Patent ID: 12252525

DETAILED DESCRIPTION

An antigen binding domain (e.g., a single-chain Fv domain (scFv)) may be fused to a CAR scaffold, wherein said scFv gene comprises a VL domain linked to a VH domain via a flexible linker (e.g., SG25spacer), wherein the CAR scaffold encodes an extracellular domain, a transmembrane domain, and a cytoplasmic domain capable of activation of an immune cell (e.g., such as a lymphocyte cell or a cytotoxic cell). The resultant construct provides antibody or antibody-like specificity to the immune cell due to the antigen binding domain (e.g., an scFv), and the ability to activate the immune cell due to the gene segment comprising the cytoplasmic domain. These techniques may be implemented in T cells, mast cells, and NK cells as well as other suitable cell types.

According to present embodiments, CAR scaffolds and codon-optimized versions of CAR scaffolds are provided for expression of said constructs in immune or other cytotoxic cells, including NK cells, wherein the CAR scaffold is coupled to an antigen binding molecule. In some aspects, these CAR scaffolds are codon optimized for expression in immune or other cytotoxic cells for improved efficacy.

In some aspects, the CAR scaffold may comprise a CD28 domain coupled to a CD3ζ domain or the complete CD3ζ domain, which is joined to the scFv. For example, the sequence may comprise a leader sequence coupled to an scFv domain coupled to a CAR scaffold, wherein the scFv comprises: VL domain—spacer—VH domain or VH domain—spacer—VL domain.

In some aspects, the spacer may be a SG spacer (e.g., SG25), however, the spacer may comprise any arrangement of amino acids providing sufficient flexibility within the scFv to allow the scFv to bind to its intended target.

In still other embodiments, the leader sequence may be a CD64 leader signal sequence. The CD64 signal sequence may comprise a signal peptide directing transport of the scFv-activation domain to the surface of the cell (e.g., immune or cytotoxic cell).

In some embodiments, the antigen binding molecule may be attached directly or through a linker to the activation region (e.g., this may be accomplished by recombinant expression of a nucleotide encoding the antigen binding molecule, an optional spacer, and a sequence encoding the activation domain). The recombinant expression system may be transfected into a host cell, wherein the fusion protein is expressed, constitutively or inductively.

The antigen binding domain may comprise a leader sequence, a variable region, a linker, and another variable region. The nucleotide sequences encoding these domains may be codon optimized for expression in a particular cell line, leading to higher expression levels of the CAR-based construct and improved efficacy over the non-codon optimized counterparts. It is noted that codon optimization does not alter the polypeptide sequence, but may change codon frequency to optimize expression in host organisms.

Any suitable antigen binding molecule may be attached to the tail activation domain. In some aspects, the antigen binding molecule may comprise a VH and VL domain. The VH and VL domain may be obtained from any suitable monoclonal antibody.

In some aspects, antigen binding molecules may include scFvs, antibodies, aptamers (short sequences of DNA, RNA, or Xeno nucleic acid (XNA) nucleotides), peptides, proteins, protein scaffolds, fusion proteins, or any other suitable molecule that may be engineered to specifically bind to target antigens. In some embodiments, protein scaffolds are related to or derived from peptide aptamers.

In other aspects, the nucleic acid sequence encoding the CD28 domain may comprise a nucleic acid encoding, partially or entirely, the CD28 ectodomain, the CD28 transmembrane domain, and the CD28 cytoplasmic domain.

In still other aspects, CD28 domain may be coupled to the CD3ζ activation domain. The nucleic acid sequence encoding the CD3ζ activation domain may encode at least two ITAMs, at least three ITAMs, etc.

Exemplary sequences are provided as follows. In one aspect, a CAR-19slh28ζ scaffold may comprise a tail end activation domain, such as SEQ ID NO.: 21. This sequence, SEQ ID NO.:21, comprises various domains, including an ectodomain (encoded by SEQ ID NO.: 27), a transmembrane domain (encoded by SEQ ID NO.: 28), and a cytoplasmic domain (encoded by SEQ ID NO.: 29).

