Antibody preloaded CD16+NK-92 cells as effective therapeutic products for tumor lysis

Abstract

The present invention relates to antibody preloaded CD16 + NK-92 cells as an effective therapeutic product for tumor lysis. Provided herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of high affinity natural killer (haNK) cells and a therapeutic antibody in the form of a combined formulation. Also provided herein are methods for treating cancer by using the pharmaceutical composition comprising the haNK cells and the therapeutic antibody.

Description

[0001] This application is a divisional application of the invention patent application entitled "Antibody Preloaded CD16+NK-92 Cells as Effective Therapeutic Products for Tumor Lysis" with international application PCT / US2020 / 043690, filed in China on July 27, 2020, and application number 202080054127.5. This application claims priority to our co-pending U.S. provisional patent application with serial number 62 / 879,111 filed on July 26, 2019, and the provisional patent application is incorporated herein by reference. Technical Field

[0002] The present disclosure relates to compositions, kits and methods for treating cancer, and in particular treating cancer with high affinity natural killer (hanK) cells preloaded with therapeutic antibodies. Background Art

[0003] The Background Description includes information that may be useful in understanding the present disclosure. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly cited is prior art.

[0004] All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0005] Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) through the reactivity of CD16 with the Fc region of human IgG1 antibodies. Several NK cell lines are known to have therapeutic effects on cancer patients, such as those with leukemias and lymphomas. NK cells can also be further engineered to enhance cancer cell killing - one such technology is high-affinity NK (haNK) cells, which are NK cells that incorporate high binding affinity receptors that bind to administered antibodies.

[0006] Combination therapy of NK cells and antibodies for treating cancer is known. PCT / US2018 / 032281 discloses the treatment of chordoma by co-administration of anti-EGFR antibodies and high-affinity NK cells (haNK). The present disclosure provides high-affinity variants of the antibody that non-covalently binds to the CD16 receptor, or administers the antibody before infusing the haNK cells to target chordoma cells for cytotoxic cell killing by haNK cells.

[0007] Most monoclonal antibodies in oncology are administered on a dosing schedule based on body size. This partially controls the variability of drug distribution between patients and eliminates both. However, dosing is a challenge in existing cell therapies such as PCT / US2018 / 032281, where a combination of engineered cells and therapeutic antibodies is administered according to different regimens. In addition, repeated boluses increase the cost of treatment.

[0008] Therefore, there is a need in the art for novel compositions and methods for controlling the viability and efficacy of cell therapeutics, and for the administration of engineered cells and therapeutic antibodies.Preferably, such combinations combine both components in one administration regimen. Summary of the invention

[0009] The inventors have discovered that it is possible to generate CD16 + Compositions, methods and apparatus for cryopreservation of NK-92 (haNK) cells Advantageously and unexpectedly, the compositions disclosed herein eliminate the increased treatment costs from repeated boluses and produce potent drug distribution in patients.

[0010] In one aspect of the present subject matter, the inventors contemplate a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of high affinity natural killer (haNK) cells and a therapeutic antibody as a combined preparation.

[0011] Preferably 5×10 5 cells / kg and 5×10 8 The envisioned haNK cells are administered at a dose of between 10 cells / kg, and it is further preferred that these haNK cells are NK92 derivatives and / or typically express recombinant IL2 intracellularly. In addition, it is generally preferred that the haNK cells are genetically engineered to have reduced expression of at least one inhibitory receptor and / or that the haNK cells are genetically engineered to express the CD16158V variant. In addition, the haNK cells can be irradiated with a radiation dose of at least 500 cGy prior to administration.

[0012] The antibody contemplated herein can be any therapeutic antibody. For example, the antibody can be an anti-VEGF, anti-HER2, anti-EGFR, anti-CTLA4, anti-CD20, anti-CD54, anti-CD33, anti-CD16 and / or anti-CD30 antibody. The antibody may also be selected from the group consisting of Atezolizumab, Ofatumumab, Ipilimumab, Ramucirumab, Olaratumab, Elotuzumab, Necitumumab, Daratumumab, Dinutuximab, Avelumab, Durvalumab, Trastuzumab, Alemtuzumab, Bevacizumab, Pertuzumab, Obinutuzumab, Rituximab and Cetuximab.

[0013] In some preferred embodiments, the haNK cells and the antibody are chemically conjugated, such as by click chemistry.

[0014] In addition, the pharmaceutical composition may further comprise a cryopreservation medium, such as CryoStor CS 10. Ideally, the cryopreservation medium is selected so that it facilitates the binding of antibodies to haNK cells.

