Antibody pre-loaded CD16+NK-92 cells as effective therapeutic product for tumor lysis

Abstract

To provide new compositions for controlling the viability and efficacy of cell therapeutics, and methods for preparing the same.SOLUTION: A pharmaceutical composition comprises a pharmaceutically acceptable carrier, therapeutically effective amounts of Natural Killer (haNK) cells and a therapeutic antibody in the form of a combined preparation, and a cryopreservation medium. The cryopreservation medium increases or stabilizes antibody binding to the haNK cells, and the therapeutic antibody is an anti-HER2 antibody, an anti-EGFR antibody, an anti-CTLA-4 antibody, or an anti-CD30 antibody.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims priority to the inventors' co-pending U.S. Provisional Patent Application No. 62 / 879,111, filed July 26, 2019, which is incorporated herein by reference.

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

[0003] The background discussion includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the invention(s) claimed herein, or that any document referenced specifically or implicitly is prior art.

[0004] All publications and patent applications herein are 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. In the event that a definition or use of a term in an incorporated reference is inconsistent or contradictory with a definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the reference shall not apply.

[0005] Natural killer (NK) cells are known to play a role in mediating innate immunity, enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses through antibody-dependent cell-mediated cytotoxicity (ADCC) due to 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 leukemia and lymphoma. NK cells can also be further engineered to enhance their cancer cell killing effects - one such technique is high affinity NK (haNK) cells, which are NK cells that have incorporated a high binding affinity receptor that binds to an administered antibody.

[0006] Combination therapy of NK cells and antibodies for the treatment of cancer is known. PCT / US2018 / 032281 discloses the treatment of chordoma by co-administration of an anti-EGFR antibody with high affinity NK cells (haNK). The disclosure provides that the antibody is non-covalently bound to a high affinity variant of the CD16 receptor, or that the antibody is administered prior to the infusion of haNK cells, such that chordoma cells are targeted 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 reduces patient-to-patient variability in both drug distribution and drug elimination. However, in existing cell therapies, such as PCT / US2018 / 032281, where combinations of engineered cells and therapeutic antibodies are administered according to different protocols, dosing is a challenge. Furthermore, repeated boluses increase the cost of treatment. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there is a need in the art for novel compositions and methods for controlling the viability and efficacy of cell therapy drugs, as well as the titration of engineered cells and therapeutic antibodies.Preferably, such combinations would combine both components into one administration protocol. [Means for solving the problem]

[0009] We have demonstrated that CD16 + We have discovered compositions, methods, and devices that are capable of producing cryopreserved preparations comprising NK-92 (haNK) cells. Advantageously, and unexpectedly, the compositions disclosed herein eliminate the increased cost of treatment associated with repeated boluses and produce effective drug distribution to patients.

[0010] In one aspect of the present subject matter, the inventors contemplate a pharmaceutical composition comprising a pharma- ceutically 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.

[0011] Contemplated haNK cells are preferably at least 5×10 5 cells / kg~5×10 8 The haNK cells are administered at a dosage of cells / kg, and further preferably the haNK cells are NK92 derivatives and / or typically express recombinant IL2 intracellularly. Moreover, it is generally preferred that the haNK cells have been genetically engineered to have reduced expression of at least one inhibitory receptor and / or that the haNK cells have been genetically engineered to express the CD16 158V mutant. Moreover, the haNK cells may 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 can 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, for example, by click chemistry.

[0014] Moreover, the pharmaceutical composition may further comprise a cryopreservation medium, such as CryoStor CS10. Ideally, the cryopreservation medium is selected such that it favors antibody binding to haNK cells.

[0015] Also disclosed herein is a method of making the composition by the steps of: (a) mixing haNK cells with a therapeutic antibody to make a combination preparation; (b) freezing the combination preparation; and (c) thawing the combination preparation. In some embodiments, the haNK cells are in a medium containing about 5% human albumin. Preferably, the mixture of haNK cells and the therapeutic antibody is mixed with an equal volume of cryopreservation medium. The combination preparation of haNK cells and the therapeutic antibody may be frozen at a temperature below -80°C, or in some cases below -120°C. The frozen combination preparation is irradiated with a certain dose of X-ray irradiation to impair the proliferation ability of the haNK cells. In some embodiments, after the thawing step, the combination preparation is incubated on ice or at room temperature.

