HK40037635B - Elimination of cd19-positive lymphoid malignancies by cd19-car expressing nk cells - Google Patents

Description

NK cells expressing CD19-CAR eliminate CD19-positive lymphoid malignancies.

This application claims priority to our co-pending U.S. provisional application filed on October 31, 2018, with serial number 62/753,719.

sequence list

An ASCII text file containing a sequence list named 104077.0008PCT Seq_ST25, which is 38KB in size, was created on July 15, 2019, and is submitted electronically with this application via EFS-Web, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The field of this invention is engineered cells in relation to cancer treatment.

Background Technology

The background description includes information that can be used to understand the invention. It is not acknowledged that any information provided herein is prior art or related to the currently claimed invention, nor is it acknowledged that any publication specifically or implicitly mentioned is prior art.

All publications and patent applications herein are incorporated by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference. Where a definition or usage of a term in an incorporated reference is inconsistent with or contrary to the definition of that term provided herein, the definition provided herein shall apply, and not the definition in that reference.

Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells typically represent about 10%–15% of circulating lymphocytes and bind to and kill target cells that are not specific to antigens and have not been previously sensitized by the immune system (including virus-infected cells and many malignant cells). Herberman et al., Science 214:24 (1981). The killing of target cells occurs through the induction of cell lysis. NK cells used for autologous NK cell transplantation are partially isolated from the peripheral blood lymphocytes (“PBL”) of the subject's blood, expanded in cell cultures to obtain a sufficient number of cells, and then re-infused into the subject. Such autologous NK cells have shown some effectiveness in in vivo therapy. However, this therapy is limited to autologous cases and is further complicated by the fact that not all NK cells are cytolytic.

This is a cytolytic cancer cell line discovered in the blood of subjects with non-Hodgkin's lymphoma and subsequently immortalized in vitro. The cells are derived from NK cells but lack the major inhibitory receptors present on normal NK cells, while retaining most of the activating receptors. However, the cells do not attack normal cells and do not elicit unacceptable immune rejection in humans. Characterization of the cell line is disclosed in WO1998/049268 and U.S. Patent Application Publication No. 2002-0068044. The cells have been evaluated as a therapeutic agent for certain cancers.

Summary of the Invention

In some embodiments, this disclosure provides a cell expressing a CD19 CAR and an Fc receptor. In some embodiments, the cell comprises a polycistronic construct encoding the CD19 CAR and the Fc receptor. In some embodiments, the Fc receptor is CD16. In some embodiments, the Fc receptor comprises SEQ ID NO:2. In some embodiments, the polycistronic construct further comprises a sequence encoding IL-2 or a variant thereof. In some embodiments, the IL-2 variant is erIL-2. In some embodiments, the coding sequences for one or more of the CD19 CAR, the Fc receptor, or erIL-2 are codon-optimized for expression in a human system.

In some embodiments, the cells are capable of killing CD19-expressing cells, such as tumor cells. In some embodiments, the tumor cells are SUP-B15 cells. In some embodiments, the CD19 CAR comprises an scFv antibody fragment. In some embodiments, the scFv antibody fragment has the amino acid sequence of SEQ ID NO:10. In some embodiments, the polycistronic construct comprises the sequence of SEQ ID NO:9, wherein the sequence encodes the scFv antibody fragment. In some embodiments, the cell comprises a sequence encoding a self-cleaving peptide, wherein the sequence is located between the CD19 CAR and CD16, and wherein the sequence allows equimolar expression of the CD19 CAR and the FcR. In some embodiments, the cell comprises an internal ribosome entry sequence (IRES) between the sequence encoding CD16 and the sequence encoding IL-2 or a variant thereof.

In some embodiments, when the effector-to-target ratio is 10, the direct cytotoxicity of the cell to CD19-expressing cells is 70%-100%. In some embodiments, when the effector-to-target ratio is 10, the ADCC activity of the cell is 30%-90%. In some embodiments, the CD19 CAR contains a sequence having at least 90% identity with SEQ ID NO:10.

In some embodiments, this disclosure provides a kit comprising a pharmaceutical composition containing the cells disclosed herein.

In some embodiments, this disclosure provides a method for generating cells, the method comprising providing a vector and introducing the vector into cells to generate the cells, wherein the vector encodes CD19CAR and CD16. In some embodiments, the vector further comprises a sequence encoding IL-2. In some embodiments, the vector comprises a sequence encoding a self-cleaving peptide, wherein the sequence is located between the CAR and CD16, and wherein the sequence allows for equimolar expression of the CAR and CD16. In some embodiments, the vector includes an internal ribosome entry sequence (IRES) between the CD16 coding sequence and the IL-2 coding sequence.

In some embodiments, this disclosure provides a method of treating a subject for cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a variety of cells as disclosed in the specification and claims. In some embodiments, the subject is administered about 1 x ^10⁸ to about ¹ x 10¹¹ modified cells/ ^m² of the subject's body surface area.

In some embodiments, the cancer is leukemia or lymphoma.

In some embodiments, the cancer is one or more of the following: B-cell malignancy, post-HSCT B-cell malignancy, CLL, B-ALL, acute lymphoblastic leukemia (ALL), post-UCBT B-lineage lymphoid malignancy, chronic lymphocytic leukemia (CLL), B-Non-Hodgkin's lymphoma (B-NHL), post-HSCT ALL; lymphoma, refractory follicular lymphoma, or lymphoblastic leukemia. In some embodiments, the B-cell malignancy is mantle cell lymphoma. In some embodiments, multiple cells are administered intravenously. In some embodiments, the multiple cells are administered intratumorally.

The foregoing general description and the following detailed description are exemplary and illustrative, and are intended to provide further explanation of this disclosure. Other objects, advantages, and novel features will be apparent to those skilled in the art.

Attached Figure Description

The purpose, features, and advantages of this disclosure will be more readily understood when considered in conjunction with the accompanying drawings and the following disclosure.

Figure 1 is a schematic diagram of the structural domains of the first, second and third generation CARs.

Figure 2 shows the components of the tricistronic plasmid, which include the CAR coding sequence, P2A sequence, CD16 coding sequence, and erIL-2 coding sequence.

Figures 3A and 3B show the results of flow cytometry analysis, which demonstrate the expression of CD16 and CD19-CAR on the surface of CD19t-haNK ^™ cells. The peaks on the right side of each inset represent populations of cells expressing either CD16 or CD19.

Figure 4A shows the cytotoxic effect of CD19 t-haNK ^™ cells on K562 cells. 16B1 and 18B1 are two CD19 t-haNK ^™ populations obtained from two electroporation events performed on two different dates. Figure 4B shows the cytotoxic effect of selected CD19 t-haNK ^™ clones on K562 cells in a cytotoxicity assay.

Figure 5A shows the cytotoxic effect of CD19 t-haNK ^™ cells on SUP-B15 cells. 16B1 and 18B1 are two CD19 t-haNK ^™ populations obtained from two electroporation events performed on two different dates. Figure 5B shows the cytotoxic effect of selected CD19 t-haNK ^™ clones on SUP-B15 cells in a cytotoxicity assay.

Figure 6A shows the ADCC activity of CD19 t-haNK ^™ cells against SKBr3 cells when combined with Herceptin (anti-Her2 antibody). Rituxan (anti-CD20 antibody) was used as a control. Figure 6B shows the ADCC activity of the selected CD19 t-haNK ^™ clone against SUP-B15 cells exhibiting CD19KO/CD20+ when combined with rituximab (anti-CD20 antibody).

Figure 7 shows the doubling time of the selected CD19 t-haNK ^™ clone.

Figure 8 shows the release of IL-2 from the selected CD19 t-haNK ^™ clone under culture conditions.

Figure 9 shows the survival curves of Raji tumor-bearing animals via intravenous infusion. Statistical analysis was performed using the Mantel-Cox test. ****, P < 0.0001.

Figure 10 shows the changes in body weight in animals in the intravenous Raji tumor model. Data are mean ± SEM. SEM values are calculated as the standard deviation divided by the square root of N.

Figure 11 shows the tumor growth curves of the subcutaneous Raji model. Data are mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by multiple comparisons via Tukey's test; ***, P < 0.001; ****, P < 0.0001.

Figure 12 shows that CD19 t-haNK ^™ reduced the metastatic disease burden in the livers of subcutaneously Raji tumor-bearing mice. (a) Whole liver image from animals in the indicated treatment group on day 13. Yellow arrows indicate metastatic lesions. The liver was fixed in 10% formalin for at least 24 hours before photography. (b) Quantitative percentage of tumor cells involved in the liver on the specified day (assessed by H&E staining). On day 13: *, P = 0.0257, by unpaired two-tailed t-test. Statistical analysis on days 11 and 15 was not possible due to limited sample size. See Table 4 for raw data.

Figure 13 shows the changes in body weight in the subcutaneous Raji tumor model. Data are mean ± SEM.

Detailed Implementation

Overview

This disclosure provides cells expressing CD19 CAR and Fc receptors. In some embodiments, these cells also express IL-2. In some embodiments, these cells comprise a tricistronic construct containing nucleic acid sequences encoding CD19 CAR, Fc, and IL2.

the term

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

In this specification and the following claims, reference will be made to numerous terms, which should be defined to have the following meanings:

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, as used herein. Thus, for example, reference to “a natural killer cell” includes a variety of natural killer cells.

All numerical references, such as pH, temperature, time, concentration, amount, and molecular weight, including ranges, are approximate values that vary in increments of 0.1 or 1.0 where appropriate, i.e., (+) or (-). It should be understood that, although not always explicitly stated, all numerical references may be preceded by the term "approximately".

As used in this article, when “+” is used to indicate the presence of a specific cell marker, it means that the cell marker is detectably present relative to the isotype control in fluorescently activated cell sorting; or that it is detectable above the background value in quantitative or semi-quantitative RT-PCR.

As used in this article, when “-” is used to indicate the presence of a specific cell marker, it means that the cell marker is undetectable relative to the isotype control in fluorescently activated cell sorting; or is undetectable above the background value in quantitative or semi-quantitative RT-PCR.

As those skilled in the art will understand, for any and all purposes, particularly in providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily identified as sufficiently descriptive and capable of being decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, middle third, and upper third, etc. Also, as those skilled in the art will understand, all language such as “at most,” “at least,” “greater than,” “less than,” etc., includes the stated numbers and refers to a scope that can subsequently be decomposed into the aforementioned subscopes. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1-3 cells means a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells means a group having 1, 2, 3, 4, or 5 cells, and so on.

As used herein, when referring to certain quantifiable characteristics of cells, such as cytotoxicity, viability, or cell doubling time, the terms “substantially the same” and “comparable” or “substantially similar” are used interchangeably, meaning that two measurements of these characteristics differ from each other by no more than 15%, 10%, 8%, or 5%.