[0015] Also disclosed herein is a method for preparing a composition by the following steps: (a) mixing haNK cells with therapeutic antibodies to prepare a combined preparation; (b) freezing the combined preparation; and (c) thawing the combined preparation. In some cases, the haNK cells are in a culture medium containing about 5% human albumin. Preferably, the haNK cells and therapeutic antibody mixtures are mixed with an equal volume of cryopreservation medium. The combined preparation of haNK cells and the therapeutic antibody can be frozen to a temperature of less than -80°C, or in some cases frozen to a temperature of less than -120°C. The frozen combined preparation is irradiated with a fixed dose of X-ray radiation to impair the proliferation capacity of the haNK cells. In some embodiments, after the thawing step, the combined preparation is incubated on ice or at room temperature.

[0016] In another aspect, disclosed herein is a method of treating a patient suffering from cancer, the method comprising administering to the patient a pharmaceutical composition comprising a combined preparation of a therapeutically effective amount of high-affinity natural killer (haNK) cells and a therapeutic antibody. The composition can be administered intravenously, intratumorally, or by infusion.

[0017] In some cases, the method may further include the step of administering another cancer treatment to the patient. The other cancer treatment may be immunotherapy, chemotherapy or radiotherapy. The immunotherapy includes administering a recombinant yeast or recombinant virus expressing a patient and tumor-specific neo-epitope. The chemotherapy may include administering at least one of aldoxorubicin, cyclophosphamide, irinotecan, gemcitabine, capecitabine, 5-FU, FOLFIRL FOLFOX and oxaliplatin. The other cancer treatment is administered alone, sequentially, co-administered simultaneously or before the composition of the haNK cells and antibodies as disclosed herein.

[0018] In another aspect of the subject matter of the present invention, a kit is disclosed herein, comprising a pharmaceutical composition, wherein the pharmaceutical composition comprises haNK cells, a therapeutic antibody, and optionally a cryopreservation medium. In this kit, the composition can be packaged in a bag or vial suitable for storage at less than -85°C. Finally, the kit can further comprise information in electronic or paper form, comprising instructions for using the pharmaceutical composition.

[0019] Various objects, features, aspects and advantages will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings, in which like numerals represent like components. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Depicted are exemplary ADCC activities of haNK cells that were thawed, incubated on ice for 30 min, and tested (with and without antibody preloading).

[0021] Figure 2A Depicted are exemplary ADCC activities of antibody preloaded haNK cells that were thawed and tested without post-thaw incubation.

[0022] Figure 2B Depicted are exemplary ADCC activities of haNK cells preloaded with antibodies that were thawed, incubated on ice for 30 min, and tested.

[0023] Figure 2C Depicted are exemplary ADCC activities of haNK cells preloaded with antibodies that were thawed, incubated on ice or at room temperature for 30 min, and tested.

[0024] Figure 3A Depicted are exemplary ADCC activities of haNK cells pre-loaded with the tested antibodies after thawing on ice for 30 min with or without a wash step.

[0025] Figure 3B Depicted are exemplary ADCC activities of haNK cells thawed on ice for 30 min followed by a wash step or without a wash step and tested in the presence of exogenous Rituxan.

[0026] Figure 4A Depicted are exemplary ADCC activities of haNK cells preloaded with the tested antibodies prepared in different media, incubated on ice for 30 min, followed by a wash step or without a wash step.

[0027] Figure 4B Depicted are exemplary ADCC activities of haNK cells thawed on ice for 30 min followed by a wash step or without a wash step and tested in the presence of exogenous Rituxan.

[0028] Figure 4C Exemplary results of ADCC activity depending on the culture medium and washing steps are depicted. DETAILED DESCRIPTION

[0029] Cell-based therapies in cancer treatment increasingly utilize various antibodies. Current treatment regimens include separate infusions of NK cells and monoclonal antibodies. However, this leads to problems with dosing, and repeated pushes that increase treatment costs. The inventors have found a solution to this problem by preparing a pharmaceutical composition of haNK cells and antibodies in a combined formulation. For example, the inventors prepare this composition in the following manner: therapeutic monoclonal antibodies such as Rituxan and haNK cells are premixed in 5% human albumin, then mixed with CS-10 (1:1) and cryopreserved using a controlled rate freezer, and stored in a vapor phase LN2 freezer until ready to thaw and use as an infusion product. This premixed combination formulation has been shown to be effective and potent in killing cancer cells. Comparable killing activity is observed if antibodies are exogenously added to cryopreserved haNK cells in an assay.