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

[0017] In some cases, the method may also include administering an additional cancer treatment to the patient. The additional cancer treatment may be immunotherapy, chemotherapy, or radiation therapy. The immunotherapy may include administration of recombinant yeast or recombinant virus (vim) expressing a patient-specific and tumor-specific neo-epitope. The chemotherapy may include administration of at least one of aldoxorubicin, cyclophosphamide, irinotecan, gemcitabine, capecitabine, 5-FU, FOLFIRL FOLFOX, and oxipiatin. The additional cancer treatment may be administered separately, sequentially, simultaneously co-administered, or prior to the haNK cell and antibody composition as disclosed herein.

[0018] In another aspect of the subject matter of the present invention, a kit is disclosed herein that includes a pharmaceutical composition, wherein the pharmaceutical composition includes haNK cells, a therapeutic antibody, and optionally a cryopreservation medium. In the kit, the composition may be packaged in a bag or vial suitable for storage below -85°C. Finally, the kit may also include information in electronic or paper form, including instructions for use of the pharmaceutical composition.

[0019] Various objects, features, aspects and advantages will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings, in which like reference numerals represent like components. [Brief description of the drawings]

[0020] [Figure 1] Exemplary ADCC activity of haNK (antibody preloaded and unloaded) cells tested after thawing and incubation on ice for 30 minutes is shown. [Figure 2A] 1 shows exemplary ADCC activity of antibody-preloaded haNK cells that were thawed and tested without post-thaw incubation. [Figure 2B] Exemplary ADCC activity of antibody preloaded haNK cells that were thawed and incubated on ice for 30 minutes and tested is shown. [Figure 2C]Exemplary ADCC activity of antibody preloaded haNK cells tested after thawing and incubation on ice or at room temperature for 30 minutes is shown. [Figure 3A] Exemplary ADCC activity of antibody preloaded haNK cells thawed on ice for 30 minutes and subsequently washed or tested without a washing step is shown. [Figure 3B] 1 shows exemplary ADCC activity of haNK cells tested in the presence of exogenous Rituxan after thawing on ice for 30 minutes and subsequent washing or without a washing step. [Figure 4A] Exemplary ADCC activity of antibody preloaded haNK cells prepared in different media, incubated on ice for 30 minutes, and subsequently washed or tested without a washing step is shown. [Figure 4B] Exemplary ADCC activity of haNK cells tested in the presence of exogenous Rituxan after thawing on ice for 30 minutes and subsequent washing or without a washing step is shown. [Figure 4C] 1 shows exemplary results of changes in ADCC activity depending on medium and washing steps. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Cell-based therapies in the treatment of cancer are increasingly utilizing a variety of antibodies. Current treatment regimens consist of separate infusions of NK cells and monoclonal antibodies. However, this leads to problems of dose setting and repeated boluses, thus increasing the cost of treatment. The inventors have found a solution to this problem by creating a pharmaceutical composition of haNK cells and antibodies as a combination formulation. For example, the inventors have created this composition by premixing a therapeutic monoclonal antibody such as Rituxan with haNK cells in 5% human albumin, followed by mixing with CS-10 (1:1), and cryopreserving it using a controlled rate freezer and storage in a vapor phase LN2 freezer until it is time to use it as an infusion formulation after thawing. This premixed combination formulation has been shown to be effective and efficacious in killing cancer cells. In assays, comparable killing activity was observed when antibodies were added exogenously to cryopreserved haNK cells.

[0022] The antibody could also be injected together with the NK cells. However, in that case, both are independent drugs, and the amount of antibody used as a drug would increase. On the other hand, the advantage of the premixed combination formulation disclosed here is that, together with the cells, a lower concentration of antibody would be effective for killing. Moreover, the haNK cells combo with the antibody could be used as a commercial drug, as a one-time injection rather than multiple injections.

[0023] The majority of monoclonal antibodies in oncology are administered on a dosing schedule based on body size. This partially suppresses the inter-patient variability in both drug distribution and drug elimination. However, in cell therapy, where the combination of engineered cells and therapeutic antibodies is administered according to different protocols, dosing is a challenge. Furthermore, repeated boluses increase the cost of treatment. Therefore, combining both components into one administration protocol is the solution sought.

[0024] Here, we investigated the CD16 +Various compositions, methods, and kits have been discovered for preloading NK-92 (haNK) cells with antibodies, such as therapeutic antibodies. The compositions, methods, and kits disclosed herein are contemplated as novel, more effective tumor lysis approaches. Preferably, the compositions comprising haNK cells and antibodies are in the form of a combined preparation, which is often stored frozen until ready for use.