It should also be understood that, although not always explicitly stated, the reagents described herein are merely exemplary, and their equivalents are known in the art.

For the purposes of this invention and unless otherwise indicated, the terminology is intended to refer to the original cell line as well as the NK-92 cell line, cell clones, and cells that have been modified (e.g., by introducing exogenous genes). Cells and their exemplary and non-limiting modifications are described in U.S. Patent Nos. 7,618,817; 8,034,332; 8,313,943; 9,181,322; 9,150,636; and published U.S. Application No. 10/008,955, all of which are incorporated herein by reference in their entirety and include wild-type -CD16, -CD16-γ, -CD16-ζ, -CD16(F176V), MI, and CI. Cells are known to those skilled in the art and are readily available to those skilled in the art from Southwest Inc.

As used herein, the term “cell” refers to a natural killer cell derived from a highly efficient and unique cell line described by Gong et al. (Leukemia, April; 8(4):652-8(1994)), the rights of which are owned by Southwest (hereinafter referred to as “cell”).

As used herein, the term “aNK cell” refers to an unmodified natural killer cell derived from a highly efficient and unique cell line described by Gong et al. (Leukemia, April; 8(4):652-8(1994)), the rights of which are owned by Southwest (hereinafter referred to as “aNK cell”).

As used herein, the term “haNK cell” refers to a natural killer cell derived from a highly efficient and unique cell line described by Gong et al. (Leukemia, April; 8(4):652-8(1994)), the rights of which are owned by Southwest, modified to express CD16 on the cell surface (hereinafter referred to as “CD16+ cell” or “haNK cell”).

As used herein, the term "taNK" refers to natural killer cells derived from a highly efficient and unique cell line described by Gong et al. (Leukemia, April; 8(4):652-8(1994)), the rights of which are owned by Southwest, modified to express chimeric antigen receptors (hereinafter referred to as "CAR-modified cells" or "taNK").

As used herein, the term “t-haNK ^™ ” cells refer to natural killer cells derived from a highly efficient and unique cell line described by Gong et al. (Leukemia, April; 8(4):652-8(1994)), owned by Southwest, modified to express CD16 and a chimeric antigen receptor on the cell surface (hereinafter referred to as “CAR-modified CD16+ cells” or “t-haNK ^™ cells”). In some embodiments, the tumor-specific antigen is CD19, and these cells are referred to as CD19t-haNK ^™ cells.

As used herein, the term "polycistronic construct" refers to a recombinant DNA construct that will be transcribed into a single mRNA molecule, and that single mRNA molecule encodes two or more transgenes. If a polycistronic construct encodes two transgenes, it is called a bicistronic construct; if it encodes three genes, it is called a tricistronic construct; and if it encodes four genes, it is called a tetracistronic construct, and so on.

As used herein, the term "chimeric antigen receptor" (CAR) refers to an extracellular antigen-binding domain fused to an intracellular signaling domain. CARs can be expressed in T cells or NK cells to increase cytotoxicity. Typically, the extracellular antigen-binding domain is a scFv specific to antigens found on the target cell. Based on the specificity of the scFv domain, cells expressing CARs target cells that express certain antigens on their cell surface. The scFv domain can be engineered to recognize any antigen, including tumor-specific antigens and virus-specific antigens. For example, a CD19 CAR recognizes CD19, a cell surface marker expressed in some cancers.

As used herein, the term "tumor-specific antigen" refers to an antigen present on cancer cells or neoplastic cells but not detectable on normal cells derived from the same tissue or lineage as cancer cells. As used herein, tumor-specific antigen also refers to tumor-associated antigens, which are antigens expressed at higher levels on cancer cells compared to normal cells derived from the same tissue or lineage as cancer cells.

As used in this article, the term "target" in reference to tumor targeting refers to the ability of cells to recognize and kill tumor cells (i.e., target cells). In this article, the term "target" refers, for example, the ability of a CAR expressed by a cell to recognize and bind to cell surface antigens expressed by a tumor.

The term "antibody" refers to any intact immunoglobulin of the same type or a fragment thereof that can competitively bind specifically to a target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies. Intact antibodies typically contain at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains, such as antibodies naturally found in camels, which may contain only heavy chains. Antibodies can be derived from a single source or can be "chimeric," meaning that different parts of the antibody originate from two different antibodies. Antigen-binding proteins, antibodies, or binding fragments can be produced in hybridomas via recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise stated, the term "antibody" includes, in addition to antibodies containing two full-length heavy chains and two full-length light chains, their derivatives, variants, fragments, and mutant proteins. In addition, unless explicitly excluded, antibodies include: monoclonal antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, the term also includes peptide bodies.

The term "subject" refers to non-human animals (including mammals such as cats, dogs, cows, horses, pigs, sheep, and goats) and humans. The term "subject" also refers to a patient requiring treatment for the disease described herein.

"Optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes examples in which the event or situation occurs as well as examples in which the event or situation does not occur.

The term "comprising" is intended to indicate that a composition and method includes the listed elements but does not exclude other elements. When used to define compositions and methods, "consistently consisting of" should mean excluding other elements that are of any significant importance to the composition. For example, a composition consisting essentially of the ingredients defined herein will not exclude other ingredients that do not substantially affect one or more of the essential and novel features of the claims. "Constitutes of" means excluding other ingredients and substantial method steps in greater than trace amounts. Examples defined by each of these transitional terms are within the scope of this disclosure.

As used herein, the terms “cytotoxicity” and “cytolysis” are intended to be synonymous when used to describe the activity of effector cells such as NK cells. Generally, cytotoxic activity involves the killing of target cells through any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of effectors that lyse the plasma membrane of target cells, thereby disrupting their physical integrity. This leads to the killing of the target cells. Without wishing to be bound by theory, the cytotoxic effect of NK cells is believed to be due to cytolysis.

The term “kill” for cells/cell populations is intended to include any type of manipulation that will cause the cell/cell population to die.

The term "cytokine" or "cytokines" refers to a general class of biomolecules that affect cells of the immune system. Exemplary cytokines include, but are not limited to, FLT3 ligands, interferons, and interleukins (ILs), particularly IL-2, IL-12, IL-15, IL-18, and IL-21.

The terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, that can be used in the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human being.

The term “treatment” encompasses the treatment of a disease or disorder as described herein in a subject, such as a human, and includes: (i) suppressing the disease or disorder, i.e., preventing its development; (ii) alleviating the disease or disorder, even if the disorder subsides; (iii) slowing the progression of the disorder; and/or (iv) suppressing, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. The term “administering” monoclonal antibodies or natural killer cells to a subject includes any route of introducing or delivering antibodies or cells to perform a desired function. Administration can be performed via any route suitable for delivering cells or monoclonal antibodies. Thus, delivery routes can include intravenous, intramuscular, intraperitoneal, or subcutaneous delivery. In some embodiments, modified cells are administered directly to a tumor, for example, by injection into the tumor. In some embodiments, modified cells as described herein are administered parenterally, for example, by injection, infusion, or implantation (subcutaneously, intravenously, intramuscularly, intracystic, intratumorally, or intraperitoneally).

The term "expression" refers to the production of gene products.

As used in this article, the term "cytotoxicity" when used to describe the activity of effector cells such as NK cells refers to the killing of target cells through any of a variety of biological, biochemical, or biophysical mechanisms.

The terms “reduction” and “reduction” are used throughout this document to mean a reduction of at least 10% compared to a reference level, such as a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a reduction of up to and including a reduction of 100% (i.e., zero level compared to the reference sample), or any reduction between 10% and 100% compared to the reference level.

The term "cancer" refers to all types of cancer, growths, or malignant tumors found in mammals, including leukemia, carcinoma, and sarcoma. Exemplary cancers include brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer, and medulloblastoma. Other examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, essential thrombocytosis, essential macroglobulinemia, primary brain tumors, cancer, malignant pancreatic islet tumors, malignant carcinoid tumors, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, urogenital tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine and exocrine pancreatic vegetations, and prostate cancer.

The term "therapeutic effective dose" or "effective dose" refers to the amount required to improve disease symptoms relative to an untreated patient. The effective dose of one or more active compounds used in practice to therapeutically treat disease varies depending on the route of administration, the subject's age, weight, and overall health. Ultimately, the attending physician or veterinarian determines the appropriate dose and dosing regimen. Such a dose is referred to as the "effective" dose.

For the convenience of the reader, headings or subheadings may be used in this specification, which are not intended to affect the scope of this disclosure. Additionally, some terms used in this specification are defined in more detail below.

cell

This is a cytolytic cancer cell line discovered in the blood of subjects with non-Hodgkin's lymphoma and subsequently immortalized in vitro. The cells are derived from NK cells but lack the major inhibitory receptors present on normal NK cells, while retaining most of the activating receptors. However, the cells do not attack normal cells and do not elicit unacceptable immune rejection in humans. Characterization of the cell line is disclosed in WO1998/049268 and U.S. Patent Application Publication No. 2002-0068044. The cells have been evaluated as a therapeutic agent for certain cancers.

carrier

This document describes vectors for transfecting cells to produce the modified cells described herein. In one embodiment, the vectors described herein are transient expression vectors. Exogenous transgenes introduced using such vectors do not integrate into the nuclear genome of the cell; therefore, in the absence of vector replication, the exogenous transgenes will degrade or dilute over time.

In one embodiment, the vector described herein allows for stable transfection of cells. In one embodiment, the vector allows for the incorporation of one or more transgenes into the genome of a cell. In one embodiment, the vector has a positive selection marker. Positive selection markers include any gene that allows cells to grow under conditions that kill cells that do not express said gene. Non-limiting examples include antibiotic resistance, such as genimycin (from the Neo gene of Tn5).

In one embodiment, the vector is a plasmid vector. In another embodiment, the vector is a viral vector. As those skilled in the art will understand, any suitable vector may be used. Suitable vectors are well known in the art.

In some embodiments, cells are transfected with mRNA encoding a target protein (e.g., CAR). Transfection of the mRNA results in transient expression of the protein. In one embodiment, the mRNA is transfected into the cells just before application. In one embodiment, "just before" application means between approximately 15 minutes and approximately 48 hours prior to application. Preferably, mRNA transfection is performed between approximately 5 hours and approximately 24 hours prior to application.

CD19

CD19 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. It has a single transmembrane domain, a cytoplasmic C-terminus, and an extracellular N-terminus. CD19 is a biomarker for normal and neoplastic B cells as well as follicular dendritic cells, and plays a crucial role in establishing the intrinsic B cell signaling threshold by regulating B cell receptor-dependent and independent signal transduction.

