Patent Application
20230372395
The present invention relates generally to the field of immunotherapy, particularly to the use of engineered immune cells that recognize a ligand previously inserted into a tumor cell.
U.S. Pat. No. 9,233,125 discloses certain CAR cells which recognize certain cancer cells marked with a tag antibody.
There is a need for more effective targeting of tumor cells with engineered immune cells, particularly for solid tumors.
In one aspect, the present disclosure provides a method of treating a tumor, comprising introducing a membrane-inserting amphiphilic ligand into a tumor of a subject in need of treatment followed by administering an engineered immune cell expressing a CAR—that specifically binds to the amphiphilic ligand. In some embodiments, the membrane-inserting amphiphilic ligand is a fluorescein isothiocyanate lipid amphiphile ligand. In some embodiments, the membrane-inserting amphiphilic ligand comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-FITC. In some embodiments, the engineered immune cell expressing a CAR that specifically binds to the amphiphilic ligand is a fluorescein isothiocyanate (FITC) lipid amphiphile-specific engineered immune cell.
In some embodiments, the membrane-inserting amphiphilic ligand comprises a therapeutic compound conjugated to an amphiphilic poly(ethylene glycol)-lipid.
In some embodiments, the membrane-inserting amphiphilic ligand is introduced into the tumor by intratumoral injection. In some embodiments, administering the engineered immune cell expressing a CAR that specifically binds to the amphiphilic ligand comprises systemic infusion of the engineered immune cell into the subject. In some embodiments, the method further comprises introducing the membrane-inserting amphiphilic ligand into dendritic cells in lymph nodes of the subject by subcutaneous injection. In some embodiments, the frequency of intratumoral injection is equal to or less than about once every 6 days.
In some embodiments, the tumor of a subject in need of treatment is a solid tumor.
In some embodiments, the engineered immune cell expressing a CAR comprises a CD28 costimulatory domain.
In another aspect, the present disclosure provides an engineered immune cell expressing a CAR that specifically binds to a membrane-inserted amphiphilic ligand of a tumor cell. In some embodiments, the membrane-inserting amphiphilic ligand is a fluorescein isothiocyanate lipid amphiphile ligand. In some embodiments, the chimeric antigen receptor recognizes the amphiphilic ligand. In some embodiments, the chimeric antigen receptor comprises amphiphilic-ligand specific scFV. In some embodiments, the chimeric antigen receptor further comprises a CD28 costimulatory domain. In some embodiments, the fluorescein isothiocyanate lipid amphiphile ligand is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-FITC.
It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.
The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. All the various embodiments of the present disclosure will not be described herein. Many modifications and variations of the disclosure can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.
In practicing the present technologies, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology.
Chimeric antigen receptor (CAR) immune cell therapy, including, for example CAR-T cells, are effective in treating certain hematological malignancies, but translation to use in solid tumors has been challenging. To date, selecting an effective target antigen is hampered by heterogeneous tumor antigen expression and target antigen expression in healthy (e.g., non-tumor, non-cancerous) tissues.
The present disclosure is based, at least in part, on technologies to promote engineered immune cell mediated destruction of any tumor, including, for example, solid tumors. Such technologies provide a solution to off-target engineered immune cell activities as a result of heterogeneous tumor antigen expression and target antigen expression in healthy tissues. In some embodiments, technologies of the present disclosure comprise the discovery and use of membrane-inserting amphiphilic ligands and engineered immune cells, wherein engineered immune cells express a CAR that specifically bind to such amphiphilic ligands. The disclosure provides, among other things, methods of treating a tumor, comprising introducing a membrane-inserting amphiphilic ligand into a tumor of a subject in need of treatment followed by administering an engineered immune cell expressing a CAR that specifically binds to the amphiphilic ligand.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
As used herein, the term “about,” when used to modify a numerical value, indicates that deviations of up to 10% above and below the numerical value remain within the intended meaning of the recited value.