[0030] The antibody can be injected side by side with the NK cells. However, in this case, the two are independent drugs, and the amount of antibody used as a drug is higher. On the other hand, the advantage of the currently disclosed premixed combination preparation is that the lower concentration of antibody together with the cells is effective in killing. In addition, the haNK cells with antibodies as a combination will be used as a ready-made drug as a single infusion rather than multiple injections.

[0031] Most monoclonal antibodies in oncology are administered in a dosing schedule based on body size. This partially controls drug distribution between patients and eliminates variability in both. However, dosing is a challenge in cell therapeutics where a combination of engineered cells and therapeutic antibodies are administered according to different regimens. In addition, repeated boluses increase the cost of treatment. Therefore, combining the two components in one dosing regimen is a desired solution.

[0032] The inventors have now discovered a method for making CD16 + Various compositions, methods and kits in which NK-92 (haNK) cells are preloaded with antibodies, such as therapeutic antibodies. It is contemplated that the compositions, methods and kits disclosed herein are novel and more effective methods for tumor lysis. Preferably, the composition comprising haNK cells and antibodies is in the form of a combined preparation, and the combined preparation is often stored frozen until ready for use.

[0033] NK cells express Fc receptors that can bind to the Fc portion of immunoglobulins, thereby initiating cell signaling within the NK cells. Once activated by antibodies bound to tumor targets through Fc receptors, NK cells are able to induce target lysis. This antibody-dependent cell-mediated cytotoxicity (ADCC) of target cells is used for cancer therapy. haNK cells are NK-92 cells engineered to express a high-affinity CD16 receptor (FcγRIIIA) to promote ADCC-mediated tumor cell lysis. The high-affinity CD16 receptor allows soluble IgG to dock. However, to date, the binding strength / affinity and the ability of soluble antibodies to induce NK activation have not been tested. NK-92 cells are well known in the art (see, e.g., Clin Cancer Res. 1998 November; 4(11): 2859-68) and are also commercially available from NantKwest, San Diego, CA.

[0034] The inventors have now unexpectedly discovered that the high affinity CD16 receptor on cryopreserved haNK cells can serve as a docking platform for soluble therapeutic antibodies and can induce ADCC when incubated with tumor target cells. Therefore, the inventive concept disclosed herein relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of high affinity natural killer (haNK) cells and a therapeutic antibody in the form of a combined preparation.

[0035] Preferably, the therapeutic antibodies contemplated herein are therapeutic antibodies that cause necrosis or apoptosis of cancer cells. In one embodiment, the therapeutic antibodies contemplated herein may be antibodies against TAAs (tumor associated antigens), antibodies against cancer-specific antigens, and antibodies against patient- and tumor-specific epitopes (neo-epitopes). Optionally or additionally, the therapeutic antibodies contemplated herein may be antibodies against antigens found in necrotic and apoptotic cells.

[0036] Non-limiting examples of antibodies contemplated herein include atezolizumab, ofatumumab, ipilimumab, ramucirumab, olatumumab, elotuzumab, nesitutumumab, daratumumab, denosumab, avelumab, durvalumab, trastuzumab, alemtuzumab, bevacizumab, pertuzumab, obinutuzumab, rituximab, and cetuximab and / or various therapeutic or diagnostic antibodies having an IgG Fc portion. For example, suitable antibodies include (a) trastuzumab (Herceptin), which targets HER2 (ErbB2) by ADCC and inhibiting HER2 signaling, and is approved for the treatment of HER2-positive breast cancer, HER2-positive gastric or gastroesophageal junction cancer; (b) bevacizumab (Avastin), which targets VEGF by inhibiting VEGF signaling, and is approved for the treatment of colorectal cancer, non-squamous non-small cell lung cancer, glioblastoma, or renal cell carcinoma; (c) cetuximab (Erbitux), which targets EGFR (ErbB1) by ADCC and inhibiting EGFR signaling, and is approved for the treatment of squamous cell carcinoma of the head and neck (SCCHN); (d) panitumumab (Vectibix), which targets EGFR (ErbB1) by inhibiting EGFR signaling, and is approved for the treatment of metastatic colorectal cancer; (e) ipilimumab (Yervoy), an antibody that targets CTLA-4 by inhibiting CTLA-4 signaling and is approved for the treatment of unresectable or metastatic melanoma; (f) rituximab (Rituxan), an antibody that targets CD20 by ADCC, direct induction of apoptosis, and CDC, and is approved for the treatment of CD20-positive B-cell non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL); (g) alemtuzumab (Campath), an antibody that targets CD52 by direct induction of apoptosis and CDC, and is approved for the treatment of B-cell CLL; (h) ofatumumab (Arzerra), an antibody that targets CD20 by ADCC and CDC, and is approved for the treatment of patients with CLL; (i) gemtuzumab ozogamicin (Gemtuzumab ozogamicin) (Mylotarg), an antibody that targets CD33 by delivering a toxic payload and is approved for the treatment of patients with CD33-positive acute myeloid leukemia; (1) 131I-Tositumomab (Bexxar), which targets CD20 through ADCC, direct induction of apoptosis, and delivery of the radioisotope iodine-131, and is approved for the treatment of patients with relapsed or refractory low-grade, follicular, or transformed NHL expressing the CD20 antigen. Those skilled in the art will recognize that any other therapeutic antibodies may also be used in the compositions disclosed herein to practice the inventive concept.