CD19 is expressed in most acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma. Most B-cell malignancies express CD19 at normal to high levels (80% in ALL, 88% in B-cell lymphoma, and 100% in B-cell leukemia). Although CD19 is a B-cell marker, it has also been observed in cases of myeloid malignancies, including 2% of AML cases. (Wang et al., Exp. Hematol. Oncol. [Experimental Hematology and Oncology], 2012 Nov 29; 1:36.). Table 1 shows non-limiting examples of CD19-associated malignancies.

Table 1: CD19 and related malignancies

CAR

Phenotypic changes that distinguish tumor cells from normal cells derived from the same tissue are typically associated with one or more changes in the expression of specific gene products, including the loss of normal cell surface components or the acquisition of other cell surface components (antigens undetectable in the corresponding normal non-cancerous tissue). Antigens expressed in neoplastic or tumor cells but not in normal cells, or antigens expressed at levels significantly higher in neoplastic cells than found in normal cells, are called “tumor-specific antigens” or “tumor-associated antigens.” Tumor-specific antigens have been used as targets for cancer immunotherapy. One such therapy utilizes chimeric antigen receptors (CARs) expressed on the surface of immune cells, including T cells and NK cells, to improve cytotoxicity against cancer cells. CARs contain a single-stranded variable fragment (scFv) linked to at least one intracellular signaling domain. The scFv recognizes and binds to antigens on target cells (e.g., cancer cells) and triggers effector cell activation. The signaling domain contains an activation domain (ITAM) based on the tyrosine residues of the immune receptor, which is crucial for intracellular signaling via the receptor.

This disclosure provides cells engineered to express at least one chimeric antigen receptor (CAR) on their cell surface. The CAR combines an extracellular antigen recognition portion (typically derived from a variable domain of a specific antibody) with an intracellular signaling domain (single or with other co-stimulatory elements) that can trigger a cell lysis response upon recognition of a specific antigen. Various types of CARs exist, all of which can be used in this application. First-generation CARs contain a cytoplasmic signaling domain. This domain can be derived, for example, from Fcε receptor γ (FcεRIγ) containing one ITAM, or from CD3ζ containing three ITAMs. CD3ζ CARs are believed to be more effective than FcεRIγ CARs in eradicating tumors. See, for example, Haynes et al., 2001, J. Immunology 166:182-187; Cartellieri et al., 2010, J. Biomed and Biotech, Vol. 2010, Article ID 956304. Second- and third-generation CARs combine multiple signal transduction domains, such as the cytoplasmic signal transduction domain of CD3ζ, and co-stimulatory signal transduction domains, such as CD28/CD134/CD137/ICOS and CD28/CD134, with a single CAR to promote cell activation and proliferation. Thus, in some embodiments, the CD19 CAR expressed by CD19t-haNK ^™ cells includes a hinge region from CD8 and/or a transmembrane domain from CD28. In some embodiments, the CD19 CAR includes a cytoplasmic signal transduction domain of FcεRIγ. In some embodiments, the CD19 CAR includes a cytoplasmic signal transduction domain of CD3ζ. Examples of hinge regions, transmembrane domains of CD28, and cytoplasmic signal transduction domains of FcεRIγ or CD3ζ are disclosed in U.S. Provisional Application No. 62/674,936, the entire contents of which are incorporated herein by reference.

Although previous publications, such as Haynes et al., 2001, J. Immunology 166:182-187 and Cartellieri et al., 2010, J. Biomed and Biotech, Vol. 2010, Article ID 956304, have disclosed that CD3ζCAR may be more effective than FcεRIγCAR in eradicating tumors, the inventors have surprisingly and unexpectedly discovered that the cells, compositions, and methods disclosed herein are not so. In fact, the inventors have found that when the cells disclosed herein have an FcεRIγCAR domain, they are as effective as those with CD3ζCAR, or even more effective in some embodiments.

Optionally, the CAR is specific for CD19. In some embodiments, CD19 is human CD19. In some embodiments, the CD19 CAR comprises an scFv fragment containing the amino acid sequence of SEQ ID NO:10. In some embodiments, the CD19 CAR comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, CD19 t-haNK ^™ cells comprise the nucleic acid sequence of SEQ ID NO:9, which encodes SEQ ID NO:10. In some embodiments, CD19 t-haNK ^™ cells comprise the nucleic acid sequence of SEQ ID NO:11, which encodes SEQ ID NO:12. In some embodiments, CD19 t-haNK ^™ cells comprise the tricistronic construct of SEQ ID NO:13.

In some embodiments, the CD19 CAR peptide comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:10. In some embodiments, epitope-tagged peptides, such as FLAG, myc, polyhistidine, or V5, may be added to the N-terminal domain of the peptide to aid cell surface detection using monoclonal or polyclonal antibodies against the epitope-tagged peptide.

In this example, variant peptides were prepared using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-direct mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985), or other known techniques can be used to generate CD16 variants (Ausubel, 2002; Sambrook and Russell, 2001).

In some embodiments, a polynucleotide mutation encoding the CD19 CAR is performed to alter the amino acid sequence encoding the CAR without changing its function. For example, a polynucleotide substitution causing an amino acid substitution at a "non-essential" amino acid residue can be made in SEQ ID NO: 9 or 11.

Conservative substitutions in SEQ ID NO: 10 or 12 (where an amino acid of one class is replaced by another amino acid of the same class) fall within the scope of the disclosed variants, provided that the substitution does not substantially alter the activity of the polypeptide. Conservative substitutions are well known to those skilled in the art. Non-conservative substitutions (which affect (1) the structure of the polypeptide backbone, such as β-sheet or α-helix conformation, (2) charge, (3) hydrophobicity, or (4) the volume of the target site side chain) can modify the functional or immunological properties of the polypeptide. Non-conservative substitutions require the exchange of a member of one class for another. Substitutions can be introduced into conserved substitution sites or, more preferably, into non-conservative sites.

In this example, variant peptides were prepared using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Direct site mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985), or other known techniques can be used to generate variants (Ausubel, 2002; Sambrook and Russell, 2001).

Optionally, CD19 t-haNK ^™ cells can be used to treat cancer, particularly cancers expressing CD19. Optionally, the cancer is selected from the group consisting of: leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including medulloblastic, promyelocytic, myelomonocytic, monocytic, and erythrocytic leukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia. Macroglobulinemia, heavy chain diseases, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial cell sarcoma, lymphangiosarcoma, lymphangioendothelial cell sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, and Wilms' tumor. Tumors, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

FC receptor

In some embodiments, cells are modified to express at least one Fc receptor, such that the at least one Fc receptor is presented on the cell surface. The Fc receptor binds to the Fc portion of an antibody. Several Fc receptors are known and vary depending on their preferred ligands, affinity, expression, and effects upon binding to the antibody.

Table 2. Illustrative Fc receptors

In some embodiments, cells are modified to express Fc receptor proteins on the cell surface.

In some embodiments, the Fc receptor is CD16. For the purposes of this disclosure, specific amino acid residues of CD16 are designated with reference to SEQ ID NO:2 or SEQ ID NO:1 (which differs from SEQ ID NO:1 at one position). Thus, when the CD16 polypeptide is maximally aligned with SEQ ID NO:2, the amino acid residue at “position 158” of the CD16 polypeptide is the amino acid residue at position 158 of SEQ ID NO:2 (or SEQ ID NO:1). In some embodiments, cells are modified to express human CD16, which has a phenylalanine residue at position 158 in the mature form of the protein, such as SEQ ID NO:1. In a typical embodiment, cells are modified to express a high-affinity form of human CD16, which has a valine residue at position 158 in the mature form of the protein, such as SEQ ID NO:2. Position 158 of the mature protein corresponds to position 176 of the CD16 sequence including the native signal peptide. In some embodiments, the CD16 polypeptide is encoded by a polynucleotide encoding the polypeptide sequence of the precursor (i.e., having a natural signal peptide) of SEQ ID NO:3 or SEQ ID NO:4. Thus, in one embodiment, the Fc receptor comprises FcγRIII-A (CD16). In some embodiments, cells are genetically modified to express an Fc receptor encoding a polypeptide having at least 90% sequence identity with SEQ ID NO:1 (FcγRIII-A or CD16 having a phenylalanine residue (F-158) at position 158); or having at least 90% identity with SEQ ID NO:2 (CD16 having a valine residue (F158V) at position 158, a higher affinity form).

In some embodiments, the polynucleotide encoding the CD16 polypeptide has at least about 70% polynucleotide sequence identity with the polynucleotide sequence encoding the full-length (including the signal peptide) naturally occurring CD16 (which has a phenylalanine residue at position 176 of the full-length CD16, corresponding to position 158 of the mature CD16 protein). In some embodiments, the polynucleotide encoding the CD16 polypeptide has at least about 70% polynucleotide sequence identity with the polynucleotide sequence encoding the full-length (including the signal peptide) naturally occurring CD16 (which has a valine residue at position 176, corresponding to position 158 of the mature protein). In some embodiments, the polynucleotide encoding CD16 has at least 70%, 80%, 90%, or 95% identity with SEQ ID NO:13 and includes a codon encoding valine at position 176 of the full-length (including the signal peptide) CD16 polypeptide. In some embodiments, the polynucleotide encoding CD16 comprises SEQ ID NO:13 but has a codon encoding valine at position 176 of the full-length CD16.

In some embodiments, the CD16 polynucleotide encodes a polypeptide having at least 70%, 80%, 90%, or 95% identity with SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 70%, 80%, 90%, or 95% identity with SEQ ID NO:2 and contains valine at position 158 as determined with reference to SEQ ID NO:2. In some embodiments, the polynucleotide encodes SEQ ID NO:2. In some embodiments, the CD16 polynucleotide encodes an extracellular domain of CD16 having or not having a signal sequence, or any other fragment of full-length CD16, or a chimeric receptor comprising at least a portion of the CD16 sequence fused to an amino acid sequence of another protein. In other embodiments, epitope-tagged peptides, such as FLAG, myc, polyhistidine, or V5, may be added to the N-terminal domain of a mature polypeptide to aid cell surface detection using monoclonal or polyclonal antibodies against the epitope-tagged peptide.

In some embodiments, the length of the homologous CD16 polynucleotide can be from about 150 to about 700, about 750, or about 800 polynucleotides, although CD16 variants having more than 700 to 800 polynucleotides are within the scope of this disclosure.

Homologous polynucleotide sequences include those sequences encoding polypeptide sequences that encode CD16 variants. Homologous polynucleotide sequences also include naturally occurring allelic variations associated with SEQ ID NO:1. Cells are transfected within the scope of this disclosure with a polypeptide having the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, its naturally occurring variants, or any polynucleotide sequence that is at least 70% identical or at least 80%, 90%, or 95% identical to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the homologous polynucleotide sequence encodes conserved amino acid substitutions in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, cells are transfected using a degenerate homologous CD16 polynucleotide sequence that is different from the natural polynucleotide sequence but encodes the same polypeptide.