As used herein, the term “administration” of an agent to a subject includes any route of introducing or delivering the agent to a subject to perform its intended function. Administration can be carried out by any suitable route, including, but not limited to, intravenously, intramuscularly, intraperitoneally, subcutaneously, and other suitable routes as described herein. Administration includes self-administration and the administration by another.
As used herein, the term “antibody” generally refers to an antibody comprising two light chain polypeptides and two heavy chain polypeptides (unless the context in which this term is used suggests otherwise). Antibodies include different antibody isotypes including IgM, IgG, IgA, IgD, and IgE antibodies. The term “antibody” includes, without limitation, a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and a fully human antibody. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, llama, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody
As used herein, the term “antibody fragment,” “antigen binding fragment,” or similar terms refer to a fragment of an antibody that retains the ability to bind to a target antigen. Such fragments include, without limitation, a single chain antibody, a single chain Fv fragment (scFv), a Fab fragment, a Fab′ fragment, and a F(ab′)2 fragment. This term also includes, e.g., single domain antibodies such as camelid single domain antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem Sci 26:230-235; Nuttall et al. (2000) Ciirr Pharm Biotech 1:253-263; Reichmann et al. (1999) J Immunol Meth 231:25-38; PCT application publication nos. WO 94/04678 and WO 94/25591; and U.S. Pat. No. 6,005,079, all of which are incorporated herein by reference in their entireties. In some embodiments, the disclosure provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed. In addition, intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibody fragments and are compatible for use in the methods described herein. See, e.g., Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J Immunol Methods 231(1):177-189; Poljak (1994) Structure 2(12): 1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology 5F257-283, the disclosures of each of which are incorporated herein by reference in their entirety.
As used herein, an “antigen” refers to a molecule to which an antibody, antibody fragment, and/or Chimeric Antigen Receptor can selectively bind. The target antigen may be a protein (e.g., an antigenic peptide), small molecule, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. An antigen may also be administered to an animal subject to generate an immune response in the subject.
As used herein, the term an “amino acid substitution” or “substituted” (when such term is referred to a substituted amino acid) refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with a different amino acid residue. The term “amino acid insertion” refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, larger “peptide insertions,” can also be made. The replaced or inserted amino acid residue(s) may be naturally occurring or non-naturally occurring (modified).
The term “amino acid deletion” refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.
By “binding affinity” is meant the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., a CAR) and its binding partner (e.g., an antigen, a ligand). Without wishing to be bound by theory, affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen-antibody bond after formation of reversible complexes (e.g., either monovalent or multivalent).
As used herein, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including: (1) hydrophobic side chains: norleucine, Met, Ala, Val, Leu; (2) neutral hydrophilic side chains: Cys, Ser, Thr, Asn, Gin; (3) acidic side chains: Asp, Glu; (4) basic side chains: His, Lys, Arg; (5) side chains that influence chain orientation: Gly, Pro; and (6) aromatic side chains: Trp, Tyr, Phe. For example, a non-conservative amino acid substitution is a substitution of an amino acid residue with an amino acid residue with a substantially different side chain (i.e., an amino acid residue that is a member of a different family). In some embodiments, a conservative amino acid substitution is made by considering the hydropathic index of the amino acid residue. Each amino acid is assigned a hydropathic index on the basic of its hydrophobicity and charge characteristics. They are: lie (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glu (−3.5); Gin (−3.5); Asp (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5). The importance of the hydropathic amino acid index in conferring interactive function on a polypeptide is understood in the art (see, e.g., Kyte et al (1982) J Mol Biol 157:105-131). In some embodiments, a conservative amino acid substitution is made by replacing one amino acid residue with another amino acid residue having a the same or similar (e.g., within about +2, +1.5, +1, +0.5, −0.5, −1, −1.5, or −2) hydropathic index. In some embodiments, a conservative amino acid substitution is made by considering the hydrophilicity of the amino acid residue. The following hydrophilicity values have been assigned: Arg (+3.0); Lys (+3.0+1); Asp (+3.0+1); Glut (+0.2); Gly (0); Thr (−0.4); Pro (−0.5+1); Ala (−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); lie (−1.8); Tyr (−2.3); Phe (−2.5); and Trp (−3.4). In some embodiments, a conservative amino acid substitution is made by replacing one amino acid residue with another amino acid residue having a the same or similar (e.g., within about +2, +1.5, +1, +0.5, −0.5, −1, −1.5, or −2) hydrophilicity. Exemplary amino acid substitutions are set forth in Table 2:
As used herein, the term, “co-stimulatory signaling domain,” or “costimulatory domain”, refers to the portion of the CAR comprising the intracellular domain of a co stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2 and a ligand that specifically binds CD83. Accordingly, while the present disclosure provides exemplary costimulatory domains derived from CD28 and 4-IBB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more co-stimulatory signaling domains can enhance the efficacy and expansion of engineered immune cells expressing CAR receptors. The intracellular signaling and co-stimulatory signaling domains can be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.