[0025] NK cells express Fc receptors, which can bind to the Fc portion of immunoglobulins to generate cell signals within the NK cells. When NK cells are activated via the Fc receptor by antibodies bound to tumor targets, they can induce target lysis. This antibody-dependent cell-mediated cytotoxicity (ADCC) of target cells is used in cancer therapy. haNK cells are NK-92 cells that have been engineered to express the high affinity CD16 receptor (FcγRIIIA) to facilitate ADCC-mediated tumor cell lysis. The high affinity CD16 receptor can be docked to soluble IgG. However, so far, the binding strength / affinity as well as 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 Nov;4(11):2859-68, and are also commercially available from NantKwest, San Diego, CA).

[0026] Here, the inventors unexpectedly found that the high affinity CD16 receptor on cryopreserved haNK cells can serve as a docking platform for soluble therapeutic antibodies and induce ADCC when co-incubated with tumor target cells.Accordingly, the inventive concept disclosed herein relates to a pharmaceutical composition comprising a pharma- ceutical 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.

[0027] Preferably, the therapeutic antibodies contemplated herein are those that induce 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-specific and tumor-specific epitopes (neoepitopes). Optionally, or in addition, the therapeutic antibodies contemplated herein may be antibodies against antigens found in necrotic and apoptotic cells.

[0028] Non-limiting examples of antibodies contemplated herein include atezolizumab, ofatumumab, ipilimumab, ramucirumab, olaratumumab, elotuzumab, necitumumab, daratumumab, dinutuximab, 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 inhibition of HER2 signaling and is approved for the treatment of HER2-positive breast cancer, HER2-positive gastric cancer, or gastroesophageal junction cancer; (b) bevacizumab (Avastin), which targets VEGF by inhibition of VEGF signaling and is approved for the treatment of colorectal cancer, non-squamous non-small cell lung cancer, glioblastoma, or renal cell carcinoma; (c) trastuzumab (Herceptin), which targets VEGF by inhibition of VEGF signaling and is approved for the treatment of colorectal cancer, non-squamous non-small cell lung cancer, glioblastoma, or renal cell carcinoma; (d) cetuximab (Erbitux) which targets EGFR (ErbB1) by inhibition of EGFR signaling and is approved for the treatment of squamous cell head and neck cancer (SCCHN); (e) panitumumab (Vectibix) which targets EGFR (ErbB1) by inhibition of EGFR signaling and is approved for the treatment of metastatic colorectal cancer; (f) ipilimumab (Yellowstone) which targets CTLA-4 by inhibition of CTLA-4 signaling and is approved for the treatment of unresectable or metastatic melanoma. (f) rituximab (Rituxan), approved for the treatment of CD20-positive B-cell non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL), which targets CD20 by ADCC, direct apoptosis induction, and CDC; (g) alemtuzumab (Campath), approved for the treatment of B-cell CLL, which targets CD52 by direct apoptosis induction and CDC; (h) rituximab (Rituxan), approved for the treatment of CD20-positive B-cell non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL), which targets CD20 by ADCC, direct apoptosis induction, and CDC; (i) gemtuzumab ozogamicin (Mylotarg), approved for the treatment of patients with CD33-positive acute myeloid leukemia, which targets CD33 by delivery of a toxic payload; (l) gemtuzumab ozogamicin (Mylotarg), approved for the treatment of patients with CD20 antigen-expressing relapsed or refractory low-grade, follicular, or transformed NHL, which targets CD20 by ADCC, apoptosis induction, and by delivery of the radioisotope iodine-131. 131I-tositumomab (Bexxar). One skilled in the art will recognize that any other therapeutic antibody may be used in the compositions disclosed herein to practice the inventive concept.

[0029] Preferably, the haNK cells as disclosed herein are derivatives of the NK-92 cell line. Furthermore, it is preferred that the haNK cells express recombinant IL2. Moreover, 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 mutant. In some desirable embodiments, the haNK cells are genetically engineered to have reduced expression of at least one inhibitory receptor.

[0030] Moreover, in further contemplated embodiments, the NK cells are irradiated prior to infusion to prevent continued cell division. Although not limiting the subject matter of the invention, irradiation of the cells typically inhibits cell division but still permits high rates of metabolic activity and NK cell function, particularly cytotoxic cell killing. Thus, suitable radiation doses for NK cells are between 50 cGy and 2,000 cGy. In a preferred embodiment, haNK cells may be irradiated with a radiation dose of at least 500 cGy prior to administration.