In other instances, cDNA sequences with polymorphisms altering the CD16 amino acid sequence are used to modify cells, such as allelic variations between individuals exhibiting genetic polymorphism in the CD16 gene. In other instances, CD16 genes from other species with polynucleotide sequences different from the sequence in SEQ ID NO:1 are used to modify cells.

Variant peptides can be prepared using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-direct mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985), or other known techniques can be used to generate CD16 variants (Ausubel, 2002; Sambrook and Russell, 2001).

In some embodiments, a polynucleotide mutation encoding CD16 is performed to alter the amino acid sequence encoding CD16 without changing its function. For example, a polynucleotide substitution causing an amino acid substitution at a "non-essential" amino acid residue can be performed in SEQ ID NO:1 or SEQ ID NO:2.

Conservative substitutions in SEQ ID NO:1 or SEQ ID NO:2 (where an amino acid of one class is replaced by another amino acid of the same class) fall within the scope of the disclosed CD16 variants, provided that the substitution does not substantially alter the activity of the polypeptide. Conservative substitutions are well known to those skilled in the art. Non-conservative substitutions (which affect (1) the structure of the polypeptide backbone, such as β-sheet or α-helix conformation, (2) charge, (3) hydrophobicity, or (4) the volume of the target site side chain) can modify the functional or immunological properties of the CD16 polypeptide. Non-conservative substitutions require the exchange of a member of one class for another. Substitutions can be introduced into conserved substitution sites or, more preferably, into non-conservative sites.

In some embodiments, the CD16 polypeptide variant is at least 200 amino acids long and has at least 70%, or at least 80%, or at least 90% amino acid sequence identity with SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the CD16 polypeptide variant is at least 225 amino acids long and has at least 70%, or at least 80%, or at least 90% amino acid sequence identity with SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, as determined with reference to SEQ ID NO:2, the CD16 polypeptide variant contains valine at position 158.

In some embodiments, the nucleic acid encoding the CD16 polypeptide may encode a CD16 fusion protein. The CD16 fusion polypeptide comprises any portion of CD16 fused to a non-CD16 polypeptide or the entire CD16. Fusion polypeptides are conveniently generated using recombinant methods. For example, a polynucleotide encoding a CD16 polypeptide, such as SEQ ID NO:1 or SEQ ID NO:2, is fused within the frame to a non-CD16-encoding polynucleotide (such as a polynucleotide sequence encoding a signal peptide of a heterologous protein). In some embodiments, fusion polypeptides may be generated in which the heterologous polypeptide sequence is fused to the C-terminus of CD16 or located within CD16. Typically, up to about 30% of the CD16 cytoplasmic domains can be replaced. Such modifications can enhance expression or cytotoxicity (e.g., ADCC responsiveness). In other instances, chimeric proteins (such as domains from other lymphocyte activation receptors, including but not limited to Ig-a, Ig-B, CD3-e, CD3-d, DAP-12, and DAP-10) replace a portion of the CD16 cytoplasmic domains.

Fusion genes can be synthesized using conventional techniques, including automated DNA synthesizers and PCR amplification using anchored primers that generate complementary overhangs between two consecutive gene fragments, which can then be annealed and re-amplified to generate a chimeric gene sequence (Ausubel, 2002). Many vectors are commercially available that facilitate the subcloning of CD16-frame subclones into the fusion region.

Cytokines

Cellular cytotoxicity depends on the presence of cytokines such as interleukin-2 (IL-2). The cost of using exogenously added IL-2, necessary for maintaining and expanding cells in commercial-scale cultures, is very high. Administering sufficient amounts of IL-2 to human subjects to sustainably activate NK92 cells can cause adverse side effects.

In one embodiment, cells are modified to express at least one cytokine. Specifically, the at least one cytokine is IL-2 (SEQ ID NO: 6), IL-12, IL-15, IL-18, IL-21, or a variant thereof. In some embodiments, the cytokine is IL-2, IL-15, or a variant thereof. In some embodiments, IL-2 is a variant targeting the endoplasmic reticulum, and IL-15 is a variant targeting the endoplasmic reticulum.

In one embodiment, IL-2 is cloned and expressed using a signaling sequence (SEQ ID NO: 7) that directs IL-2 to the endoplasmic reticulum (erIL-2). This allows IL-2 to be expressed at a level sufficient for autocrine activation, but without releasing IL-2 extracellularly. See Konstantinidis et al., “Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to cells”, Exp Hematol. 2005 Feb; 33(2):159-64. The continued activation of FcR-expressing cells can be prevented, for example, by the presence of suicide genes.

Suicide gene

The term "suicide gene" refers to a transgenic gene that allows negative selection of cells expressing a suicide gene. Suicide genes are used as a safety system, allowing cells expressing the gene to be killed by the introduction of a selector. This is ideal when recombinant genes cause mutations leading to uncontrolled cell growth or when the cells themselves are capable of such growth. Many suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, cytosine deaminase gene, varicella-zoster virus thymidine kinase gene, nitroreductase gene, *E. coli* gpt gene, and *E. coli* Deo gene. Typically, the proteins encoded by suicide genes have no adverse effects on cells but kill them in the presence of specific compounds. Therefore, suicide genes are typically part of a system.

In one embodiment, the suicide gene is active in the cell. In one embodiment, the suicide gene is the thymidine kinase (TK) gene. The TK gene can be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Ganciclovir can be used to kill cells expressing the TK protein.

In another embodiment, the suicide gene is cytosine deaminase, which is toxic to cells in the presence of 5-fluorocytosine. Garcia-Sanchez et al., “Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential purging method for autologous transplantation.” Blood. July 15, 1998; 92(2):672-82.

In another embodiment, the suicide gene is cytochrome P450, which is toxic in the presence of ifosfamide or cyclophosphamide. See, for example, Touati et al., “A suicide gene therapy combining the improvement of cyclophosphamide tumor cytotoxicity and the development of an anti-tumor immune response.” Curr Gene Ther. 2014; 14(3):236-46.

In another embodiment, the suicide gene is iCasp9. Di Stasi, (2011) “Inducible apoptosis as a safety switch for adoptive cell therapy.” N Engl J Med 365:1673-1683. See also Morgan, “Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic” Molecular Therapy (2012); 20:11-13. iCasp9 induces apoptosis in the presence of the small molecule AP1903. AP1903 is a biologically inert small molecule that has shown good tolerability in clinical studies and has been used in the context of adoptive cell therapy.

Codon optimization

In some embodiments, the sequence codons of the construct used to transform aNK cells are optimized to maximize the expression efficiency of CD19 CAR, CD16, and/or erIL-2 in the human system. Codon optimization is typically performed by modifying the nucleic acid sequence by replacing at least one, more than one, or a large number of codons in the natural sequence with codons that are more commonly or most frequently used in the expression system gene. Codon optimization can be used to evaluate translation or to generate recombinant RNA transcripts with desired characteristics, such as a longer half-life, compared to transcripts generated using unoptimized sequences. Methods for codon optimization are readily available, for example, GeneArt ^™ from Thermo Fisher Scientific (Waltham, MA); Optimizer, freely available from http://genomes.urv.es/OPTIMIZER; and GeneGPS expression optimization technology from DNA 2.0 (Newark, California). In a particular embodiment, the coding sequence of the CD19 CAR is codon-optimized and comprises the sequence shown in SEQ ID NO:9.

Transgenic expression

Transgenic engineering can be performed into expression vectors using any mechanism known to those skilled in the art. In the case of inserting multiple transgenes into cells, the transgenes can be engineered into the same expression vector or different expression vectors.

In some embodiments, cells are transfected with mRNA encoding the transgenic protein to be expressed.

Transgenic genes and mRNA can be introduced into cells using any transfection method known in the art (including but not limited to infection, electroporation, lipid transfection, nuclear transfection, or "gene gun").

Expressing CD19 CAR cell

This disclosure provides modified cells expressing CD19 CAR and FcR. Optionally, the modified cells further express IL-2.

In some embodiments, the modified cells contain a polycistronic transgene that encodes a chimeric antigen receptor and an Fc receptor, and optionally encodes IL-2.

In some embodiments, FcR is CD16. In some embodiments, CD16 is high-affinity CD16, which comprises or consists of SEQ ID NO:2. In some embodiments, IL-2 is erIL-2, which comprises or consists of SEQ ID NO:7.

In some embodiments, the CD19 CAR coding sequence and the CD16 coding sequence are separated by a sequence encoding a self-cleaving peptide to produce equimolar expression levels of CD19 CAR and CD16 encoded by the same mRNA. Self-cleaving peptides and their coding sequences are well known, for example, as disclosed in Wang et al., Scientific Reports 5, article number 16273 (2015), the disclosure of which is incorporated herein by reference. Non-limiting examples of self-cleaving peptides include porcine tetanus virus-12A (P2A), thosea asigna virus 2A (T2A), equine rhinitis virus A 2A (E2A), cytoplasmic polyhedrosis virus (BmCPV 2A), and silkworm (B. mori) softening disease virus (BmIFV 2A). In some embodiments, the self-cleaving peptide is a P2A peptide encoded by SEQ ID NO:8: ggaagcggagctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacct.

In some embodiments, the CD16 coding sequence and the erIL-2 coding sequence are separated by an internal ribosome entry sequence (IRES), which allows translation to be initiated from an internal region of the mRNA transcribed from the nucleic acid sequence.

In some embodiments, the cell comprises a tricistronic construct expressing CAR, high-affinity CD16, and erIL-2 from a single mRNA. CAR integration enables effector cells to specifically engage with and kill target cells expressing the target recognized by the CAR; CD16 integration enables ADCC when combined with a therapeutic monoclonal antibody; and erIL2 allows cells to expand and maintain the selective pressure of transgene expression in the absence of exogenous IL-2. An illustrative tricistronic construct is shown in Figure 2.

To generate cells expressing CAR and modified CD16 (e.g., high-affinity CD16) and erIL-2, a polycistronic plasmid is introduced into aNK cells via, for example, electroporation. The transformed cells are grown in an IL-2-free medium, and individual clones can be selected from the transformed cells by limiting dilution clones and characterized according to criteria including, for example, high levels of CAR and CD16 expression, cytotoxicity, ADCC, growth rate, and/or IL-2 secretion. Suitable clones may also express surface markers such as CD3, CD16, CD54, CD56, NKG2D, and/or NKp30 at levels substantially similar to those of aNK cells. Optionally, whole-genome sequencing (WGS) is performed to determine transgene integration sites. Clones meeting one or more of these criteria can be selected for further development and for use in treating clinical patients.