In another aspect, the CAR scaffold contains a CD3ζ domain that is codon optimized, such as SEQ ID NO.: 23. This sequence, SEQ ID NO.: 23, also comprises various domains, including an ectodomain (encoded by SEQ ID NO.: 24), a transmembrane domain (encoded by SEQ ID NO.: 25), and a cytoplasmic domain (encoded by SEQ ID NO.: 26). Codon optimization may improve expression and thereby improve activity/efficacy of the CAR-based therapeutic composition in its target host cell (e.g., NK cells).

In other aspects, either of the CAR scaffolds (encoded by SEQ ID NO.: 5 or SEQ ID NO.: 22) may be attached to an antigen binding domain comprising a CD64 leader signal sequence (encoded by SEQ ID NO.: 1), a variable heavy (VH) or variable light (VL) chain (see figures for various VH and VL domains), and a GS spacer (encoded by SEQ ID NO.: 3, SEQ ID NO.: 10, SEQ ID NO.: 13, or SEQ ID NO.: 30), wherein the GS spacer joins the VH and VL domains.

In some aspects, sequences having 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over the full sequence or a specified region of the sequence (e.g., nucleotide sequences encoding VH and/or VL domains, CAR scaffold, etc.) are contemplated to fall within the scope of the embodiments provided herein.

Percent identity refers to a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, over the entire length of the sequence or over a designated region of the sequence. A region may comprise at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, or more. A region may comprise at least 10 amino acids, at least 20 amino acids, at least 50 amino acids, or more.

As used herein, “antigen binding domain” refers to a molecule that specifically binds to an antigen on a cell surface (e.g., a malignant or cancer cell). The antigen binding domain includes but is not limited to single-chain Fvs (scFvs) and other antibody fragments as well as affimers, aptamers, peptides, proteins, small molecules, etc. Other antibody fragments include but are not limited to F(ab′)2, Fab2, Fab′, Fab, Fv, scFv-Fc, VhH, disulfide-linked Fvs (sdFv), etc. or any active fragment thereof, i.e. antibody fragments or other molecules that immunospecifically bind to an antigen (e.g., EGFR, IGFR-1, FRP5, PD-L1, etc.) or a variant thereof.

It is also understood that any one or more of the CDRs of any antibody fragment may be grafted onto the antigen binding domains described herein. The heavy chains and light chains have a general structure of relatively conserved framework regions (FR) joined by three hyper variable regions or CDRs (CDR1, CDR2, CDR3). The CDRs from the heavy and the light chains, which are aligned by the framework regions, enable binding to the antigen. One of skill in the art would be able to ascertain the CDRs based on known techniques.

The VH and VL domains may be derived from any suitable antibody, including but not limited to monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, murine antibodies, conjugated antibodies (e.g., to a chemotherapeutic agent, to a radionuclide, to another protein, etc.), synthetic antibodies, bispecific antibodies, chimeric antibodies, single chain antibodies, antibody fragments produced by a Fab expression library, antibody fragments produced by mRNA display or phage display, and monovalent immunoglobulins (e.g., IgG).

Antibodies or fragments thereof may be generated using any suitable technique known in the art, including hybridoma technology, generation of phage displayed scFvs, generation of mRNA displayed scFvs or peptides, or isolation and screening of antisera, chemical synthesis, or through the use of recombinant expression systems.

Antibody fragments (e.g., scFvs) may be screened for binding to a suitable antigen according to techniques known in the art. Affinity maturation may be employed to improve the affinity of a scFv for its desired target. Therapeutic compositions that are administered to a patient include complexes comprising the antibody fragments (e.g., scFvs, etc.) described herein and are usually human or humanized.

Antibody fragments (e.g., scFvs) may be of any origin including but not limited to human, murine (e.g., mouse and rat), donkey, rabbit, goat, guinea pig, bird, camel, horse, or chicken. For therapeutic purposes, antibodies that are human or that have been humanized are preferred.

Any methodology known in the art for screening large combinatorial libraries to identify antibody fragments that bind to an antigen may be applied, including but not limited to phage display, yeast surface display, ribosome display, or mRNA display, or any combination thereof (see, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas (1981) 563-681; WO 98/31700).

