of the nanoparticle composition when administered to the patient alone.

[0206] In some aspects, the second binding agent is in an effective amount. In some aspects, the second binding agent is in an amount that is less than the effective amount when administered to the patient alone.

[0207] In one embodiment, a therapeutically effective amount of the nanoparticles described herein is about 1 mg / m 2 ~about 200mg / m 2 Antibody, approximately 2mg / m 2 ~about 150mg / m 2 , about 5mg / m 2 ~about 100mg / m 2 , about 10mg / m 2 ~about 85mg / m 2 , about 15mg / m 2 ~about 75mg / m 2 , about 20mg / m 2 ~about 65mg / m 2 , about 25mg / m 2 ~about 55mg / m 2 , about 30mg / m 2 ~about 45mg / m 2 or about 35 mg / m 2 ~about 40mg / m 2 Other embodiments include antibodies of the In such a case, the therapeutically effective amount is about 20 mg / m 2 ~about 90mg / m 2 In one embodiment, the therapeutically effective amount is 30 mg / m 2 ~about 70mg / m 2 In one embodiment, the therapeutically effective amount of the nanoparticles described herein is about 50 mg / m 2 ~about 200mg / m 2 or a carrier protein and a therapeutic agent. In such a case, the therapeutically effective amount is about 75 mg / m 2 ~about 175mg / m 2 Carrier protein or includes a carrier protein and a therapeutic agent. Possible values ​​include any value, subrange, or range within any of the recited ranges, including the endpoints.

[0208] In one embodiment, the therapeutically effective amount of the nanoparticle composition is about 20 mg / m of binding agent, e.g., antibody, aptamer, or Fc fusion. 2 ~about 90mg / m 2 In a preferred embodiment, the therapeutically effective amount is 30 mg / m of a binding agent, e.g., an antibody, an aptamer, or an Fc fusion. 2 ~about 70mg / m 2 Possible values ​​include any value, subrange or range within any of the recited ranges, including the endpoints.

[0209] Cancers or tumors that can be treated by the compositions and methods described herein include biliary tract cancer; brain cancer, including glioblastoma and medulloblastoma; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer, gastric cancer; hematological tumors, including acute lymphocytic leukemia and myeloid leukemia; multiple myeloma; AIDS-related leukemia and adult T-cell leukemia-lymphoma; These include, but are not limited to, intradermal tumors, such as intraepithelial neoplasia including Bowen's disease and Paget's disease; liver cancer (hepatocellular carcinoma); lung cancer; lymphomas, such as Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; oral cancer, such as squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, such as leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma; testicular cancer, including germinal tumors (seminoma, non-seminoma [teratoma, choriocarcinoma]), stromal tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and kidney cancer, including adenocarcinoma and Wilms' tumor.

[0210] In general, the compounds of the present invention will be administered in a therapeutically effective amount by any of the accepted administration methods for agents serving similar purposes. The actual amount of the compounds of the present invention, i.e., the nanoparticles, will depend on many factors, such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors well known to those skilled in the art.

[0211] The effective amounts of such agents, as well as the most effective and convenient routes of administration and the most suitable formulations, can be readily determined by routine experimentation. A variety of formulations and drug delivery systems are available in the art. See, e.g., Gennaro, AR et al. (1995) Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co.

[0212] An effective amount or therapeutically effective amount or dose of an agent, such as a compound of the present invention, refers to the amount of the agent or compound that results in amelioration of symptoms or prolongation of survival in a subject. The toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Agents that exhibit a high therapeutic index are preferred.

[0213] An effective amount or therapeutically effective amount is that amount of a compound or pharmaceutical composition that elicits the biological or medical response of a tissue, system, animal or human that is being sought by a researcher, veterinarian, physician or other clinician. Dosage amounts can vary within this range depending on the dosage form used and / or the route of administration utilized. The exact formulation, route of administration, dosage and interval should be selected according to methods known in the art, taking into account the specifics of the subject's condition.

[0214] Dosage amount and interval may be adjusted individually to provide a plasma level of the active moiety that is sufficient to achieve the desired effect, i.e., the minimal effective concentration (MEC). The MEC will vary from compound to compound, but may be based on, for example, in vitro data. The dosage required to achieve the MEC can be estimated from individual studies and animal experiments. In cases of local administration or selective uptake, the effective local concentration of drug may not be related to plasma concentration.

[0215] Working Example

[0216] The present disclosure is illustrated using nanoparticles comprised of albumin-bound paclitaxel (i.e., Abraxane®) or cisplatin as a core, and an antibody that recognizes PD-L1 (e.g., atezolizumab). Those skilled in the art will appreciate that the preparation and use of the nanoparticles in the examples are for illustrative purposes only. It will be understood that this disclosure is not limited by this example only.