[0031] In some embodiments, the antibody is an anti-CD20 antibody (rituximab, ofatumumab, and / or 131 I-tositumomab), or anti-CD16 antibodies such as MB311. The term "MB311" as used herein contemplates a fully humanized monoclonal antibody that recognizes the tumor-associated antigen Lewis Y. This carbohydrate antigen is expressed in 60-90% of all epithelial cancers, but has limited expression on normal tissues, and therefore represents an attractive target for cancer immunotherapy.

[0032] In some cases, the haNK cells and the antibody are chemically conjugated. Chemical conjugation may be performed by any method known to a skilled chemist. One preferred chemical conjugation method 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 International Edition. 40(11):2004-2021. The term "click chemistry" as used herein therefore refers to such cycloaddition reactions, particularly the cycloaddition of azides with alkynes - a reaction that may be performed in some embodiments under the catalysis of Cu(I) or exposure to microwaves. The Click reaction occurs in one pot, is water-free, produces minimal and harmless by-products, and is spring-loaded - characterized by a high thermodynamic driving force that drives it rapidly and irreversibly to high yields of a single reaction product. These qualities make the Click reaction particularly well suited to the problem of isolating and targeting molecules in complex biological environments.

[0033] The pharmaceutical composition disclosed herein may further comprise a cryopreservation medium. The cryopreservation medium is intended to be a cell-specific, optimized freezing medium that is designed to prepare and maintain cells in a cryogenic environment (-70°C to 120°C). Furthermore, the cryopreservation medium is intended to provide a safe protective environment for cells and tissues during freezing, storage, and thawing, and to provide enhanced cell viability and functionality while eliminating the need for serum, proteins, or high levels of cytotoxic drugs. In a preferred embodiment, the cryopreservation medium is CryoStor CS10. Furthermore, the cryopreservation medium is selected such that it favors antibody binding to haNK cells.

[0034] In another aspect of the subject matter of the invention, the inventors disclose a method of treating a patient with cancer, comprising administering to the patient a combination formulation comprising a therapeutically effective amount of high affinity natural killer (haNK) cells and a therapeutic antibody. By using the compositions and methods disclosed herein, any type of cancerous cell can be treated. Thus, the methods disclosed herein are suitable for treating mixed types of cancer, such as carcinoma, sarcoma, myeloma, leukemia (liquid or blood cancer), lymphoma (solid cancer), or adenosquamous carcinoma, mixed mesodermal tumor, carcinosarcoma, and teratoma.

[0035] Preferably, the pharmaceutical compositions and methods disclosed herein may be formulated for delivery to a patient by any route of administration. "Route of administration" may refer to any route of administration known in the art, including, but not limited to, aerosol, nasal, oral, mucosal, transdermal, or parenteral. Preferred routes of administration include inhalation, ocular, nasal instillation, parenteral, dermal, transdermal, buccal, rectal, sublingual, perilingual, nasal, topical, or oral administration. "Parenteral" refers to routes of administration generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. When via a parenteral route, the composition may be in the form of a solution or suspension for infusion or injection. In some particularly preferred embodiments, the compositions may also be injected locally directly into a tumor (intratumoral administration).

[0036] Optionally, the method of treating cancer as disclosed herein may include administering to the patient a further cancer treatment. The further cancer treatment includes immunotherapy, chemotherapy, or radiation therapy. When immunotherapy is the further cancer treatment, it includes administration of recombinant yeast or recombinant virus expressing patient-specific and tumor-specific neo-epitopes. The chemotherapy may include administration of at least one of aldoxorubicin, cyclophosphamide, irinotecan, gemcitabine, capecitabine, 5-FU (5-fluorouracil), FOLFIRI (folinic acid, fluorouracil and irinotecan), FOLFOX (oxaliplatin, fluorouracil and leucovorin), and oxipiatin. The further cancer treatment may be administered separately, sequentially, simultaneously co-administered, or prior to the composition comprising haNK cells and therapeutic antibodies disclosed herein.