As used herein, “specifically binds” typically refers to non-covalent interactions between a target entity (e.g., a cell) and the antigen binding domain, and usually refers to the presence of such an interaction with a particular structural feature (e.g., such as an antigenic determinant on the cell surface) of the target entity with the antigen binding domain. As understood by one of skill in the art, an interaction is considered to be specific if it occurs in the presence of other alternative interactions.

As used herein, “composition” or “pharmaceutical composition” refers to a formulation comprising a therapeutic cell that is delivered to a patient comprising the CAR scaffold coupled to an antigen binding domain and may include one or more additional ingredients (e.g., buffers, excipients, stabilizers, diluents, emulsifiers, preservatives, etc.).

As used herein, and unless the context dictates otherwise, the term “linked to” or “linked with” is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements).

In other embodiments, assays may be performed to determine the effectiveness of the compositions. For such detection, an assay that includes the steps of culturing tumor cells under conditions suitable for growth and in a tumor microenvironment, contacting the cells with the CAR-based therapeutic as described herein, and then evaluating whether the CAR-based therapeutic reduces or inhibits the size of the mass may be used.

Components of the compositions disclosed herein can be organized in nearly any manner provided that functional activity for which the complex was designed is maintained. Additionally, the complexes described herein may include one or more tags, e.g., to facilitate modification, identification and/or purification of the components of the complex.

Linkers/Spacers

Linker sequences may be used to link the VH and VL domains of the scFvs, while maintaining desired functional activity of the VH and VL domains. As used herein and unless otherwise noted, the terms “linker sequence,” “linker,” “spacer,” and “G-S spacer” are interchangeable and refer to sequences that connect the VH and VL domains. Where a particular linker sequence is contemplated, it will be associated with a corresponding SEQ ID NO. The linker sequence should allow effective positioning of the VH and VL domains to allow functional activity including binding to their respective antigenic targets.

The linker is preferably encoded by a nucleotide sequence resulting in a peptide that can effectively position the VH and VL domains or other antibody fragment for recognition of its intended target.

In some aspects, the linker sequence is flexible so as to not constrain the VH and VL domains in an undesirable conformation. In some embodiments, the linker comprises amino acids with small side chains, e.g., glycine, alanine and serine, to provide for flexibility. Preferably about 80 or 90 percent or greater of the linker sequence comprises such residues. Exemplary nucleotide sequences encoding linkers are provided in the specification (SEQ ID NO.: 3, SEQ ID NO.: 10, SEQ ID NO.: 13, and SEQ ID NO.: 30), although any suitable linker sequence may be used with the embodiments provided herein. Different linker sequences can be used including any number of flexible linker designs that have been used successfully to join antibody variable regions together, see Whitlow, M. et al., (1991) Methods: A Companion to Methods in Enzymology 2:97-105. Suitable linker sequences can be identified empirically, or determined by computer modeling techniques.

The compositions described herein may be administered in combination with any anti-cancer therapy, including but not limited to, chemotherapeutic agents (such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin), surgery, radiation therapy, and/or chemotherapy.

Recombinant Expression Systems

Methods for introducing a polynucleotide into a host cell for expression are well known in the art, and include but are not limited to, integration of the foreign nucleotide sequence into the genome of the cell, vector-based methods wherein the foreign polynucleotide sequence is not integrated into the genome of the cell, and virus-mediated methods.

The sequence of interest (SOI), in this case, the nucleotide sequence encoding the antigen binding domain and the CAR scaffold may be recombinantly expressed in a host cell by inserting the nucleotide sequence into a suitable vector for expression in a mammalian cell and transfecting the vector into the host mammalian cell.

Vectors include DNA molecules into which a genetic insert has been introduced, allowing replication and expression of the insert in a host cell. Vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. Expression vectors typically comprise an origin of replication (ORI), a multicloning site comprising various restriction sites into which an insert can be cloned, and one or more selectable markers (e.g., ampicillin or tetracycline, etc.) for selection. Additionally, the vector can include one or more transcription units, with a transcription unit including a promoter, a polyA signal sequence, and a transcription termination sequence. In some embodiments, the promoter is a mammalian promoter. In other embodiments, the promoter is a viral promoter. In still other embodiments, the promoter is associated with the gene of interest. Promoters can be constitutive or inducible. If induced, a chemical such as IPTG is added to the cell culture in order for the system to recombinantly express the desired protein.