Express

IL-2 expression can be confirmed by the ability of modified cells to grow under IL-2-free conditions. CAR and CD16 expression can be measured by flow cytometry. For cells transformed with a tricistronic construct containing coding sequences for CD19 CAR, CD16, and IL-2 (e.g., erIL-2), the transformed cells that are able to grow under IL-2-free conditions typically also show high levels of both CAR and CD16 expression in at least 70%, at least 80%, and at least 85% of the cells.

Optionally, methods well known in the art, such as ELISA, can be used to measure the IL-2 secretion level of transformed cells at various time points.

In some embodiments, the IL-2 level in the culture supernatant is measured to determine the level of IL-2 released into the cell culture medium. In some embodiments, the IL-2 level in the cell pellet is measured to assess the total intracellular level of IL-2. In some embodiments, both the amount of IL-2 in the supernatant and the amount of IL-2 in the cell pellet are measured to determine the total amount of IL-2 produced by the transformed cells.

Optionally, other surface markers of transformed cells can be measured by flow cytometry. These markers include, but are not limited to, CD54, CD56, NKG2D, NKp30, and CD3. Suitable clones are those that have demonstrated expression levels of these markers substantially similar to those of aNK cells under the same growth conditions.

Cytotoxicity

Optionally, the cytotoxicity of cells transformed with a tricistronic plasmid can be assessed using a flow-based cytotoxicity assay. Effector cells (cells) are mixed with fluorophore-labeled target cells, such as tumor cells, at different effector-to-target ratios. Propidium iodide (PI) can be added to the cells, and the sample can be analyzed by flow cytometry. Preferably, the fluorophore used to label the target cells can be distinguished from the PI in flow cytometry. In some embodiments, the fluorophore is CFSE. In some embodiments, the fluorophore is PKHGL67. Cytotoxicity can be determined by the percentage of PI-positive cells in a fluorophore-positive target population.

Optionally, the cytotoxicity of cells transformed with the tricistronic plasmid can also be tested using methods well known in the art. Cellular cytotoxicity can be reflected by its direct cytotoxicity or ADCC activity. The direct cytotoxicity of the resulting cells, their ability to target and kill abnormal cells such as tumor cells, can be assessed using methods well known in the art, such as a ^51Cr release assay performed using the procedure described by Klingemann et al. (Cancer Immunol. Immunother. [Cancer Immunology Immunotherapy] 33:395-397 (1991)) (Gong et al. (Leukemia [Leukemia], April; 8(4):652-8 (1994))). In some embodiments, target cells express an antigen that can be recognized by a CAR expressed on the surface of t-haNK ^™ cells. Briefly, ^51Cr -labeled target cells are mixed with cells and lysed. The percentage of a specific cytotoxicity can be calculated based on the amount of ^51Cr released. See US Patent Publication No. 20020068044.

Alternatively, a calcein release assay can be used to assess the direct cytotoxicity of the cells produced. For example, cells (referred to as effectors in the assay) can be mixed with target cells loaded with calcein (referred to as targets in the assay) at certain ratios. After incubation for a period of time, the amount of calcein released from the target cells can be assessed, for example, using a fluorescent plate reader.

The effector-to-target ratio used in each assay can vary; optionally, the effector:target ratio can be 20:1, 15:1, 10:1, 8:1, or 5:1; preferably, the effector:target ratio is 10:1. Target cells can be any cell expressing an antigen molecule that can be recognized by a CAR on the cell (t-haNK ^™ cell). For example, SUP-B15 cells can be recognized by a CD19 CAR and are target cells for CD19 t-haNK ^™ cells. The cytotoxicity value of the cells may vary depending on the type of target cells used and the effector:target ratio. Typically, cells produced using the methods described herein can have 60%–100%, for example, 70–100% or 80%–100% cytotoxicity. In some cases, when using an effector:target ratio of 1:10, cytotoxicity of cells can be determined by calcein release assays, for example, 82%-100%, 85%-100%, 87%-100%, 88%-100%, or 89%-100%.

Optionally, the cytotoxicity of the cells being evaluated, such as t-haNK ^™ cells, is antibody-dependent cytotoxicity (ADCC). The method for measuring ADCC activity of the cells is similar to the method described above for measuring direct cytotoxicity, but with the addition of an antibody that can recognize the target cells. The Fc receptors of NK cells recognize the cell-bound antibody and trigger a cell lysis reaction that kills the target cells. In an illustrative example, t-haNK ^™ cells can be incubated with Herceptin (an anti-Her2 antibody) and SKBr3 (target cells), and the killing of SKBr3 cells can be measured by releasing internal components of the target cells, such as ^51Cr or calcein described above, or by PI staining of the target cells.

Doubling time

The growth rate of cells, such as t-haNK ^™ cells, can be assessed by cell doubling time, which is the time required for cells to multiply to twice their initial number. Doubling time is inversely correlated with cell growth rate; the longer the doubling time, the lower the growth rate.

WGS

Optionally, whole-genome sequencing (WGS) of the transformed cells is performed to identify the insertion sites of the polycistronic construct.

Therapeutic applications

This disclosure also provides a method for treating any type of cancer in a subject at any stage of disease. Non-limiting examples of suitable cancers include carcinoma, melanoma, or sarcoma. In some embodiments, the invention is used to treat hematopoietic cancers, such as leukemia or lymphoma. In some embodiments, the cancer is a solid tumor.

In some embodiments, a method of treating a subject with any type of cancer includes administering a therapeutically effective amount of the aforementioned cells to the patient, thereby treating the cancer. In some embodiments, the cells express an Fc receptor, such as a high-affinity Fc receptor having the sequence shown in SEQ ID NO:2. In some embodiments, the cells express CD19CAR, the Fc receptor, and IL-2. In some embodiments, the modified cells comprise a polycistronic construct, and wherein the polycistronic construct encodes a chimeric antigen receptor and an Fc receptor.

Methods for treating subjects in need with the modified cells described herein are also provided. In some embodiments, the subject or patient has cancer or an infectious disease, such as a viral infection.

The modified cells can be administered to an individual in absolute numbers, for example, from about 1,000 cells/injection to up to about 10 billion cells/injection, such as about, at least about or at most about ¹ x 10⁸, ¹ x 10⁷, ⁵ x 10⁷, 1 x 10⁶, ⁵ x 10⁶, ¹ x 10⁵, ⁵ x 10⁵, ¹ x 10⁴, ⁵ x ^10⁴ , ¹ x 10³, 5 x ^10³ (etc.) cells per injection, or any range (including endpoints) between any two of these numbers. Therefore, this disclosure also provides a composition comprising a variety of cells, wherein the number of cells is ^1x10⁸ , ^1x10⁷ , ^5x10⁷ , ^1x10⁶ , ^5x10⁶ , ^1x10⁵ , ^5x10⁵ , ^1x10⁴ , ^5x10⁴ , ^1x10³ , or ^5x10³ (etc.).

In other embodiments, the individual may be administered at a rate of approximately 1,000 cells/injection/ ^m² to up to approximately 10 billion cells/injection/ ^m² , such as approximately, at least approximately, or at most approximately ¹ x 10⁸ cells/ ^m² , ¹ x 10⁷ cells/ ^m² , ⁵ x 10⁷ cells/ ^m² , ¹ x 10⁶ cells/ ^m² , ⁵ x 10⁶ cells/ ^m² , ¹ x 10⁵ cells/ ^m² , ⁵ x 10⁵ cells/ ^m² , ¹ x 10⁴ cells/ ^m² , ⁵ x 10⁴ cells/ ^m² , ¹ x 10³ cells/ ^m² , ⁵ x 10³ cells/ ^m² (etc.), or any range (including endpoints) between any two of these numbers.

In other embodiments, cells may be administered to the individual in relative quantities, such as from about 1,000 cells to up to about 10 billion cells/kg individual, such as about, at least about or at most about about ^1x10⁸ , ^1x10⁷ , ^5x10⁷ , ^1x10⁶ , ^5x10⁶ , 1x10⁵, ^5x10⁵ , ^1x10⁴ , ^5x10⁴ , ^1x10³ or ^5x10³ cells/kg individual, or any range (including ^endpoints ) between any two of these numbers.

In other embodiments, the total dose can be calculated in ^m² body surface area, including approximately ¹ x 10¹¹, ¹ x 10¹⁰, ¹ x 10⁹, ¹ x 10⁸, ¹ x 10⁷ cells/ ^m² , or any range between any two of these numbers (including the endpoints). The average human body surface area is approximately 1.6 to approximately 1.8 ^m² . In a preferred embodiment, between approximately 1 billion and approximately 3 billion cells are administered to the patient. In other embodiments, the amount of cells injected per dose can be calculated in ^m² body surface area, including ¹ x 10¹¹, ¹ x 10¹⁰, ¹ x 10⁹, ¹ x 10⁸, ¹ x 10⁷ cells/ ^m² . The average human body surface area is 1.6–1.8 ^m² .

In other embodiments, cells may be administered to the individual in relative quantities, such as from about 1,000 cells to up to about 10 billion cells/kg individual, such as about, at least about or at most about about ^1x10⁸ , ^1x10⁷ , ^5x10⁷ , ^1x10⁶ , ^5x10⁶ , 1x10⁵, ^5x10⁵ , ^1x10⁴ , ^5x10⁴ , ^1x10³ or ^5x10³ cells/kg individual, or any range (including ^endpoints ) between any two of these numbers.

Cells may be administered to a patient with cancer once during therapy, or they may be administered multiple times, such as once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks or more, or any range between any two of these numbers (including the endpoints).

In some embodiments, cells are applied in the form of a composition comprising cells and a culture medium, such as human serum or an equivalent thereof. In some embodiments, the culture medium comprises human serum albumin. In some embodiments, the culture medium comprises human plasma. In some embodiments, the culture medium comprises about 1% to about 15% human serum or a human serum equivalent. In some embodiments, the culture medium comprises about 1% to about 10% human serum or a human serum equivalent. In some embodiments, the culture medium comprises about 1% to about 5% human serum or a human serum equivalent. In a preferred embodiment, the culture medium comprises about 2.5% human serum or a human serum equivalent. In some embodiments, the serum is human AB serum. In some embodiments, a serum substitute acceptable for human therapeutics is used instead of human serum. Such serum substitutes may be known in the art or may be developed in the future. Although concentrations of more than 15% human serum may be used, concentrations greater than about 5% are contemplated to be costly. In some embodiments, cells are applied in the form of a composition comprising cells and an isotonic fluid solution supporting cell viability. In some embodiments, cells are applied in the form of a composition reconstituted from a cryopreserved sample.