[0037] The pharmaceutical composition of the present disclosure is preferably delivered in a therapeutically effective amount. The exact therapeutically effective amount is the amount of the composition that will produce the most effective results in terms of efficacy of cancer treatment in a given subject. This amount may vary depending on a variety of factors, including, but not limited to, the characteristics of the haNK cells, antibodies, media composition, etc., as well as the physiological condition of the subject (including age, sex, type and stage of disease, general health, responsiveness to a given dosage, and type of drug therapy). Those skilled in the clinical and pharmacological fields will be able to determine the therapeutically effective amount through routine experimentation, for example, by monitoring the subject's response to administration of the pharmaceutical composition and adjusting the dosage accordingly. In a preferred embodiment, the haNK cells are administered in a concentration of 5×10 5 cells / kg~5×10 8 It is contemplated that the cells will be administered in a dosage of cells / kg.

[0038] The compositions disclosed herein can be made by various techniques. In one preferred method, haNK cells are mixed with a therapeutic antibody to make a combined preparation. The combined preparation is then frozen and subsequently thawed before use. Preferably, the haNK cells are in a 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 mixture of haNK cells and therapeutic antibody 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.

[0039] The composition is frozen to a temperature of -50°C to -100°C. Once the cells reach a temperature of -50°C to -100°C, they are further cooled, for example to a temperature compared to -120°C, by placing the cells in liquid nitrogen. The combined preparation is frozen until it is ready for use, or in some cases for at least one month, or at least two weeks, or at least one week, or at least five days, or at least three days, or at least one day, or at least 12 hours, or at least six hours, or at least two hours, or at least one hour. In some preferred embodiments, the frozen combined preparation is irradiated with a certain dose of X-ray radiation to impair the proliferation ability of haNK cells. After the thawing step, the combined preparation is incubated on ice or at room temperature. The thawing step can be on ice or at room temperature, or at any other temperature between 0°C to 100°C, or more preferably between 0°C to 40°C, or even more preferably between 0°C to 25°C. Once the composition is thawed, it is allowed to incubate on ice or at room temperature (0°C to 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.

[0040] In another aspect of the present disclosure, the 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 is useful for carrying out the method of the present invention for treating cancer or tumor. The kit is a collection of materials or components, including at least one of the compositions of the present invention as described throughout this disclosure.

[0041] The exact nature of the components that are configured in the kit of the present invention depends on its intended purpose. For example, some embodiments are configured for the purpose of treating tumors and / or cancer. In that case, the antibody used is specific for that cancer, such as trastuzumab for breast cancer. Moreover, the composition may further comprise a pharma- ceutically acceptable carrier that favors the binding of trastuzumab to haNK cells.

[0042] In one embodiment, the kit is configured specifically for treating a mammalian subject. In another embodiment, the kit is configured specifically for treating a human subject. In a further embodiment, the kit is configured for veterinary applications to treat subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.

[0043] The kit may include instructions for use. "Instructions for use" typically include tangible language describing the techniques to be used in using the components of the kit to achieve a desired result, such as reducing or killing a tumor. Optionally, the kit also contains other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipetting or metering equipment, dressings, or other useful accessories that one of skill in the art would readily recognize.

[0044] The materials or components assembled in the kit can be provided to the practitioner and stored in any convenient and suitable manner that ensures their operability and usefulness. For example, as contemplated herein, the components are more preferably provided at freezing temperatures, typically below -85°C. The components are typically housed in one or more suitable packaging materials. As used herein, the phrase "packaging material" refers to one or more physical structures used to contain the contents of the kit, such as the composition of the present invention. The packaging material is preferably constructed by well-known methods to provide a sterile and contamination-free environment. As used herein, the term "package" refers to a suitable solid matrix or material capable of holding individual kit components, such as glass, plastic, paper, foil, etc. The packaging material generally has an external indicia that indicates the contents and / or purpose of the kit and / or its components.

[0045] The following examples further describe embodiments of the present disclosure, which are merely illustrative and are not intended to limit in any way the scope of the invention as claimed. EXAMPLES

[0046] Example 1 In one exemplary embodiment, we mixed haNK cells (commercially available from NantKwest, San Diego) in 5% albumin (human) with anti-CD20 antibody (Rituxan). An equal volume (1:1) of CryoStor10 (CS10) was then added, and the mixture was transferred into CellFreeze infusion bags and vials. The study also included haNK cells without antibody as a negative control. The filled infusion bags and vials were then frozen and stored at -85°C or below using a controlled rate freezer. The frozen preparations were then transferred to a liquid nitrogen vapor phase (-120°C or below) freezer for storage. The frozen preparations were irradiated with a fixed dose of X-ray radiation to impair the proliferation ability of the haNK cells. After thawing, the cells were incubated with calcein-labeled Ramos (target) cells expressing CD20, and lysis was assessed using a calcein release assay.