Viral promoters include but are not limited to promoters from adenovirus (such as Adenovirus 2 or 5), cytomegalovirus (CMV), herpes simplex virus (thymidine kinase promoter), retroviral promoters (e.g., MoMLV or RSV LTR), ubiquitin C (UBC), EF1α, PGK, CAGG, and simian virus 40 (SV40). Many other viral promoters are suitable, and all such viral promoters are contemplated herein.

These techniques utilize, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, virology, microbiology, recombinant DNA, and immunology, all of which are within the skill of an ordinary artisan. Such techniques are explained more fully in the literature. For a description of the functional components of expression vectors, including specific examples of promoters, enhancers, terminal signals, splicing signals, polyA signals, etc., reference is made to the following laboratory manuals that describe standard techniques of molecular biology, and are known by one of skill in the art (see, e.g., Green and Sambrook eds., Molecular Cloning: A Laboratory Manual, 4thedition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012; Ausubel et al., Current Protocols in Molecular Biology, 3rded. 1995; Bothwell et al. Methods for Cloning and Analysis of Eukaryotic Genes, Jones and Bartlett Publ. 1990; Wu, Grossman, Moldave eds. Recombinant DNA Methodology, Academic Press 1989; Adams ed., Cell Culture for Biochemists, Elsevier/North-Holland Biomedical Press, 1990; Butler ed., Mammalian Cell Biotechnology, IRL Press, 1991; Griffiths, et al., recombinant DNA technology in eukaryotes, in An Introduction to Genetic Analysis (2000), New York.

Examples of mammalian expression vectors include but are not limited to adenoviral vectors, adeno-associated vectors, baculovirus vectors, coronavirus, herpes simplex vectors, lentiviruses, pCMV series of plasmid vectors, pSV series of plasmid vectors, retroviral vectors, vaccinia, etc. Various vectors suitable for mammalian expression are derived from viruses, there are many such suitable vectors for expression in mammalian cells and all are contemplated herein.

In embodiments in which integration of the SOI into the host cell genome is desired, lentiviral expression systems may be selected. In embodiments in which integration into the host genome is not desirable, adenovirus expression systems or adeno-associated viruses expression systems may be selected.

Various methods are known in the art for transfection of mammalian expression vectors into host cells for expression, including viral transfection, lipofection, electroporation, calcium phosphate co-precipitation, rubidium chloride or polycation (such as DEAE-dextran)-mediated transfection, protoplast fusion and microinjection, see, e.g., Sambrook et al., for a description of such techniques. Preferably, the transfection method will provide an optimal transfection frequency and expression of the construct in the particular host cell line. Optimization may be performed using well-known techniques in the art.

In some embodiments, bacterial host cells are utilized to propagate mammalian expression vectors for preparation of DNA stocks for subcloning or for introduction into host cells. In other embodiments, bacterial host cells are utilized to produce large quantities of the protein encoded by the SOI (e.g., complexes). Bacterial host cells include but are not limited toE. coli. Yeast host cells include but are not limited toPichia pastoris.

Suitable expression vectors for bacterial cells include but are not limited to bacterial expression vectors (e.g.,E. coliexpression vectors such as pGEX and pET series). In still other embodiments, suitable expression vectors include but are not limited to yeast expression vectors (e.g., pPIC series). In still other embodiments, cell-free systems containing the components needed for transcription and translation are provided.

In preferred embodiments, adenoviral expression systems are used. In such systems, an insert comprising a nucleotide encoding the SOI may be cloned into a shuttle vector. The shuttle vector and adenovirus backbone vector (e.g., commercially available systems such as RAPAd® Adenoviral Expression System from Cell Biolabs), are both linearized and are cotransfected into 293 cells to generate a viral stock solution in about 2-3 weeks. The viral stock solution may then be used to transfect target host cells with the nucleotide encoding the SOI(s).