Pharmaceutically acceptable compositions containing cells can contain a variety of carriers and excipients. A variety of aqueous carriers can be used, such as buffered saline solutions. These solutions are sterile and generally free of unwanted substances. Suitable carriers and excipients and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st edition, edited by David B. Troy, Lippicott Williams & Wilkins (2005). A pharmaceutically acceptable carrier is a material that is not biologically or otherwise unsuitable, i.e., that administration to a subject will not cause undesirable biological effects or interact with other components contained in the pharmaceutical composition in a harmful manner. If administered to a subject, the carrier is optionally selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject. As used herein, the term pharmaceutically acceptable is used synonymously with physiologically acceptable and pharmacologically acceptable. Pharmaceutical compositions typically contain reagents for buffering and preservation during storage, and may include buffers and carriers for appropriate delivery, depending on the route of administration.

These compositions for in vivo or in vitro use can be sterilized using cell-specific sterilization techniques. These compositions may contain acceptable adjuvants close to those required for physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The cell concentration in these formulations and/or other agents can vary and will be selected based on the specific administration method chosen and the needs of the subject, primarily according to fluid volume, viscosity, body weight, etc.

In one embodiment, the cells are administered to a patient along with one or more other treatments or agents for the cancer being treated. In some embodiments, one or more other treatments for the cancer being treated include, for example, antibodies, radiation, chemotherapy, stem cell transplantation, or hormone therapy.

In some embodiments, cells and other cancer agents/treatments are administered simultaneously or nearly simultaneously (e.g., within approximately 1, 5, 10, 15, 20, or 30 minutes of each other). In some embodiments, cells and other cancer agents/treatments are administered sequentially. In some embodiments, other cancer treatments/agents are administered one, two, or three days after cell administration.

In one embodiment, the other cancer agent is an antibody. In one embodiment, cells are administered together with an antibody that targets diseased cells. In one embodiment, cells and antibodies are administered to the patient in such a manner as: together, for example, in the same formulation; separately, for example, in separate formulations, simultaneously; or may be administered separately, for example, at different dosing schedules or at different times of the day. When administered separately, the antibody may be administered via any suitable route, such as intravenous or intratumoral injection.

In some embodiments, the cells disclosed herein are used in combination with therapeutic antibodies and/or other anticancer agents. Therapeutic antibodies can be used to target cells expressing cancer-related or tumor-related biomarkers. Table 4 shows examples of cancer therapeutic monoclonal antibodies. In some embodiments, the cells express Fc receptors, such as high-affinity Fc receptors having the sequence shown in SEQ ID NO:2. In some embodiments, these cells are cellular organisms. In one embodiment, the therapeutic antibody is avelumab.

Table 3. Illustrative Therapeutic Monoclonal Antibodies

Examples of FDA-approved therapeutic monoclonal antibodies

Examples of FDA-approved therapeutic monoclonal antibodies

The administration of such cells can be performed simultaneously with the administration of monoclonal antibodies, or sequentially. In some embodiments, the cells are administered to the subject after the subject has been treated with monoclonal antibodies. Alternatively, the cells can be administered concurrently with the administration of monoclonal antibodies, for example, within 24 hours.

In some embodiments, the cells are administered intravenously. In some embodiments, the cells are infused directly into the bone marrow.

Therefore, this disclosure provides a method for treating cancer or viral infection in patients in need, the method comprising administering a therapeutically effective amount of the cells disclosed herein to the patient, thereby treating cancer.

Reagent test kit

Kits for treating cancer or infectious diseases using compositions comprising the various cell types described herein are also disclosed. In some embodiments, the kits disclosed herein may further comprise at least one monoclonal antibody. The cells included in the kit express CAR and Fc receptors. In some embodiments, the cells further express IL-2, such as erIL-2, or IL-15, such as erIL-15. In some embodiments, the cells comprise a polycistronic construct, wherein the polycistronic construct encodes a chimeric antigen receptor, an Fc receptor, and optionally encodes IL-2 or IL-15.

In some embodiments, the kit may contain other compounds, such as therapeutically active compounds or drugs intended to be administered before, during, or after cell administration. Examples of such compounds include antibodies, vitamins, minerals, fludrocortisone, ibuprofen, lidocaine, quinidine, chemotherapeutic agents, etc.

In various embodiments, the kit's instructions for use will include guidance on using the kit components in the treatment of cancer or infectious diseases. The instructions may further contain information on how to handle cells (e.g., thawing and/or culturing). The instructions may further include guidance on dosage and frequency of administration.

In some embodiments, the kit further comprises one or more containers filled with one or more compositions described herein, such as compositions containing cells described herein. A label indicating the kit is intended for the treatment of cancer, such as the cancer described herein, may optionally be attached to such containers. Optionally, the label may also include notification of the manufacture, use, or sale of the drug or biological product in a form prescribed by a government agency, reflecting approval for human administration by the agency manufacturing, using, or selling it.

The following materials, compositions, and components are disclosed, which can be used in the disclosed methods and products of the compositions, can be used in combination with these products, can be used to prepare these products, or these products are disclosed. These and other materials are disclosed herein, and it should be understood that when combinations, subgroups, interactions, groups, etc., of these materials are disclosed, each is specifically contemplated and described herein, although specific references to the individual and overall combinations of these compounds and variations thereof may not be explicitly disclosed. For example, unless specifically indicated to the contrary, if a method is disclosed and discussed, and multiple modifications (including the method) that can be made to multiple molecules are discussed, each and every combination and variation of the method, and possible modifications thereof, are specifically contemplated and disclosed. Similarly, any subgroups or combinations thereof are also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure, including but not limited to steps in methods using the disclosed compositions. Therefore, if multiple other steps can be performed, it should be understood that each of these other steps can be performed by any particular method step or combination of method steps of the disclosed method, and each such combination or subgroup of combinations is specifically contemplated and should be considered disclosed.

Example

The following examples are for illustrative purposes only and should not be construed as limiting. Those skilled in the art can use various alternative techniques and procedures that will similarly allow one to successfully perform the following examples.

Example 1 : Producing CD19 CAR-modified cells

The CD19 CAR was cloned into the tricistronic plasmid pNEUKv l FcR_IL-2 vector, which also contained CD16 and erIL-2 transgenes. These tricistronic plasmids were electroporated into aNK cells. Cells expressing the CD19 CAR were selected using IL-2-deficient medium, as IL-2-dependent untransformed aNK cells could not survive in IL-2-deficient medium.

Restrictive dilution clone

Aliquots of polyclonal CD19 t-haNK ^™ cultures were diluted to a density of 3 cells/ml in growth medium without IL-2. This cell suspension was aliquoted into 96-well plates at 200 μl per well, equivalent to an average of 0.6 cells per well. The plates were incubated at 37°C for 10 days, followed by visual inspection of cell growth. A total of 20 cultures (now referred to as clones) were picked and transferred to larger containers, numbered according to their initial growth rate, with clones #1 through #10 being passaged first.

Example 2. Bioanalytical Methods

Cell culture:

Polyclonal and cloned CD19 t-haNK ^™ cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum (from CMV-negative test donors) and free of IL-2.

aNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum (from a CMV-negative test donor) and 500 IU/ml recombinant human IL-2.

haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum (from CMV-negative test donors) and free of IL-2.

K562 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and an antibiotic/antifungal agent mixture. K562 cells were passaged every 2–5 days or whenever the medium turned yellow.

SUP-B15 and SUP-B15 ^CD19KO/CD20+ cells were cultured in RPMI-1640 culture medium supplemented with 20% heat-inactivated fetal bovine serum, 55 μM β-mercaptoethanol, and an antibiotic/antifungal agent mixture. K562 cells were passaged as described above.

Antibody staining for flow cytometry analysis:

Cells were collected by centrifugation, washed twice in FACS buffer (5% FBS in 1X D-PBS), and resuspended in 1 ml of FACS buffer. For antibody staining with direct fluorophores conjugated to surface proteins, cells were incubated with an appropriate conjugated antibody (or isotype control) in the dark at 4°C for 20 min, followed by washing twice with FACS buffer. To detect CAR proteins, cells were incubated with biotinylated anti-F(ab') ₂ fragment antibody, followed by incubation with anti-streptolysin-APC antibody. Samples were analyzed using a MACSQuant flow cytometer.

Growth measurement:

Starting at an initial concentration of 1 x ^10⁵ cells/mL in growth medium supplemented with 5% heat-inactivated human AB serum (day 1), cultures were counted using an automated cell counter on days 3, 5, and 7. The growth rate was calculated using the following formula:

Doubling time (hours) =

[Duration (hours) x log(2)] / [log(final cell density) - log(initial cell density)]

Cytotoxicity:

Cell lines growing in suspension were resuspended by pipetting the cell culture. Cell viability was determined by automated counting (trypan blue exclusion method). Target cells were labeled with CFSE dye, and target and effector cells were diluted to the desired cell concentrations in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotic/antifungal agent. Effector and target cells were mixed in 96-well plates at different effector-to-target ratios (E:T of 20:1, 10:1, 5:1, 2.5:1, 1.25:1, 0.62:1, 0.31:1, and 0.15:1) and incubated for 4 h in a 5% CO2 atmosphere at 37°C. PI was then added to fluorescently label dead cells, and the assay was analyzed on a MACSquant flow cytometry device.

ADCC:

Cell lines growing in suspension were resuspended by pipetting the cell culture. Cell viability was determined by automated counting (trypan blue exclusion method). Target cells were labeled with PKH67-GL dye and diluted to the desired cell concentrations in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotic/antifungal agent. Target cells were pre-incubated for 30 min at room temperature with the monoclonal antibody trastuzumab, rituximab, or no antibody. The antibody-labeled target cells (and the antibody-free control) and effector cells were then mixed in 96-well plates at different effector-to-target ratios (E:T of 20:1, 10:1, 5:1, 2.5:1, 1.25:1, 0.62:1, 0.31:1, and 0.15:1) and co-incubated for 4 h in a 5% _CO2 atmosphere at 37°C. PI was then added to fluorescently label dead cells, and the assay was analyzed on a MACSquant flow cytometry device.

Quantitative analysis of IL-2

Cells for analysis were washed once in D-PBS to remove residual culture medium, then resuspended in fresh growth medium and aliquoted into triplicate into two 96-well plates at a density of ^10⁵ cells/well (200 μl/well). The plates were incubated at 37°C in a humidified incubator with 5% _CO₂ . After 24 h of incubation, one set of plates was analyzed, and the other set was analyzed at 48 h. Sample supernatant for analysis was prepared by a first centrifugation at 500 x g for 5 min to remove cells, followed by a second centrifugation at 2000 x g for 5 min to remove cell debris. The sample supernatant was frozen at -80°C until analysis. The cell pellet from the 500 x g centrifugation was resuspended, collected in triplicate, and the cell density was recorded. Using a human IL-2 ELISA kit (available from Thermo Fisher Scientific, Waltham, MA), measure the IL-2 concentration in the sample supernatant according to the manufacturer's instructions and compare it with the provided standards. Normalize the IL-2 concentration to cell counts at 24 and 48 h and express it as pg/ml/ ^10⁵ cells.