[0217] Any abbreviations used herein have their usual scientific meaning. All temperatures are in °C unless otherwise specified. As used herein, the following terms have the following meanings unless otherwise defined:

[0218] [Table 2]

[0219] Example 1: Antigen binding of lyophilized AR160

[0220] CD20-positive Daudi lymphoma cells were labeled with fluorescently tagged anti-human CD20 or an isotype-matched control in panels F and A, respectively, and analyzed by flow cytometry. In other panels, the Daudi cells were pretreated with ABX, AR160, AR160L (AR160 was lyophilized and resuspended in a solution suitable for injection) or Rituxan prior to CD20 labeling. Figure 1 demonstrates that CD20 binding was specifically blocked by AR particles and Rituxan, but not by ABX alone. These results support the conclusion that AR particles and Rituxan are the only effective means of CD20 binding. This suggests that IL-1 binds to its CD20 ligand on these cells and blocks the binding of fluorescent anti-CD20.

[0221] FIG. 2 is a histogram overlay of the data presented in FIG.

[0222] Figures 3A and 3B show the ABX agonist activity compared to freshly produced lyophilized AR (Figure 3A) and AT (Figure 3B). A single particle size is shown.

[0223] 4 shows the results of a Daudi proliferation assay comparing the toxicity of ABX and AR particles. The data demonstrate that lyophilized and non-lyophilized nanoparticles have essentially the same toxicity in the Daudi assay.

[0224] Example 2: Fluorescence analysis of tumor accumulation of AlexaFluor750-labeled nanoparticles

[0225] Mice were treated with either labeled Abraxane®, nonspecific antibody-coated labeled Abraxane® (AB IgG), or rituximab-coated laminar IgG. Patients were administered an intravenous (IV) injection of either 100 mg or 150 mg of Abraxane® (AR160) in a dose-equivalent dose. Regions of interest (ROIs) 2, 3, and 4 (Figure 5A) were selected based on the fluorescence threshold. The tumor accumulation was tracked by ROIs 1, 5, and 6 (FIG. 5A) serving as background references. Fluorescence was determined in the ROIs 24 hours after injection. FIG. 5B is a bar graph of the mean fluorescence per unit of tumor area in mice in all three treatment groups, determined to provide total tumor delivery. FIG. 5C is a bar graph of the mean fluorescence per unit of tumor area, normalized by the background ROI to give the percentage of drug-to-body delivered to the tumor. The data show that administration of AR160 nanoparticles significantly increased the efficacy of Abraxane® alone or nonspecific anti-inflammatory drugs. It is demonstrated that the antibody results in increased fluorescence compared to body coated Abraxane®.

[0226] Example 3: In vivo efficacy of ABX-rituximab nanoparticles with a size of 225 nm

[0227] To produce nanoparticles having a size of 225 nm, the particles were prepared according to International Publication WO 2017 / 031368, which is incorporated herein by reference in its entirety, with a ratio of BEV to Abraxane® of 4:5, i.e., 4 parts BEV and 5 parts Abraxane. This ratio produced nanoparticles (AB225) with a size of 225 nm. The survival rates of mice treated with a single dose of saline, BEV, ABX, AB160, and AB225, and with AB160 with BEV pretreatment, were assayed in animals as described in WO 2017 / 031368. Figure 6 shows the survival rates of mice treated with saline, BEV, ABX, AB160, and AB225, and with AB160 with BEV pretreatment. At 30 days post-treatment, the survival rates of mice treated with AB225 and with AB1605 with or without BEV pretreatment were significantly higher than those of mice treated with Abraxane. The survival rate of mice treated with BEV alone far exceeds that of mice treated with BEV alone.

[0228] Example 4: Preparation of Atezolizumab-Abraxane® Nanoparticles

[0229] Atezolizumab and Abraxane® (ABX) were co-incubated at room temperature for 30 minutes at concentrations of 4 mg / mL and 10 mg / mL, respectively, to form nanoparticles, AA130. Ta.

[0230] Whether atezolizumab and ABX can interact to form nanoparticle complexes To determine whether or not atezolizumab binds to the streptavidin probe, biolayer interferometry (BLItz) (Forte Bioscience) was performed using a streptavidin probe. Biotinylated atezolizumab at 100 μg / ml in 1x PBS was bound to the streptavidin probe. After washing unbound Atezolizumab from the probe, the antibody-bound probe was exposed to ABX at concentrations of 100, 500, and 1000 μg / mL in 1X PBS. The antibody probe exposed to PBS was used as background and the background was subtracted. The BLItz software was used to calculate the dissociation constant (Figure 7). Kd was 1.462x10 -9 It was determined that.