[0037] Preferably, the haNK cells as disclosed herein are NK-92 cell line derivatives. It is further preferred that the haNK cells express recombinant IL2. In addition, it is generally preferred that the haNK cells are genetically engineered to have reduced expression of at least one inhibitory receptor and / or that the haNK cells are genetically engineered to express the CD16 158V variant. In some desired embodiments, the haNK cells are genetically engineered to have reduced expression of at least one inhibitory receptor.

[0038] In addition, in other contemplated embodiments, NK cells are irradiated prior to infusion to prevent continued cell division. Although not limiting the subject matter of the present invention, cells are typically irradiated to eliminate cell division, but still allow rapid metabolic activity and NK cell function, especially cytotoxic cell killing. Therefore, a suitable radiation dose for NK cells is between 50 cGy and 2,000 cGy. In a preferred embodiment, haNK cells can be irradiated with a radiation dose of at least 500 cGy prior to administration.

[0039] In some embodiments, the antibody is an anti-CD20 antibody (such as rituximab, ofatumumab, and / or 131 As used herein, the term "MB311" contemplates a fully humanized monoclonal antibody that recognizes the tumor-associated antigen Lewis Y. This carbohydrate antigen is expressed on 60%-90% of all epithelial cancers, has only limited expression on normal tissues, and therefore represents an attractive target for cancer immunotherapy.

[0040] In some cases, the haNK cells and antibodies are chemically conjugated. Chemical conjugation can be performed by any method known to skilled chemists. A preferred method for chemical conjugation is the Huisgen 1,3-dipolar cycloaddition reaction ("click chemistry"), as disclosed in HC Kolb; MG Finn; KB Sharpless (2001). "Click Chemistry: Diverse Chemical Function from a Few Good Reactions". Angewandte Chemie [Applied Chemistry] International Edition. 40 (11): 2004-2021. As used herein, the term "click chemistry" therefore refers to such cycloaddition reactions and particularly to the cycloaddition of azides and alkynes - a reaction that can be performed in some embodiments under the catalysis of Cu (I) or under exposure to microwaves. Click reactions occur in one pot, are not disturbed by water, produce minimal and harmless byproducts, and are spring-loaded, characterized by high thermodynamic driving forces that drive them rapidly and irreversibly to high yields of single reaction products, and have high reaction specificity (in some cases, both regio- and stereo-specificity). These qualities make click reactions particularly well suited to the problem of separating and targeting molecules in complex biological environments.

[0041] The pharmaceutical composition disclosed herein may further include a cryopreservation medium. It is envisioned that the cryopreservation medium is a cell-specific optimized cryopreservation medium designed to prepare and preserve cells in very low temperature environments (-70°C to -120°C). In addition, it is envisioned that the cryopreservation medium provides a safe protective environment during the freezing, storage and thawing processes of cells and tissues, and provides enhanced cell viability and functionality while eliminating the need for serum, protein or high levels of cytotoxic agents. In a preferred embodiment, the cryopreservation medium is CryoStor CS10. In addition, the cryopreservation medium is selected so that it is conducive to the combination of antibodies with haNK cells.

[0042] In another aspect of the inventive subject matter, the inventors have discovered a method of treating a patient suffering from cancer, the method comprising administering to the patient a combined formulation of a therapeutically effective amount of high affinity natural killer (haNK) cells and a therapeutic antibody. All types of cancer cells can be treated by using the compositions and methods disclosed herein. Thus, the methods disclosed herein are suitable for treating carcinomas, sarcomas, myelomas, leukemias (liquid cancers or blood cancers), lymphomas (solid cancers), or mixed types of cancers (such as adenosquamous carcinomas, mixed mesodermal tumors, carcinosarcoma, and teratocarcinomas).