[0047] As shown in Figure 1, which depicts the ADCC activity of haNK cells after thawing, Ramos lysis, which was not induced by haNK cells alone, was potently induced by haNK cells preloaded with Rituxan. Here, haNK cells were either preloaded with 1 μg / mL of Rituxan antibody and then cryopreserved, or were cryopreserved without preloading. Cryopreserved cells were then irradiated at 15 Gy using an RS-2000 X-ray irradiator. Cells were thawed, incubated on ice for 30 min, and added directly to the assay plate. Ramos lysis rate was assessed using a standard calcein release assay.

[0048] In further examples, we investigated the timing of ADCC after thawing. Interestingly, we found that a post-thaw holding time of 10-30 minutes (or even longer in some cases) on ice or at room temperature significantly affected the potency of the ADCC function of haNK cells. For example, haNK cells were cryopreserved with or without mixing with 1-2 μg / mL of Rituxan antibody. Cryopreserved cells were irradiated with 15 Gy using an RS-2000 X-ray irradiator. Thawing was then performed with various protocols as follows: Figure 2A shows the results of cells that were thawed, washed and tested with no post-thaw incubation time, and virtually no ADCC-based cell killing was observed. In contrast, a substantial improvement in ADCC cytotoxicity was observed when cells were thawed and incubated on ice (Figure 2B) or at room temperature (RT) or on ice (Figure 2C) for 30 minutes. Such activity was also observed after a washing step intended to remove unbound Rituximab. Again, in Figures 2A-C, % Ramos lysis was assessed using a standard calcein release assay.

[0049] In yet another example, we also evaluated the contribution of free antibodies in thawed haNK preparations. To that end, antibody preloaded cells were washed once after thawing by centrifugation at 336×g (1200 RPM) for 5 minutes, followed by ADCC assay. As illustrated in FIG. 3A-B, haNK cells were cryopreserved with or without mixing with 1 μg / mL Rituxan antibody. Cryopreserved cells were irradiated at 15 Gy using an RS-2000 X-ray irradiator. FIG. 3A illustrates the ADCC results after thawing, where Rituxan preloaded haNK cells were either washed or added directly to the assay plate without a washing step. For comparison, after thawing, haNK cells (not preloaded with antibodies) were either washed or added directly to the assay plate containing Rituxan-bound Ramos cells. The results are illustrated in FIG. 3B. As can be easily seen, when the cells were not preloaded with antibodies, the washing step by itself had no effect on the cells.

[0050] In yet further experiments, to assess the contribution of the components of the freezing medium in Rituxan preloaded haNK preparations, the cells were tested to induce ADCC prior to freezing. 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 shows the activity of fresh preparations with or without a washing step. Similarly, Figure 4B shows the results where haNK cells were either washed after thawing or added directly to the assay plate. Figure 4C shows the reduced rate of change in %ADCC activity when cells were suspended in albumin (HA) versus complete growth medium. Interestingly, these results indicate a beneficial contribution of the medium type (here: complete growth medium and human albumin medium) to improved binding of the antibody to the haNK cells.

[0051] Example 2 In a further example, we developed a novel technique based on the premise that tumor cells have aberrant carbohydrate glycosylation and that such structures, i.e. Lewis antigens, can be used to specifically target mAbs. In this method, we used the moss expression system to generate mAbs with increased FcR affinity. Currently, there are several techniques focused on generating mAbs with modified FcRs that bind with higher affinity to NK cells, and the preferred technique to be used here is one that is safe and effective in humans.

[0052] Preferably, the mAb formulation is combined with systemic CD-16 expressing NK-92 cell infusion (two-agent therapy). It is noteworthy that a portion of the NK cell Fc receptors would be occupied by human serum IgG before the therapeutic mAb can bind to the NK cells. Pre-binding the NK cells to MB311 before administration could be a possible solution. Such a construct would be large enough to become retained in the pulmonary capillary bed when administered into a peripheral vein. The same could be expected when administered via the arterial route. MB311 is a fully humanized monoclonal antibody that recognizes the tumor-associated antigen Lewis Y.

[0053] Another limitation may be that pre-bound NK92 complexes, whose CD16 receptors are saturated with MB311 or other similar mAbs, easily dissociate and rebind with plasma IgG. In general, Fc affinity is not very high, even on NK92 cells expressing 176V. Thus, the antibodies may be free to elicit toxicity, possibly resulting in a loss of activity. This may be addressed in part by utilizing moss reactor-grown MB311 (which has up to 40-fold higher affinity for Fc than MB311), as the Abs are more likely to remain bound.