[0231] Example 5: Size Measurement of Atezolizumab-Abraxane® Nanoparticles

[0232] A Mastersizer NS300 was used to determine the particle size of atezolizumab-bound ABX compared to ABX alone. Nanosight used dynamic light scattering and Brownian motion to determine the particle size of atezolizumab-bound ABX compared to ABX alone. , calculate the particle size.

[0233] As described above, atezolizumab and ABX were incubated together to form nanoparticles. ,AA130, was formed. ABX was diluted 1:200, and atezolizumab-bound ABX was diluted 1:800. Diluted; three 30-second video clips were captured to determine particle size. The mean particle size of the atezolizumab-ABX nanoparticles was determined to be approximately 129 nm. ; the average size of ABX alone is approximately 90 nm.

[0234] Example 6: AA130 binds PD-L1

[0235] Flow cytometry was performed to compare the efficacy and safety of atezolizumab and atezolizumab against the ligand, PD-L1. The binding of Abraxane conjugated with IgE was investigated. The PD-L1 positive melanoma cell line, C8161, was used for this study. AA130 was prepared as described above, and aliquots of NaCl were The nanoparticles were spun at 6000 rpm for 10 min to remove any unbound atezolizumab. C8161 cells were stained with FITC-labeled isotype control and anti-human PD-L1 as negative and positive controls, respectively. The C8161 cells were incubated with ABX and atezolizumab alone and AA130 nanoparticles for 30 min. After incubation, the cells were labeled with FITC-labeled anti-human PD-L1 for 30 min and washed with FACS buffer (1xPBS + 0.5% BSA and 0.05% sodium azide). After washing, cells were analyzed by flow cytometer on Guava 8HT and data analysis was performed using Gauvasoft software (Millipore).

[0236] C8161 cells were pretreated with isotype control antibody (Figure 9A), no treatment (Figure 9B), Abraxane® (Figure 3C), atezolizumab (Figure 9D) or AA130 (Figure 9E) and then labeled with fluorescently labeled anti-PD-L1 antibody. Atezolizumab in the context of 130 nm particles retains its ability to bind to its ligand, PD-L1.

[0237] Example 7: Cytotoxicity of AA130

[0238] C8161 melanoma cells were exposed to ABX and AA130 overnight at paclitaxel concentrations ranging from 0 to 200 μg / mL to determine cytotoxicity. The cells were also treated with the thymidine analog EdU and The next day, the cells were harvested, fixed with 2% paraformaldehyde, and permeabilized with 1% saponin. After permeabilization, the cells were incubated with FITC-labeled anti-EdU antibody for 30 min to determine the percentage of cell proliferation. After washing, the cells were analyzed by flow cytometer on a Guava 8HT and data analysis was performed using Gauvasoft software (Millipore). The proliferation index was calculated by normalization to the no treatment positive control.

[0239] Figure 10 shows the dose-dependent toxicity of ABX (solid line) and AA130 (dashed line) against C8161 cells. AA130 has similar cytotoxicity to ABX alone.

[0240] Example 8: In vivo efficacy of AA130 nanoparticles

[0241] Athymic nude mice (Harlan Sprague Dawley) were cultured at 2x10 6 The tumors were injected with 100 μg of PD-L1 positive C8161 melanoma tumor cells. The tumors were approximately 600 mm 3 The mice were grown to 100 ng / mL and then treated with saline, atezolizumab alone (18 mg / kg), ABX alone (45 mg / kg), and AA130 (18 mg / kg atezolizumab and 45 mg / kg ABX). ABX) once via 100 μl IV tail vein injection (Figures 11A-11D). Tumor growth was monitored 3 times / week. Tumor size was calculated using the following formula: (length x width) 2 ) / 2.