[0043] Preferably, the pharmaceutical composition and method disclosed herein can be formulated for delivery to the patient via any route of administration." route of administration " can refer to any route of administration known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral. Preferred routes of administration include suction, ocular administration, nasal instillation, parenteral administration, dermal administration, transdermal administration, buccal administration, rectal administration, sublingual administration, perilingual administration, nasal administration, topical administration or oral administration. " Parenteral " refers to the route of administration usually associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intravertebral, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal or transtracheal. Via parenteral route, the composition can be in the form of a solution or suspension for infusion or for injection. In some particularly preferred embodiments, the composition can also be directly injected locally into the tumor (intratumoral administration).

[0044] Optionally, the method for treating cancer as disclosed herein may include the step of administering another cancer treatment to the patient. Another cancer treatment includes immunotherapy, chemotherapy or radiotherapy. When immunotherapy is another cancer treatment, it includes administering a recombinant yeast or recombinant virus expressing a patient- and tumor-specific new epitope. Chemotherapy may include administering at least one of odoxorubicin, cyclophosphamide, irinotecan, gemcitabine, capecitabine, 5-FU (5-fluorouracil), FOLFIRI (folinic acid, fluorouracil and irinotecan), FOLFOX (oxaliplatin, fluorouracil and leucovorin) and oxaliplatin. Another cancer treatment may be administered alone, sequentially, co-administered simultaneously, or administered before a composition comprising haNK cells and therapeutic antibodies as disclosed herein.

[0045] The pharmaceutical compositions disclosed herein are preferably delivered in a therapeutically effective amount. The precise therapeutically effective amount is the amount at which the composition will produce the most effective results in terms of cancer treatment efficacy in a given subject. This amount will vary according to a variety of factors, including but not limited to the characteristics of haNK cells, antibodies, culture medium composition, etc., as well as the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug). Technicians in the clinical and pharmacological fields will be able to determine the therapeutically effective amount by routine experiments, such as by monitoring the subject's response to the administration of the drug compound and adjusting the dose accordingly. In a preferred embodiment, it is envisioned that the amount of the drug is administered at 5×10 5 cells / kg and 5×10 8 haNK cells were administered at a dose between 10 cells / kg.

[0046] The compositions disclosed herein can be prepared by a variety of techniques. In a preferred method, haNK cells are mixed with therapeutic antibodies to prepare a combined preparation. The combined preparation is then frozen and then thawed before use. Preferably, the haNK cells are in a culture medium containing about 10% albumin, or at least 8% albumin, or at least 6% albumin, or at least 5% albumin, or at least 3% albumin, or at least 1% albumin. The haNK cell and therapeutic antibody mixture is mixed with a cryopreservation medium. The ratio of the combined preparation to the cryopreservation medium can be at least 3:1, or at least 2:1, or at least 1:1, or at least 1:2, or at least 1:3.

[0047] The composition is frozen to a temperature between -50°C and -100°C. Once the cells have reached a temperature between -50°C and -100°C, the cells are even further cooled by placing them in liquid nitrogen, for example, to a temperature of less than -120°C. The combined preparation is frozen until it is ready for use, or in some cases, frozen for at least 1 month, or at least 2 weeks, or at least 1 week, or at least 5 days, or at least 3 days, or at least one day, or at least 12 hours, or at least 6 hours, or at least 2 hours, or at least 1 hour. In some preferred embodiments, the frozen combined preparation is irradiated with a fixed dose of X-ray radiation to impair the proliferation capacity of the haNK cells. After the thawing step, the combined preparation is incubated on ice or at room temperature. The thawing step can be in ice or at room temperature, or between 0°C and 100°C, or more preferably between 0°C and 40°C, or even more preferably at any other temperature between 0°C and 25°C. Once the composition has been thawed, it is incubated in ice or at room temperature (between 0°C and 25°C) for at least 5 hours, or more preferably at least 4 hours, or at least 3 hours, or at least 2 hours, or at least 1 hour, or at least 30 minutes, or at least 15 minutes, or at least 10 minutes.

[0048] In another aspect of the present disclosure, the present inventive concept relates to the preparation and use of a kit comprising a pharmaceutical composition of haNK cells and a therapeutic antibody. Optionally, the pharmaceutical composition in the kit may also comprise a cryopreservation medium. The kit can be used to practice the methods of the present invention for treating cancer or tumors. A kit is a collection of materials or components comprising at least one composition of the present invention as described throughout the present disclosure.

[0049] The exact nature of the components configured in the kit of the present invention depends on its intended use. For example, some embodiments are configured for the purpose of treating tumors and / or cancer. In this case, the antibody used is specific for cancer, such as trastuzumab for breast cancer. In addition, the composition may further include a pharmaceutically acceptable carrier that is conducive to the combination of trastuzumab and haNK cells.