[0054] The route of administration may be local administration (pleural / abdominal effusion of metastatic cancer) or local injection into the tumor. In the case of local injection, tumor infiltration may be a concern, but this may be addressed by including collagenase / proteinase to mechanically break down "hard" tumors for improved penetration / delivery.

[0055] Blood group-related antigens refer to a group of carbohydrate determinants carried by both glycolipids and glycoproteins. They are usually of the mucin type and are found on erythrocytes, certain epithelial cells, and secretions of certain individuals. This group of antigens includes 16 genetically and biosynthetically distinct, but interrelated specificities, including the A, B, H, Lewis a, Lewis b, Lewis x, Lewis y, and precursor type 1 chain antigens. The Lewis y (type 2 chain) antigen is a difucosylated tetrasaccharide found in blood group 2 oligosaccharides of glycolipids and glycoproteins. It is expressed in colon tumors and colorectal cancer. The Lewis y antigen may also serve as a clinical marker for the diagnosis and prognosis of cholangiocarcinoma, hepatocellular carcinoma, and breast cancer.

[0056] Example 3 In one embodiment, a Phase I / II open-label study of the Lewis Y-specific monoclonal antibody IGN311 evaluated its safety and efficacy in patients with malignant effusions. Briefly, treatment of CRC patients with the humanized mAb IGN311 targeting the glycan Lewis Y eliminated circulating tumor cells in the blood, thereby confirming the clinical profile of the parent murine antibody ABL364, which showed elimination of Lewis Y and cytokeratin positive cells in the bone marrow of breast cancer patients.

[0057] An open-label, single-arm, uncontrolled study of IGN311 (100 mg per dose, intravenously on days 1 and 7) in patients with malignant effusions (ascites or pleural effusion) is ongoing with the primary objective of investigating safety and tolerability. Secondary objectives are to obtain data on the volumetric measurements of the malignant effusion and on several immunological parameters.

[0058] As of December 2005, four patients (two with gastric cancer and malignant ascites, two with breast cancer and malignant pleural effusion / ascites) have completed the study. IGN311 was well tolerated, with only one patient experiencing side effects of up to grade 2 nausea, vomiting, and skin rash after the first application, which were easily managed. Significant levels of IGN311 were measured in all patients, followed by an increase in CD45-positive cells in the effusion. The patient with the highest levels of tumor cells expressing Lewis Y showed a decrease in effusion volume during treatment.

[0059] These experiments therefore showed that IGN311 was well tolerated, penetrated into malignant effusions, attracted immune cells, and led to a reduction in the number of tumor cells in the effusion. In cases of strong Lewis Y expression in the malignant cells in the effusion, a reduction in the effusion volume could be demonstrated.

[0060] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct and indirect administration of the pharmaceutical composition or drug, where direct administration of the pharmaceutical composition or drug is typically performed by a medical professional (e.g., a doctor, a nurse, etc.), and indirect administration includes providing or making available the pharmaceutical composition or drug to a medical professional for direct administration (e.g., by injection, infusion, oral delivery, topical delivery, etc.). Most preferably, the cells or exosomes are administered by subcutaneous or subdermal injection. However, in other contemplated aspects, administration may also be intravenous injection. Alternatively or in addition, antigen-presenting cells may be isolated or grown from the patient's cells infected in vitro and then infused into the patient. It should therefore be understood that the contemplated systems and methods may be considered as a complete drug discovery system (e.g., drug discovery, treatment protocol, validation, etc.) for highly personalized cancer treatment.

[0061] The description of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each individual value contained within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually described herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any examples provided herein with respect to specific embodiments, or the use of exemplary language (e.g., "etc.") are intended merely to better outline the full scope of the disclosure and do not impose limitations on the scope of the invention as originally claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the claimed invention.

[0062] It should be apparent to one skilled in the art that many more variations beyond those already described are possible without departing from the full scope of the concepts disclosed herein. Thus, the disclosed subject matter should not be limited, except as provided in the appended claims. Moreover, in interpreting both this specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted to refer to elements, components, or steps in a non-exclusive manner, indicating that the referenced element, component, or step may be present or utilized or may be combined with other elements, components, or steps not expressly mentioned. When a claim herein refers to at least one selected from the group consisting of A, B, C, and N, the sentence should be interpreted as requiring only one element from that group, and not A+N, or B+N, etc.