[0242] Tumor growth curves (Figure 12) showed that AA130-treated tumors showed significantly increased tumor growth compared to saline and individual drugs alone. Kaplan-Meier curves show slowed tumor growth in mice. Graph Pad software was used to generate the median survival times for each group. The median survival times for each group were 14, 13, 16, and 21.5 days for saline, atezolizumab, ABX, and AA130, respectively. The difference in survival between AA130 and all other groups was significant with p-values ​​of 0.0008 for saline, 0.0015 for atezolizumab, and 0.0113 for Abraxane. The present invention provides, for example, the following items. (Item 1) 1. A method for treating a patient suffering from a cancer that expresses PD-L1 or PD-L2, comprising administering to the patient: (a) a nanoparticle composition comprising a plurality of nanoparticles, each of the nanoparticles being an albumin. a binding agent capable of binding to PD-L1 or PD-L2, and paclitaxel, wherein the nanoparticles are capable of binding to PD-L1 or PD-L2; and (b) PD-1 immunotherapy The above method, comprising administering (Item 2) 1. A method for increasing the therapeutic efficacy of an immunotherapeutic treatment in a patient suffering from a cancer that expresses PD-L1 or PD-L2, comprising administering to the patient (a) a therapeutically effective amount of a nanoparticle composition, and (b) a PD-1 immunotherapeutic agent, wherein the nanoparticle composition comprises one or more nanoparticles comprising albumin, a binding agent capable of binding to PD-L1 or PD-L2, and paclitaxel; The method, wherein the nanoparticles are capable of binding to PD-L1 or PD-L2. (Item 3) Item 1 or 2, wherein the PD-1 immunotherapeutic agent comprises a second binding agent capable of binding to PD-1. The method according to (Item 4) 4. The method of claim 3, wherein the second binding agent is a free binding agent. (Item 5) The second binding agent may comprise a carrier protein and the second binding agent and, optionally, a second therapeutic agent. The method of claim 3, wherein the agent is incorporated on and / or in the nanoparticles comprising the agent. (Item 6) 4. The method of claim 3, wherein the second binding agent is an anti-PD-1 antibody. (Item 7) 7. The method of item 6, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, PDR001, or a derivative thereof. (Item 8) 4. The method of claim 3, wherein the second binding agent is a fusion protein. (Item 9) 9. The method of claim 8, wherein the fusion protein is AMP-224 or AMP-514. (Item 10) 3. The method of item 1 or 2, wherein the binding agent capable of binding to PD-L1 or PD-L2 is an antibody. (Item 11) 11. The method of claim 10, wherein the antibody comprises atezolizumab, avelumab, durvalumab, BMS936559 (MDX1105), or a derivative thereof. (Item 12) The method according to any one of items 1 to 11, wherein the nanoparticle composition is lyophilized and the method further comprises reconstituting the nanoparticle composition in an aqueous solution prior to administration. 3. The method of claim 1 or 2, wherein the nanoparticle composition and the second binding agent are administered sequentially. (Item 14) 14. The method of claim 13, wherein the nanoparticle composition is administered prior to administration of the second binding agent. (Item 15) 14. The method of claim 13, wherein the second binding agent is administered prior to administration of the nanoparticle composition. (Item 16) 3. The method of claim 1 or 2, wherein the nanoparticle composition and the second binding agent are administered simultaneously. . (Item 17) 17. The method of any one of items 1 to 16, further comprising an additional therapeutic agent selected from abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine, or cyclophosphamide. (Item 18) a nanoparticle, such that the nanoparticle is capable of binding to PD-1, PD-L1 or PD-L2; Albumin, b. a binding agent capable of binding to PD-1, PD-L1 or PD-L2, and C. Paclitaxel The nanoparticles described above. (Item 19) 19. The nanoparticle according to item 18, wherein the binding agent is a fusion protein. (Item 20) 19. The nanoparticle according to item 18, wherein the binding agent is an antibody. (Item 21) 21. The nanoparticle according to item 20, wherein the antibody binds to PD-1 and is selected from nivolumab, pembrolizumab, pidilizumab, PDR001 or a derivative thereof. (Item 22) 21. The nanoparticle according to item 20, wherein the antibody binds to PD-L1 and is selected from atezolizumab, avelumab, durvalumab, BMS936559 (MDX1105) or a derivative thereof. (Item 23) 23. A nanoparticle composition comprising the nanoparticles according to any one of items 18 to 22. (Item 24) 24. The nanoparticle composition of item 23, which has been lyophilized and, upon reconstitution in an aqueous solution, the nanoparticles are capable of binding to PD-1, PD-L1 or PD-L2. (Item 25) and an additional therapeutic agent selected from abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine, or cyclophosphamide. 23. The nanoparticle according to any one of items 18 to 22, comprising: (Item 26) 24. A kit comprising the nanoparticle composition of item 23 and a PD-1 immunotherapeutic agent. (Item 27) 27. The method according to claim 26, wherein the PD-1 immunotherapeutic agent comprises a second binding agent capable of binding to PD-1. Kit included. (Item 28) 28. The kit of item 27, wherein the second binding agent is a free binding agent. (Item 29) 28. The kit of item 27, wherein the second binding agent is incorporated on and / or in nanoparticles comprising albumin and the second binding agent. (Item 30) 28. The kit of item 27, wherein the second binding agent is an anti-PD-1 antibody. (Item 31) 31. The kit of item 30, wherein the antibody comprises pidilizumab, PDR001, pembrolizumab or nivolumab, or a derivative thereof. (Item 32) 27. The kit of item 26, wherein the PD-1 immunotherapeutic agent is a fusion protein. (Item 33) 33. The kit according to item 32, wherein the fusion protein is AMP-224, AMP-514 or a derivative thereof. (Item 34) 27. The kit of item 26, wherein the nanoparticle composition is lyophilized.

Claims

[Claim 1] The invention as depicted in the drawings.

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