[0050] In one embodiment, the kit is specifically configured for the purpose of treating a mammalian subject. In another embodiment, the kit is specifically configured for the purpose of treating a human subject. In a further embodiment, the kit is configured for veterinary applications for treating subjects such as, but not limited to, livestock, domestic animals, and laboratory animals.

[0051] Instructions for use may be included in the kit. "Instructions for use" typically include a clear statement describing the techniques employed in using the components of the kit to achieve the desired results (such as reducing or killing tumors). Optionally, the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, tying materials, or other useful accessories as will be readily recognized by those skilled in the art.

[0052] The materials or components assembled in the kit can be provided to the practitioner in any convenient and suitable manner to store them for their operability and effectiveness. For example, as contemplated herein, the components are more preferably provided at a freezing temperature typically less than -85°C. The components are typically contained in one or more suitable packaging materials. As used herein, the phrase "packaging material" refers to one or more physical structures for containing the contents of the kit (such as the compositions of the present invention, etc.). The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term "packaging" refers to a suitable solid matrix or material capable of containing individual kit components, such as glass, plastic, paper, foil, etc. The packaging material typically has an external label indicating the contents and / or purpose of the kit and / or its components.

[0053] Embodiments of the present disclosure are further described in the following examples. The examples are merely illustrative and do not in any way limit the scope of the claimed invention.

[0054] Examples

[0055] Example 1

[0056] In an exemplary embodiment, the inventors mixed haNK cells (commercially available from Nankvest, San Diego) in 5% albumin (human) with an anti-CD20 antibody (Rituxan). Subsequently, an equal volume (1:1) of CryoStor1 0 (CS1 0) was added, and the mixture was transferred to a cell freezing infusion bag or vial. HaNK cells without antibodies were also included in this study as a negative control. The filled infusion bags and vials were then cryopreserved to ≤-85°C using a controlled rate freezer. The cryopreserved product was then transferred to a liquid nitrogen vapor phase (≤-120°C) freezer for storage. The frozen product was irradiated with a fixed dose of X-ray radiation to impair the proliferation capacity of the haNK cells. After thawing, the cells were incubated with calcein-labeled Ramos (target) cells expressing CD20, and lysis was evaluated using a calcein release assay.

[0057] Efficient Ramos lysis was induced by preloading of haNK cells with Rituxan but not by haNK cells alone, as shown by the ADCC activity of post-thaw haNK cells. Figure 1 As shown in . Here, haNK cells were preloaded with 1 μg / mL of Rituxan antibody or not preloaded and then cryopreserved. The cryopreserved cells were then irradiated with 15Gy using an RS-2000 X-ray irradiator. The cells were thawed and incubated on ice for 30 minutes and added directly to the assay plate. The Ramos lysis percentage was evaluated using a standard calcein release assay.

[0058] In another example, the inventors studied the timing of ADCC after thawing. Interestingly, the inventors found that the post-thawing holding time of 10-30 minutes (or longer in some cases) on ice or at room temperature had a significant effect on the effectiveness of the ADCC function of haNK cells. For example, haNK cells were mixed with or without 1-2 μg / mL of Rituxan antibodies and cryopreserved. The cryopreserved cells were irradiated with 15Gy using an RS-2000 X-ray irradiator. Then thawed with different schemes as follows: Figure 2A Results are shown for cells that were thawed, washed, and tested without any post-thaw incubation time, and essentially no ADCC-based cell killing was observed. In contrast, when cells were thawed and incubated on ice ( Figure 2B ) or at room temperature (RT) or on ice ( Figure 2C ) for 30 minutes, much improved ADCC cytotoxicity was observed. This activity was also observed after a wash step designed to remove unbound rituximab. Again, Figures 2A-2C Ramos lysis % was assessed using a standard calcein release assay.

[0059] In yet another example, the inventors also evaluated the contribution of free antibodies in thawed haNK products. To this end, antibody preloaded cells were washed once after thawing by centrifugation at 336×g (1200 RPM) for 5 minutes before performing ADCC assays. Figure 3A-3B As indicated in Figure 2, haNK cells were mixed with or without 1 μg / mL of Rituxan antibody and cryopreserved. The cryopreserved cells were irradiated with 15 Gy using an RS-2000 X-ray irradiator. Figure 3A The results of post-thaw ADCC are depicted. Here, Rituxan pre-loaded haNK cells were washed or added directly to the assay plate without a wash step. For comparison, haNK cells (without antibody pre-loading) were washed or added directly to the assay plate containing Rituxan-bound Ramos cells after thawing, and the results are shown in Figure 3B As can be easily seen, when the cells were not preloaded with antibodies, the washing steps did not affect the cells themselves.