In some embodiments, the transfected cells may be cultured under conditions to express the recombinant protein. In some embodiments, the cells may be subjected to suspension culture. In other embodiments, the cells may be subject to tissue culture. In still other embodiments, expression of the recombinant protein may be induced. In still other embodiments, expression of the recombinant protein may be constitutively expressed.

In some embodiments, the SOI is co-expressed with co-stimulatory molecules, such as cytokines. Immune stimulatory cytokines are added to promote or trigger an immune response. Cytokines include but are not limited to IL2, IL4, IL7, IL11, IL15, IL21, TNF-alpha, IFN-gamma, etc.

Use with Cytotoxic Cells

Genetically engineered cells may express the constructs provided herein (antigen binding domain coupled to CAR scaffold). These cells can be administered to the patient to recognize and eliminate tumorigenic and/or cancerous cells. Immune response assays may be performed to validate whether or not the engineered cells have activity against the tumor or cancer cell.

NK cells are deemed particularly suitable for use herein, especially where the NK cells are autologous NK cells, obtained from the same individual from which the tumor is obtained.

Alternatively, NK cells may also be grown from monoclonal sources, such as NK-92 or NK-92 derivatives, in which the cells are modified to have a reduced or abolished expression of at least one killer cell immunoglobulin-like receptor (KIR) to render such cells constitutively activated. Such modified cells may be prepared using protocols well known in the art.

Of course, it should also be appreciated that multiple different cell populations may be prepared that have different combinations or sub-combinations of Fc receptors and signaling moieties to so even further increase the anticipated therapeutic effect. For example, two different populations of NK cells may be administered where the first type of Fc receptor is CD16a and where the cell overexpresses Fcγ-signaling subunits, and where the second type of Fc receptor is CD32a and where the cell overexpresses Fcγ-signaling subunits. In another example, two different populations of cells may be administered where the first cell is an NK cell with expressing CD16a and overexpressing Fcγ-signaling subunits, and where the second cell is an CD8+ T-cell expressing CD16a and overexpressing Fcγ-signaling subunits. Regardless of the source of the cell, it is generally contemplated that the cell is a mammalian cell, and especially a human cell.

Additionally, and particularly where the cells are not obtained from the mammal that is to receive the subsequently modified cells, it is contemplated that the cells are rendered less immunogenic to the mammal (e.g., via HLA grafting or deletion of MHC complexes).

In yet another example, suitable NK cells for administration may be (or may be derived from) previously established therapeutic cell lines, which are well known in the art. For example, suitable cell lines include aNK cells, haNK cells, taNK cells, NK-92 cells (e.g., commercially available from Nantkwest, 9920 Jefferson Blvd. Culver City, CA 90232) or TALL 104 cells (e.g., commercially available from ATCC, CRL-11386, 10801 University Boulevard, Manassas, Va. 20110 USA).

In some cases, the administered cells may be allogeneic, and may be rejected by the recipient's immune system. Thus, in some embodiments, allogeneic cytotoxic cells are modified to be resistant to immunosuppressive agents (e.g., inactivating a gene that is a target for an immunosuppressive agent, e.g., a cyclophilin gene member, a CD52, a FKBP receptor, a glucocorticoid receptor, etc.) so that the cytotoxic cell is capable of functioning in the presence of the immune suppressive agent. The cytotoxic cells are administered in conjunction with immunosuppressive agents including but not limited to an immunosuppressive antimetabolite, a calcineurin inhibitor, a corticosteroid, a dihydrofolic acid reductase inhibitor, an inosine monophosphate dehydrogenase inhibitor, an interleukin-2 ot-chain blocker, or a rapamycin target.

NK cells express a variety of activating receptors, including NKG2D, Ly49 (some are activating, most are inhibitory), KIR (both activating and inhibitory), CD94-NKG2C, and CD94-NKG2E, and inhibitory receptors including Ly49 and KIR. These receptors recognize cellular stress ligands as well as MHC class I and related molecules. (see, Pegram et al., Immunology and Cell Biology (2011) 89:216-224)

Immune Activation

Once the CAR-based therapeutic (e.g., an antigen binding domain coupled to a CAR scaffold) binds to the antigen expressed by the cancer cell, the cytotoxic cell can trigger destruction of the cancer cell. While it is generally contemplated that all cytotoxic cells are deemed suitable for use herein, especially preferred cytotoxic cells include NK cells, activated NK cells, high affinity NK cells, CD8+ T-cells, and CD4+ T-cells that have been modified to recombinantly express the CAR-based therapeutic, any of which may be of different origins.