Example 3 : Phenotype of modified cells

The expression of CD19 CAR in CD19 t-haNK ^™ cells was measured by flow cytometry, and the results showed that CD19 t-haNK ^™ cells were able to grow in the absence of IL-2, and more than 80% of these cells expressed high levels of both CD16 (Fig. 3A) and CAR (Fig. 3B).

In a separate experiment, twenty selected clones were screened by flow cytometry for surface expression of CD19CAR (detected by biotinylated F(ab') ₂ fragment-specific primary antibody and streptavidin-APC secondary antibody) and CD16 (detected by 3G8 monoclonal antibody). Clones showing multiple positive populations, low CD16 staining intensity, or high background were discarded.

Table 4. Determination of the percentage and intensity of CD19CAR and CD16 expression on CD19 t-haNK ^™ clones

The expression profiles of six NK cell markers in the selected CD19 t-haNK ^™ clones were determined by antibody staining and flow cytometry and compared with aNK. CD3 expression was negative in all clones and aNK, while CD16 expression was negative only in aNK. CD54, CD56, NKp30, and NKG2D expression were positive in all clones, and their expression levels were similar to those of the aNK control.

Table 5. Percentage and intensity of NK cell surface marker expression on CD19 t-haNK ^™ clone.

Example 4 : Cytotoxicity of CD19 t-haNK ^™ cells to target cell lines

The cytotoxicity of CD19 t-haNK ^™ cells was analyzed by incubating them with target cells K562 cells, SUP-B15 cells, and SKBr cells. Figure 4A shows that CD19 t-haNK ^™ cells maintained cytotoxicity comparable to parental aNK cells upon killing K562 cells (target cells). 16B1 and 18B1 are two CD19 t-haNK ^™ populations obtained from two electroporation events performed on different dates.

In a separate experiment, the selected CD19 t-haNK ^™ clones were used as effectors in a flow cytometry-based in vitro cytotoxicity assay against the K562 target cell line (CD19-, NK-sensitive). In the 4-hour cytotoxicity assay, all clones exhibited potent cytolytic activity against K562. At a 10:1 ratio, the mean maximum killing efficiency of the CD19 t-haNK ^™ clones ranged from 70.9% ± 10.1% to 84.4% ± 0.6%, compared to 84.1% ± 2.4% for the aNK control (n = 2 to 5). See Figure 4B.

Figure 5A shows that CD19 t-haNK ^™ cells exhibit resistance to aNK ^™ , with enhanced specific killing of CD19-positive SUP-B15 cell lines – at an effector-to-target ratio of 10, approximately 80%-90% of cells were killed by CD19 t-haNK ^™ cells, compared to only approximately 10%-20% of cells being killed by aNK cells.

In a separate experiment, selected CD19 t-haNK ^™ clones were used as effectors against SUP-B15 target cell lines (CD19+, NK-resistant) in an in vitro cytotoxicity assay based on flow cytometry. All clones were able to effectively target and kill resistant SUP-B15 cells in a 4-hour cytotoxicity assay. At a 10:1 ratio, the mean maximum killing efficiency of the CD19 t-haNK ^™ clones ranged from 85.7 ± 0.1% to 92.2 ± 1.2%, compared to 10.8 ± 7.4% for the aNK control (n = 2 to 5). See Figure 5B.

Figure 6A shows that when combined with the anti-Her2/neu antibody Herceptin, the ADCC activity of CD19 t-haNK ^™ cells against SKBr3 cells (CD19-, Her2/neu+) was comparable to that of cells expressing only the CD16 (158V) receptor.

In another experiment, selected CD19 t-haNK ^™ clones were used as effectors against modified SUP-B15 target cell lines (CD19-, CD20+, Her2-neu-, NK-resistant) in an in vitro ADCC assay based on flow cytometry, in combination with either anti-CD20 rituximab or anti-Her2-neu trastuzumab monoclonal antibodies. In a 4-hour cytotoxicity assay, all clones effectively targeted and killed resistant SUP-B15 CD19KO/CD20+ cells when combined with the anti-CD20 antibody rituximab. At a 10:1 ratio, the maximum killing efficiency of the CD19 t-haNK ^™ clones ranged from 63.7% to 77.8%, compared to 67.1% for the control (n = 1 to 2). When combined with the anti-Her2/neu control antibody trastuzumab, neither the CD19t-haNK ^™ clone nor the CD19t-haNK™ clone could kill the target SUP-B 15CD19KO/CD20+ cells (at a 10:1 ratio, the maximum killing efficiency of the CD19t-haNK ^™ clone was between 7.7% and 21.9%, while the control was 4.1%). At a 10:1 ratio, the ADCC-mediated killing efficiency of the CD19t-haNK ^™ clone was between 46.4% and 65.2%, compared to 62.7% for the control. See Figure 6B.

Example 5 : Other characteristics of CD19 t-haNK ^™ cells

Without altering the culture medium, the population doubling time of selected CD19 t-haNK ^™ clones was determined by 7-day cell growth assays, and the mean doubling time was calculated. Compared to the 34.5 hours for the aNK control, the population doubling time for all clones ranged from 33.1 to 54.5 hours. See Figure 7.

CD19 t-haNK ^™ clones were cultured at a density of 10e5 cells/ml in IL-2-free 6-well plates, and the culture supernatant was harvested after 24 and 48 h. The supernatant was analyzed by ELISA to detect and measure the potential ERIL-2 release from CD19 t-haNK ^™ cells. At 24 h of culture, CD19 t-haNK ^™ clones released between 16.1 and 1278.5 pg/ml/10⁵ cells. See Figure 8.

Example 6 : CD19 t-haNK ^™ : Evaluation of the antitumor activity of CD19-targeted t-haNK ^™ cells in intravenous and subcutaneous models of Raji human Burkitt's lymphoma in NSG mice.

CD19 t-haNK ^™ is a natural killer cell that expresses a chimeric antigen receptor (CAR) targeting CD19 to treat B-cell lineage hematologic malignancies. In this study, the antitumor effect of repeated intravenous (IV) administration of CD19 t-haNK ^™ was evaluated in intravenous and subcutaneous (SC) Raji xenograft models in NSG mice. In both models, CD19 t-haNK ^™ cells demonstrated significant therapeutic efficacy. Specifically, in the intravenous tumor model, CD19 t-haNK ^™ cells significantly improved animal survival compared to the mediator control. In the subcutaneous tumor model, CD19 t-haNK ^™ significantly inhibited tumor growth, reduced the number of morbidity/mortality events, and significantly reduced the metastatic disease burden in the liver.

Previous studies have shown that targeted aNK cells expressing a chimeric antigen receptor (CAR) against CD19 are effective in Raji tumor-bearing NSG mice, likely due to target-specific cytotoxicity in CAR-expressing cells (see, e.g., Oelsner et al., Cytotherapy, 2017). In this study, the efficacy of CD19 t-haNK ^™ cells was evaluated in two different variants of the Raji xenograft model: 1) intravenously (IV) seeded Raji cells; and 2) subcutaneously (SC) seeded Raji tumors, both models receiving repeated intravenous administrations of CD19 t-haNK ^™ cells. Note that other animal groups (groups B and E) were also included in the original study protocol for evaluation in this model, but are not included in this report as they were not relevant to the determination of CD19 t-haNK ^™ efficacy (see Table 6 for a brief experimental design).

Example 7 : Materials used in CD19 t-haNK ^™ research

CD19 t-haNK ^™ cells (clone 19.6): CD19 t-haNK™ cells were cultured in growth medium supplemented with 5% heat-inactivated human AB ^serum , following a protocol provided by the Process Development, Inc., Torrey Pines.

Test animals: The test animals used were female NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ (NSG) mice, 9–10 weeks old at the start of the study (after isolation) and weighing between 20–27 grams at the start of the study. Twenty animals were used for the intravenous tumor model, and 12 for the subcutaneous tumor model. The animals were supplied by The Jackson Laboratory (610 Main Street Bar Harbor, ME04609, USA). Sterile stainless steel ear tags were applied to each mouse using a handheld applicator for identification. Additionally, each cage had a cage card containing the study number and animal identification number.

Raji Cancer Cell Line: Raji cells were initially purchased from ATCC (Catalogue No. CCL-86 ^™ ; Lot No. 61723871) and then expanded and prepared by the Preclinical Development, Inc. of Southwest. These cells were validated by IDEXX on March 18, 2018 (see Appendix 2 for the validation report). The cell culture medium was ATCC-prepared RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin (100 U/mL) and streptomycin (100 μg/mL). Raji cells in the exponential phase (passage 12) were collected by centrifugation. Cells were washed and resuspended in serum-free medium at a concentration of ⁵ x 10⁵ viable cells/mL for intravenous seeding, and then resuspended in Matrigel (1:1 v/v) at a concentration of 2.5 x ^10⁶ viable cells/mL for subcutaneous implantation. Cells were stored on ice prior to animal injection. The cells used in the in vivo study had 96% viability.

Raji intravenous model: 0.2 mL of Raji cell suspension (1 x ^10⁵ cells inoculum) was injected intravenously into 20 animals via the lateral caudal vein using a 27-gauge needle.

Raji subcutaneous model: 0.1 mL of Raji cell suspension (2.5 x ^10⁵ cells inoculum) was implanted subcutaneously into the two flanks of 12 animals using a 25-gauge needle.

Example 8 : Experimental Procedure for CD19 t-haNK ^™ Research

Intravenous Raji model: Within 24 hours of cancer cell inoculation (defined as day 1), 20 animals were pseudo-randomized into two groups of 10 each to achieve similar mean body weight between groups. On days 2, 5, 8, 10, 12, and 17, CD19 t-haNK ^™ cells in the exponential growth phase were harvested by centrifugation and prepared in growth medium at a concentration of ⁵ x 10⁷ cells/mL. The cells were then administered intravenously at a dose of ¹ x 10⁷ cells per mouse via an injection volume of 200 μL. As shown in Table 6, animals in group A received a mediator control, while animals in group C received CD19 t-haNK ^™ cells.

Weigh the animals before injecting tumor cells and twice a week. Observe the animals daily for mortality/morbidity (G0 to G4) and clinical signs of toxicity (T1 to T12; see Table 6). Euthanize paralyzed or dying animals. Euthanize animals by inhaling _CO2 followed by cervical dislocation. Record death events (euthanasia or spontaneous death) in a death log and calculate to determine the survival curve.