[0060] In still additional experiments, to evaluate the contribution of freezing medium components in Rituxan preloaded haNK products, cells were tested prior to freezing to induce ADCC. Fresh haNK cells suspended in complete growth medium (CGM) or albumin (human) were mixed with or without 1 μg / mL of Rituxan antibody. Figure 4A The activity of fresh product with and without a washing step is shown. Similarly, Figure 4B Post-thaw results are shown, where haNK cells were either washed or added directly to the assay plate. Figure 4C Shown is the % change in reduction of ADCC activity when cells are suspended in albumin (HA) versus complete growth medium. Interestingly, these results suggest that the type of medium (here: complete growth medium and human albumin medium) beneficially contributes to better binding of antibodies to haNK cells.

[0061] Example 2

[0062] In another example, the inventors developed a new technology based on the premise that tumor cells have abnormal carbohydrate glycosylation and these structures can be used to specifically target mAbs, namely Lewis system antigens. In this method, a mAb with increased FcR affinity is produced using a moss expression system. There are currently several technologies focused on preparing mAbs with modified FcRs that bind to NK cells with higher affinity, and suitable technologies for use herein are those that are safe and effective in humans.

[0063] Preferably, the mAb product is combined with a systemic CD-16 expressing NK-92 cell infusion (dual therapy). It is noteworthy that before the therapeutic mAb can bind to NK cells, a portion of the NK cell Fc receptor will be occupied by human serum IgG. Pre-binding NK cells to MB311 before administration can be a solution. When administered in a peripheral vein, this construct will likely be large enough to stay in the pulmonary capillary bed. If administered by an arterial route, similar means will be expected. MB311 is a fully humanized monoclonal antibody that recognizes the tumor-associated antigen Lewis Y.

[0064] In another view, the pre-bound complex of NK92, in which its CD16 receptor is saturated with MB311 or other similar mAbs, may have the limitation of being easily dissociated and reconstituted with plasma IgG. In general, even on 176V expressing NK92 cells, the Fc affinity is not very high. The antibody will then become free, potentially causing toxicity. This can be partially addressed by utilizing MB311 grown in moss reactors, as this Ab has a better chance of staying bound (affinity for Fc is up to 40 times greater than MB311).

[0065] The route of administration may be local administration (pleural / peritoneal effusion for metastatic cancer) or local injection into the tumor. In the case of local injection, tumor penetration may be a problem which may be addressed by including collagenase / protease to mechanically break down 'hard' tumors to improve penetration / entry.

[0066] Blood group-related antigens represent a group of carbohydrate determinants carried on both glycolipids and glycoproteins. They are usually mucin types and are detected on erythrocytes, certain epithelial cells and in the secretions of certain individuals. Sixteen different but mutually related specificities belong to this group of antigens, including A, B, H, Lewis a, Lewis b, Lewis x, Lewis y and precursor type 1 chain antigens. Lewis y (type 2 chain) antigen is a difucosylated tetrasaccharide found on type 2 blood group oligosaccharides of glycolipids and glycoproteins. It is expressed in colorectal tumors and colorectal cancer. Lewis y antigen can also serve as a clinical marker for diagnosis and prognosis of cholangiocarcinoma, hepatocellular carcinoma and breast cancer.

[0067] Example 3

[0068] In one example, a Phase I / II open-label trial of the Lewis Y-specific monoclonal antibody IGN311 evaluated safety and efficacy in patients with malignant effusions. In brief, treatment of CRC patients with the humanized mAb IGN311 targeting the carbohydrate Lewis Y eliminated circulating tumor cells in the blood and thereby confirmed the clinical profile of the parental murine antibody ABL364, which showed elimination of Lewis Y and cytokeratin-positive cells in the bone marrow of patients with breast cancer.

[0069] An open-label, single-arm, non-controlled study of IGN311 (100 mg / dose, intravenously on Days 1 and 7) is ongoing in patients with malignant effusions (ascites or pleural effusions) with the primary objective of examining safety and tolerability. Secondary objectives are volumetric measurement of malignant effusions and obtaining data on several immune parameters.

[0070] Four patients (2 patients with gastric cancer and malignant ascites, 2 patients with breast cancer and malignant pleural effusion / ascites) completed the study until December 2005. IGN311 was well tolerated, with only one patient showing up to grade 2 nausea, vomiting and rash as side effects after the first application, which were easily manageable. In all patients, significant levels of IGN311 were measured, followed by an increase in CD45-positive cells in the effusion. The patient with the highest level of Lewis Y-expressing tumor cells showed a decrease in the volume of the effusion during treatment.