However, it should be appreciated that in other aspects, the cytotoxic cell may also be a macrophage, a monocyte, a neutrophil cell, a basophile, or eosinophil cell. Therefore, and viewed from a different perspective, the cells contemplated herein may effect cytotoxic action via phagocytosis, pore formation, induction of antibody-dependent cell-mediated cytotoxicity (ADCC), by triggering TNF or fas mediated killing pathways, etc.

Cytotoxic cells may release various types of cytotoxic granules (e.g., granulysin, perforin, granzymes) as part of this process. A variety of assays are available for monitoring cell-mediated cytotoxicity, including flow cytometric assays, e.g., based on presence of lytic granules such as perforin, granzymes, or production of TNF family members, e.g., TNF-α, FasL or TRAIL (Zaritskaya 2010, Clay, T. et al., Clin. Cancer Res. (2001) &:1127-1135).

In one embodiment, a bodily fluid is obtained, wherein the bodily fluid comprises cellular components, e.g., tumorigenic or cancer cells displaying an antigen to which the CAR scaffold expressing cells described herein bind to, and cytotoxic cells expressing the antigen binding moiety are contacted with the cells. Assays are then performed to detect immune responses, e.g., indicating that an ADCC response or an ADCP response has been triggered by the patient's own immune cells.

Assays for detecting an immune response are known in the art and are described herein. For example, assays for detecting such a response may detect a release of cytotoxic granules (e.g., granulysin, perforin, granzymes), or phagocytosis, or receptor-ligand mediated cytolysis (e.g., as mediated by the Fas/APO pathway). A variety of flow cytometric assays are available for monitoring cell-mediated cytotoxicity, e.g., based on presence of lytic granules such as perforin, granzymes, or production of TNF family members, e.g., TNF-α, FasL or TRAIL (Zaritskaya 2010, Clay, T. et al., Clin. Cancer Res. (2001) &:1127-1135).

In other embodiments, immune stimulatory cytokines are administered to a patient in combination with the host cell (expressing a CAR-based therapeutic (e.g., an antigen binding domain coupled to a CAR scaffold)) to promote or trigger an immune response. Cytokines include but are not limited to IL2, IL4, IL7, IL11, IL15, IL21, TNF-alpha, IFN-gamma, etc. In some embodiments, cytokines can reactivate exhausted T cells. In other cases, immune competent cells may be engineered to recombinantly express one or more cytokines.

Techniques to treat cancer include surgery, radiation therapy, chemotherapy, immunosuppressive reagents (e.g., azathioprine, cyclosporin, methotrexate, mycophenolate, etc.), immunotherapy, targeted therapy, hormone therapy, stem cell transplant, or other precision methods. Any of these techniques may be combined with embodiments of the present invention to treat cancer.

It is understood that present invention embodiments may be administered to a patient using appropriate formulations, indications, and dosing regimens suitable by government regulatory authorities such as the Food and Drug Administration (FDA) in the United States.

In some embodiments, a cytotoxic cell expressing a CAR-based therapeutic (e.g., an antigen binding domain coupled to a CAR scaffold) is administered to a patient as a pharmaceutical composition. In another embodiment, a method of treating cancer by administration of the cytotoxic cell to a subject is contemplated. In still another embodiment, a method inhibiting the proliferation or reducing the proliferation of a cell that is expressing the corresponding antigen (to which the antigen binding region binds to) on the surface of its cell by administration of the cytotoxic cell to a subject is contemplated.

In some embodiments, the cytotoxic cell expressing a CAR-based therapeutic (e.g., an antigen binding domain coupled to a CAR scaffold) reduces the amount (e.g., number of cells, size of mass, etc.) by at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, or at least 99% in a subject with cancer associated with expression of the corresponding antigen on the surface of the cells relative to a negative control.