Claims

1. A pharmaceutical composition comprising a pharma- ceutically 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.

2. The pharmaceutical composition of claim 1, wherein the haNK cells are a derivative of the NK-92 cell line.

3. The pharmaceutical composition of claim 1 , wherein the haNK cells further express recombinant IL2.

4. 2. The pharmaceutical composition of claim 1, wherein the haNK cells are genetically engineered to have reduced expression of at least one inhibitory receptor.

5. 2. The pharmaceutical composition of claim 1, wherein the haNK cells are irradiated with a radiation dose of at least 500 cGy prior to administration.

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

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

8. 2. The pharmaceutical composition of claim 1, wherein the antibody is selected from the group consisting of atezolizumab, ofatumumab, ipilimumab, ramucirumab, olaratumumab, elotuzumab, necitumumab, daratumumab, dinutuximab, avelumab, durvalumab, trastuzumab, alemtuzumab, bevacizumab, pertuzumab, obinutuzumab, rituximab, and cetuximab.

9. The pharmaceutical composition according to any one of claims 1 to 8, further comprising a cryopreservation medium.

10. 10. The pharmaceutical composition of claim 9, wherein the cryopreservation medium is CryoStor CS10.

11. 10. The pharmaceutical composition of claim 9, wherein the cryopreservation medium increases or stabilizes antibody binding to the haNK cells.

12. A method of making a composition according to any one of claims 1 to 11, comprising the steps of: mixing the NK cells with a therapeutic antibody to form a combination formulation; Freezing the combined formulation; and Thawing the combined preparation. The method includes:

13. The method of claim 12, wherein the haNK cells are in a medium containing about 5% albumin.

14. The method of claim 12, wherein the mixture of haNK cells and therapeutic antibody is mixed with an equal volume of cryopreservation medium.

15. The method of claim 12, wherein the combined preparation is frozen at a temperature below -80°C.

16. The method of claim 12, wherein the combined preparation is frozen at a temperature below -120°C.

17. The method according to any one of claims 12 to 16, wherein the frozen combined preparation is irradiated with a certain dose of X-ray irradiation to impair the proliferation ability of the haNK cells.

18. 13. The method of claim 12, wherein after the thawing step, the combined preparation is incubated on ice or at room temperature.

19. A method for treating a patient having cancer, comprising administering to the patient a pharmaceutical composition comprising a combination formulation of (a) a therapeutically effective amount of high affinity natural killer (haNK) cells and (b) a therapeutic antibody.

20. 20. The method of claim 19, wherein the administration is by infusion.

21. 20. The method of claim 19, wherein the administration is intratumoral administration.

22. 20. The method of claim 19, wherein the administration is intravenous administration.

23. The method of any one of claims 19 to 22, further comprising the step of administering to the patient a further cancer treatment.

24. 24. The method of claim 23, wherein the additional cancer treatment comprises immunotherapy.

25. 25. The method of claim 24, wherein the immunotherapy comprises administration of a recombinant yeast or a recombinant virus expressing a patient-specific and tumor-specific neoepitope.

26. 24. The method of claim 23, wherein the additional cancer treatment comprises chemotherapy.

27. 27. The method of claim 26, wherein the chemotherapy comprises administration of at least one of aldoxorubicin, cyclophosphamide, irinotecan, gemcitabine, capecitabine, 5-FU, FOLFIRI, FOLFOX, and oxipiatin.

28. 24. The method of claim 23, wherein the additional cancer treatment comprises radiation therapy.

29. The method of any one of claims 19 to 28, wherein said further cancer treatment is administered separately, sequentially, simultaneously co-administered with, or prior to, said pharmaceutical composition.

30. The haNK cells are 5×10 ５ cells / kg~5×10 ８ 20. The method of claim 19, administered in a dosage of cells / kg.

31. A pharmaceutical composition according to any one of claims 1 to 11 for use in the treatment of cancer.

32. A kit comprising a pharmaceutical composition, said pharmaceutical composition comprising haNK cells, a therapeutic antibody, and optionally a cryopreservation medium.

33. 33. The kit of claim 32, wherein the composition is packaged in a bag or vial suitable for storage below -85°C.

34. 34. The kit of claim 32 or 33, further comprising information in electronic or paper form, said information comprising instructions for use of said pharmaceutical composition.

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