Subcutaneous Raji model: After subcutaneous tumor implantation, tumor formation in animals was examined at least twice a week. When the tumor became visible, tumor volume (TV) was measured once or twice a week using digital handheld calipers and calculated using the formula: TV = length x ^width² /2 [length is the maximum diameter of the tumor, and width is the shortest diameter]. When the average tumor volume reached an injectable size (195 ^mm³ in this case; 24 days post-implantation), 12 tumor-bearing animals were pseudo-randomized into two groups of six each to achieve similar tumor volumes between groups. This was defined as day 0. On days 1, 4, 7, 9, 11, and 13, CD19 t-haNK ^™ cells in the exponential growth phase were harvested by centrifugation, irradiated with 1000 cGy γ, and prepared in growth medium at a concentration of 5 x ^10⁷ cells/mL. The cells were then administered intravenously in 200 μL injection volumes at a dose of 1 x ^10⁷ cells per mouse. As shown in Table 6, animals in group D received the mediator solution, while animals in group F received CD19 t-haNK ^™ cells. Animals were weighed before injection of tumor cells and then twice weekly.

Animal mortality/morbidity (G0 to G4) and clinical signs of toxicity (T1 to T12) were observed daily. Paralyzed or dying animals were euthanized. Once dying animals showed signs of illness, they were euthanized, while surviving animals were euthanized on a pre-planned basis for tissue collection. Specifically, on day 13, 6 hours after the last dose of the test substance, half of the surviving animals (up to 3 mice/group) were euthanized. On day 15, 48 hours after the last administration, the remaining animals were euthanized. Euthanasia was performed by cervical dislocation while the animals were under deep anesthesia following eventual intracardiac hemorrhage. Blood/serum samples were not analyzed in this part of the study and are therefore not included in this report.

After termination, an autopsy was performed, and organs with obvious gross lesions were collected, fixed in 10% formalin, and sent to a contract pathology laboratory (Seventh Wave Laboratories) for histological evaluation of tumor/metastatic disease burden.

Table 6: Research Design (Simplified)

IR, irradiation (1000 cGy); non-IR, non-irradiation; IV, intravenous; SC, subcutaneous; Tx, treatment.

Example 9 : Data Analysis of the CD19 t-haNK ^™ Study

The tumor volume is calculated using the following equation: Tumor volume = length x ^width² / 2 (where length and width are the longest and shortest diameters of the tumor, respectively).

Tumor growth inhibition (TGI) was calculated as follows: TGI = ( _TC - _Tt ) / _ΔTC x 100%, where _TC and _Tt are the mean tumor volumes of the control and treatment groups, respectively, at the end of the study, and _ΔTC is the change in mean tumor volume in the control group.

Tumor growth curves were analyzed using a two-factor ANOVA followed by multiple comparisons using the Tukey test. Survival curves were analyzed using the Mantel-Cox log-rank test. Differences in liver metastatic disease burden across days were analyzed using unpaired two-tailed t-tests. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 7.

Example 10 : Results of the intravenous Raji model from the CD19 t-haNK ^™ study

The primary readout in the intravenous tumor model was animal survival. Mortality events were counted when animals died or were euthanized due to disease-related morbidity and/or paralysis. As shown in Figure 9, CD19 t-haNK ^™ cell treatment significantly improved animal survival compared to the vector control, resulting in a median survival of 27 days, compared to 21.5 days in the vector control group (P < 0.0001). Animal weight changes were also monitored throughout the study. As shown in Figure 10, animals treated with CD19 t-haNK ^™ exhibited moderate (less than 10%) and short-term weight loss at the start of initial treatment, which is uncommon in animals receiving intravenous NK infusions and is not specific to CD19 t-haNK ^™ cells. Weight was able to recover after the first week of treatment, but then declined again due to disease progression.

Example 11 : Results of the subcutaneous Raji model from the CD19 t-haNK ^TM study

The primary readout in the subcutaneous tumor model was tumor growth. As shown in Figure 11, CD19t-haNK ^™ cells exhibited significant and statistically significant inhibition of tumor growth on and after day 7, with a TGI of 49% at the end of the study (day 13).

Furthermore, because Raji is an aggressive lymphoma model, cancer cells were able to spread and generate multiple metastatic sites even during subcutaneous inoculation, ultimately leading to morbidity and/or death in the animals. In the vector group, three animals (50%) were near death between days 11 and 13 and were therefore euthanized. In contrast, no unplanned deaths occurred in the CD19 t-haNK ^™ cell group (Table 7).

Furthermore, a qualitative reduction in liver metastases was observed in animals treated with CD19 t-haNK ^™ during autopsy (Fig. 12a). Semi-quantitative estimates of disease burden were performed on representative samples of H&E-stained liver sections by a contract pathology laboratory (Seventh Wave Laboratory). As summarized in Fig. 12b and Table 8, a clear trend of increasing disease burden was observed as the study progressed. Animals treated with CD19 t-haNK ^™ exhibited a significantly lower percentage of cancer-infiltrated areas in their livers compared to the mediator control. Due to the small sample size and unplanned early death in the control group, statistical analysis was only possible for data from day 13. This analysis revealed a significant difference in disease burden, with a mean infiltration of 10% in CD19 t-haNK ^™ -treated animals compared to 30% in the control group. Body weight changes were monitored throughout the study, and similar to the intravenous Raji model, animals treated with CD19 t-haNK ^™ exhibited moderate (less than 10%) and transient weight loss at the start of the treatment regimen (Fig. 13).

Table 7: Mortality/Death Records of Animals in the Subcutaneous Raji Model

Scheduled: Scheduled euthanasia for the purpose of tissue collection.

Table 8: Percentage of liver tumor cells involved

Example 12 : Conclusions of the CD19 t-haNK ^™ study

To evaluate the antitumor efficacy of CD19 t-haNK ^™ cells in repeated intravenous administration regimens, two variants of the Raji xenograft model, involving intravenous and subcutaneous tumor inoculation, were used in this study. In the intravenous tumor model, CD19 t-haNK ^™ cells significantly improved animal survival compared to the vector control, extending median survival by 5.5 days (an increase of 26%). In the subcutaneous tumor model, CD19 t-haNK ^™ cells significantly inhibited tumor growth, resulting in a TGI of 49% at the end of the study. Furthermore, CD19 t-haNK ^™ treatment reduced the number of morbidity/mortality events (0/6 in CD19 t-haNK ^™ -treated animals compared to 3/6 in the control group) and significantly reduced the metastatic disease burden in the livers of subcutaneously Raji tumor-bearing animals. Overall, CD19 t-haNK ^™ cells demonstrated significant therapeutic efficacy in both variants of the Raji xenograft model compared to the vector control.

It will be apparent to those skilled in the art that further modifications are possible beyond those already described without departing from the inventive concept of this document. Therefore, the subject matter of this invention is limited only to the scope of the appended claims. Furthermore, in interpreting the specification and claims, all terms should be interpreted in the broadest possible way, consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the mentioned element, component, or step may be present, used, or combined with other elements, components, or steps not explicitly mentioned. When a claim in the specification relates to at least one element selected from the group consisting of A, B, C, ..., and N, the word should be interpreted as requiring only one element from that group, rather than A plus N, or B plus N, etc.

Claims (21)

1. An NK-92 cell expressing a CD19 CAR and an Fc receptor for suppressing metastatic burden, wherein the NK-92 cell comprises a polycistronic DNA construct, and wherein the polycistronic DNA construct encodes: 1) the CD19 CAR, wherein the CD19 CAR consists of the amino acid sequence of SEQ ID NO: 12; 2) CD16; and 3) IL-2 or erIL-2. 2. The NK-92 cell of claim 1, wherein the CD16 comprises SEQ ID NO: 2. 3. The NK-92 cell of claim 1, wherein the coding sequences of one or more of the CD19 CAR, CD16, and erIL-2 are codon-optimized for expression in a human system. 4. The NK-92 cells as described in any one of claims 1-3, wherein the NK-92 cells are capable of killing CD19-expressing cells. 5. The NK-92 cells as described in claim 4, wherein the CD19-expressing cells are tumor cells. 6. The NK-92 cells of claim 5, wherein the tumor cells are SUP-B15 cells. 7. The NK-92 cells of claim 1, wherein the polycistronic DNA construct comprises the sequence of SEQ ID NO: 9, wherein the sequence encodes an anti-CD19 scFv antibody fragment. 8. The NK-92 cell of claim 1, wherein the NK-92 cell comprises a sequence encoding a self-cleaving peptide, wherein the sequence is located between the CD19 CAR and CD16, and wherein the sequence allows for equimolar expression of the CD19 CAR and the CD16. 9. The NK-92 cell of claim 1, wherein the NK-92 cell contains an internal ribosome entry sequence (IRES) between the sequence encoding CD16 and the sequence encoding IL-2 or erIL-2. 10. The NK-92 cells of claim 4, wherein when the ratio of effector to target is 10, the direct cytotoxicity of the NK-92 cells to CD19-expressing cells is 70%-100%. 11. A kit comprising a pharmaceutical composition containing NK-92 cells as described in claim 1. 12. A method for generating NK-92 cells, said NK-92 cells for suppressing metastatic burden, said method comprising: Provided a polycistronic vector, wherein the polycistronic vector encodes a CD19 CAR, CD16, and IL-2 consisting of the amino acid sequence of SEQ ID NO: 12, and The vector was introduced into NK-92 cells to generate the NK-92 cells. 13. The method of claim 12, wherein the vector comprises a sequence encoding a self-cleaving peptide, wherein the sequence is located between CAR and CD16, and wherein the sequence allows for equimolar expression of CAR and CD16. 14. The method of claim 12, wherein the vector contains an internal ribosome entry sequence (IRES) between the CD16 coding sequence and the IL-2 coding sequence. 15. Use of the composition in the preparation of a medicament for treating a subject with B-cell malignancies, wherein the composition comprises a therapeutically effective amount of the NK-92 cells as claimed in claim 1. 16. The use as claimed in claim 15, wherein the composition comprises 1 x ^10⁸ to 1 x ^10¹¹ modified cells per ^m² of the subject's body surface area. 17. The use as claimed in claim 15, wherein the B-cell malignancy is one or more of the following: B-acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-non-Hodgkin's lymphoma, refractory follicular lymphoma, and mantle cell lymphoma. 18. The use as described in claim 15, wherein the B-cell malignancy is a B-cell malignancy following hematopoietic stem cell transplantation. 19. The use as described in claim 15, wherein the B-cell malignancy is a B-lineage lymphatic system malignancy following unrelated cord blood transplantation. 20. The use as described in claim 15, wherein a plurality of the NK-92 cells are formulated for intravenous administration. 21. The use as described in claim 15, wherein a plurality of the NK-92 cells are formulated for intratumoral administration.