Examples of cancer that are treatable by the cytotoxic cells contemplated herein include any cancer expressing or overexpressing a cancer-associated antigen on its cell surface. Examples of cancer that can be treated with a cytotoxic cell expressing a CAR-based therapeutic (e.g., an antigen binding domain coupled to a CAR scaffold) include but are not limited to breast cancer, colon cancer, leukemia, lung cancer, melanoma, neuroblastoma, pancreatic cancer, pediatric intracranial ependymoma, and prostate cancer.

Pharmaceutical Compositions

Pharmaceutical compositions may comprise cytotoxic cells comprising an antigen binding domain coupled to or linked to a CAR scaffold, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Additionally, pharmaceutical compositions may comprise one or more adjuvants (e.g., aluminum hydroxide), antioxidants, bacteriostats, buffers, carbohydrates, chelating agents such as EDTA or glutathione; coloring, flavoring and/or aromatic substances, emulsifiers, excipients, lubricants, pH buffering agents, preservatives, salts for influencing osmotic pressure, polypeptides (e.g., glycine), proteins, solubilizers, stabilizers, wetting agents, etc., which do not deleteriously react with the active compounds (e.g., antigen binding domain coupled to a CAR scaffold, etc.) or otherwise interfere with their activity. Buffers include but are not limited to neutral buffered saline, phosphate buffered saline, etc. Carbohydrates include but are not limited to dextrans, glucose, mannose, mannitol, sucrose, etc.

Pharmaceutical compositions may be formulated for a particular mode of administration. Modes of administration may include but are not limited to: intraarticular, intradermal, intranasal, intraperitoneal, intrathecally, intratumoral, intravenous, intraventricularly, subcutaneous, transdermal, transmucosal or topical routes.

In preferred embodiments, the cytotoxic cells are administered by intravenous infusion. Such formulations may be prepared according to standard techniques known by one of ordinary skill in the art. For example, a composition that is to be administered intravenously may have one or more ingredients (e.g., a diluent, a suspension buffer, saline or dextrose/water, other components such as cytokines, etc.) prior to infusion in the patient.

Many such techniques for formulating and administering pharmaceutical compositions are known in the art, e.g., U.S. Patent Application Publication No. 2014/0242025, and all such references are incorporated by reference herein in their entirety.

In some embodiments, the cytotoxic cells proliferate in vivo, thereby persisting in the patient for months or even years after administration to provide a sustained mechanism for inhibiting tumor growth or recurrence. In some aspects, the cytotoxic cells persist at least for three months, six months, nine months, twelve months, fifteen months, eighteen months, two years, three years, four years, or five years after administration of the cytotoxic cells to the patient.

Cytotoxic cells may be obtained from any of a variety of sources, (e.g., isolated from a human, from commercially available cytotoxic cells, from a cell repository, etc.). Procedures for ex vivo expansion of NK cells, T cells or other types of cytotoxic cells are known in the art (e.g., Smith et al., Clinical & Translational Immunology (2015) 4: e31). The examples presented herein are not intended to be limited to any particular method of ex vivo expansion of cytotoxic cells.

Pharmaceutical compositions comprising cytotoxic cells, as described herein, may be administered at a dosage of 104to 109cells/kg body weight, of 105to 106cells/kg body weight, or any integer values within these ranges. Cytotoxic cell compositions may be administered one time or serially (over the course of days or weeks or months) at these dosages. Infusion techniques for cytotoxic cells, such as T cells, are known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

In other embodiments, the pharmaceutical compositions are administered in a therapeutically effective amount, which is the amount effective for treating the specific indication. Administration may occur as a one-time dose or based on an interval. As used herein, “interval” indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The administration interval for a single individual need not occur at a fixed interval, but can vary over time. The term, “in combination with” or “co-administered” indicates that a composition can be administered shortly before, at or about the same time, or shortly after another composition.