[0071] Thus, these experiments show that IGN311 is well tolerated, penetrates into malignant effusions and attracts immune cells, resulting in a reduction in tumor cell counts in the effusions. In the case of strong Lewis Y expression by malignant cells in the effusions, a reduction in effusion volume can be demonstrated.

[0072] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct and indirect administration of a pharmaceutical composition or drug, wherein direct administration of a pharmaceutical composition or drug is typically performed by a health care professional (e.g., a physician, a nurse, etc.), and wherein indirect administration includes providing the pharmaceutical composition or drug to a health care professional or making the pharmaceutical composition or drug available to a health care professional for direct administration (e.g., via injection, infusion, oral delivery, local delivery, etc.). Most preferably, the cells or exosomes are administered via subcutaneous or subdermal injection. However, in other contemplated aspects, administration may also be intravenous injection. Alternatively or additionally, antigen presenting cells may be isolated or grown from cells of a patient, infected in vitro, and then infused into the patient. Therefore, it should be understood that the envisioned systems and methods may be viewed as complete drug discovery systems (e.g., drug discovery, treatment regimens, validation, etc.) for highly personalized cancer therapy.

[0073] The description of the range of values ​​herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were individually described herein. Unless otherwise indicated herein or otherwise clearly contradictory to the context, all methods described herein can be performed in any suitable order. The application of any and all examples or exemplary language (e.g., "such as") provided with respect to certain embodiments herein is intended only to better illustrate the full scope of this disclosure, without limiting the scope of the invention claimed in addition. Any language in this specification should not be interpreted as indicating that any unclaimed element is necessary for practicing the claimed invention.

[0074] It should be clear to those skilled in the art that, without departing from the full scope of the concept disclosed herein, in addition to those already described, many other modifications may also be made. Therefore, the present disclosure subject matter is only limited to the scope of the appended claims. In addition, when interpreting both this specification and the claims, all terms should be interpreted in a manner as broad as possible consistent with the context. In particular, the term "comprises" and "comprising" should be interpreted as referring to elements, components or steps in a non-exclusive manner, thereby indicating that the mentioned elements, components or steps can exist, or utilize, or combine with other elements, components or steps not explicitly mentioned. When the specification claims refer to at least one of something selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element in the group, rather than A plus N, or B plus N, etc.

Claims

1. A pharmaceutical composition, comprising: A pharmaceutically acceptable carrier and a therapeutically effective amount of CD16+NK-92 cells; wherein the CD16+NK-92 cells are genetically engineered to have reduced expression of at least one inhibitory receptor and / or are genetically engineered to express the CD16 158V variant, and wherein the CD16+NK-92 cells are preloaded with a therapeutic antibody; wherein the pharmaceutically acceptable carrier comprises a cryopreservation medium for cryopreserving the pharmaceutical composition to a temperature below -85°C, and wherein the cryopreservation medium increases or stabilizes the binding of the antibody to the haNK cells; and The CD16+NK-92 cells have abolished cell division while maintaining metabolic activity and NK cell function.

2. The pharmaceutical composition of claim 1, wherein the CD16+NK-92 cells further express recombinant IL-2.

3. The pharmaceutical composition of claim 1, wherein the CD16+ NK-92 cells are genetically engineered to have reduced expression of at least one inhibitory receptor and are genetically engineered to express the CD16 158V variant.

4. The pharmaceutical composition of claim 1, wherein the therapeutic antibody non-covalently binds to CD16.

5. The pharmaceutical composition of claim 1, wherein the antibody is an anti-CD20 antibody.

6. The pharmaceutical composition of claim 5, wherein the anti-CD20 antibody is rituximab.

7. The pharmaceutical composition of claim 1, wherein the antibody is selected from the group consisting of: Atezolizumab, ofatumumab, ipilimumab, ramucirumab, olatumumab, elotuzumab, nesitutumumab, daratumumab, denosumab, avelumab, durvalumab, trastuzumab, alemtuzumab, bevacizumab, pertuzumab, obinutuzumab, rituximab, and cetuximab.

8. The pharmaceutical composition of claim 1, wherein the cryopreservation medium is CryoStor CS10.

9. The pharmaceutical composition of claim 1, wherein the carrier further comprises human albumin.

10. The pharmaceutical composition of claim 1, wherein the NK cell function comprises antibody-dependent cell-mediated cytotoxicity (ADCC).

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