It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the concepts herein. The present subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

EXAMPLES

Example 1. Generation of Antigen Binding Domain—CAR Scaffold

A variety of techniques are available for generation of the constructs provided herein. These constructs comprise an antibody fragment (e.g., a scFv, one or more CDRs, etc.) fused to a linker, which is fused to a CAR scaffold. Various protocols for generating these constructs are known to one of ordinary skill in the art.

In one embodiment, the nucleotide sequence encoding for a scFv may be obtained according to the techniques presented herein (e.g., phage display, mRNA display, from monoclonal antibodies, etc.). In some cases, the VH and VL domains may be known in the art, and linked together using known techniques to form an scFv. The nucleotide sequence corresponding to the scFv may be coupled to a CAR scaffold. Once the full nucleotide sequence is obtained, it may ligated into a suitable vector. Immune cells are then transfected with the vector, the construct is expressed, and the engineered cells are subjected to the assays disclosed herein.

CAR constructs direct NK cells to a particular target as NK cells without relying on HLA matching (unlike T cells) (see, e.g., Hermanson et al., Front Immunol (2015) 6:195; and Carlsten et al., Front Immunol (2015) 6:266). Various NK cell lines contemplated herein include but are not limited to aNK, HaNK, NK-92, NKG, NKL, NK-YS, TaNK, YT, and YTS cells.

Also contemplated herein is transfection with genes encoding for one or more cytokines, e.g., IL2, IL4, IL7, IL11, IL15, IL21, TNF-alpha, IFN-gamma. In some embodiments, IL-2 and/or IL-15 are transfected to promote in vivo expansion and persistence.

Cytotoxic cells may be genetically modified (i.e., transduced or transfected in vitro) with a vector encoding the constructs disclosed herein. The cytotoxic cell may be administered to a mammalian recipient to provide a therapeutic benefit, namely directing cytotoxic cells to destroy cells expressing the antigen that the antigen binding moiety binds to on their surface, e.g., cancer cells.

The mammalian recipient may be a human and the cytotoxic cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient. In some embodiments, allogeneic cells are administered with an immune suppressant.

Example 2. Cell Killing Assays

Killings assays are known in the art, e.g., at U.S. Pat. No. 7,741,465. For example, as provided in the '465 patent, the ability of the transfected cells to mediate specific target cell killing was determined by a51Cr release assay.

For example, engineered cells may be transformed with constructs provided herein, and a level of cell lysis measured, based on a51Cr release signal when compared to an untransfected control. Transfected cells will generally induce a significantly higher specific51Cr release from human tumors or cancer cells than the corresponding control cells.

Example 3. Detection of an Immune Response

The ability of genetically engineered cells to elicit an immune response may be tested. A variety of assays for monitoring cellular immune responses in vivo and in vitro are available (see, e.g., Clay T. et al., Clin. Cancer Res. (2001) p 1127-1135).

In some embodiments, cytotoxic cells expressing the constructs provided herein (antigen binding domain coupled to a CAR scaffold) can be tested to determine whether an immune response is triggered, e.g., by detecting a release of cytotoxic granules, phagocytosis, or receptor-ligand mediated cytolysis, from lysed tumor cells.

Traditional assays for measuring cell lysis include addition of a radioisotope, e.g.,51Cr, to cell culture, which is trapped in the interior of living cells. The radioisotope is released upon cell lysis into the extracellular fluid, providing an indicator of the amount of lysis occurring.

Other assays exist in which levels of Granzyme B are measured. Granzyme B is secreted by activated cytotoxic T cells or NK cells. Granzyme B is released through exocytosis, and in conjunction with perforin, is able to enter target cells to help trigger cell death. Enzyme linked immunoassays (e.g., ELISpot, an ELISA sandwich assay) are known in the art for quantifying the amount of secreted Granzyme B. Essentially, cells are incubated in the presence of antibodies specific for Granzyme B. The cells are removed, and a second Granzyme B specific antibody is added with a detectable marker (e.g., biotin/alkaline phosphatase streptavidin complex). Based on the intensity of color formation, the amount of Granzyme B can be quantified (see, www.rndsystems.com/products/human-granzyme-b-elispot-kit_e12906#product-details; Malyguine A. et al., Cells (2012) 1(2): 111-126).