As used herein, the term “effective amount” or “therapeutically effective amount” refers to a quantity of an agent sufficient to achieve a beneficial or desired clinical result upon treatment. In the context of therapeutic applications, the amount of a therapeutic agent administered to the subject can depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, sex, body weight, effective concentration of the engineered immune cells administered, and tolerance to drugs. It can also depend on the degree, severity, and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. As used herein the term “KD” or “Kd” has the same meaning as commonly understood by one of ordinary skill in the art, and refers to the equilibrium dissociation constant of a binding reaction, for example, between an antibody (or antigen binding fragment thereof or CAR) and an antigen or ligand. The value of KD is a numeric representation of the ratio of the antibody off-rate constant (koff) to the antibody on-rate constant (kon). The value of KD is inversely related to the binding affinity of an antibody (or antigen binding fragment thereof or CAR) to an antigen or ligand. The smaller the KD value the greater the affinity of the antibody (or antigen binding fragment thereof or CAR) for its antigen or ligand. Affinity can be measured by any method known in the art.
As used herein, the term “immune cell” refers to any cell that plays a role in the immune response of a subject. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. As used herein, the term “engineered immune cell” refers to an immune cell that is genetically modified. As used herein, the term “native immune cell” refers to an immune cell that naturally occurs in the immune system.
As used herein, the term “isolated,” “purified,” or “biologically pure” refers to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or polypeptide of the presently disclosed subject matter is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
As used herein, the term “ligand” refers to a molecule that binds to a receptor (e.g., a CAR). In particular, the ligand binds a receptor on another cell, allowing for cell-to-cell recognition and/or interaction.
The term “linker” refers to synthetic sequences (e.g., amino acid sequences) that connect or link two sequences, e.g., that link two polypeptide domains. In some embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.
As used herein, the term “percent (%) amino acid sequence identity” or “percent sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known in the art, for instance, using publicly available computer software such as BFASTp, BFAST-2, AFIGN (e.g., AFIGN-2) or Megalign (DNASTAR) software. To obtain gapped alignments for comparison purposes, Gapped BEAST can be utilized as described in Altschul el ah, (1997) Nucleic Acids Res. 25(17):3389-3402. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are a non-naturally occurring amino acid, e.g., an amino acid analog. The terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
As used herein, the term “reduce” or “decrease” means to alter negatively by at least about 5% including, but not limited to, alter negatively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.
As used herein, the terms “specific binding,” “specifically binds,” “selective binding,” and “selectively binds,” are intended to mean that an antibody or antigen-binding fragment thereof or a CAR that exhibits appreciable affinity for a particular antigen or ligand (e.g., a membrane-inserting amphiphilic ligand described herein) and, generally, does not bind to, or substantially does not bind to, other antigens or ligands. “Appreciable” or preferred binding includes binding with a KD of 107, 108, 109, or 1010 M or better. The KD of an antibody, antigen-binding fragment or CAR—antigen or ligand interaction (the affinity constant) indicates the concentration of antibody, antigen-binding fragment, or CAR at which 50% of antibody, antigen-binding fragment, or CAR and antigen or ligand molecules are bound together. Thus, at a suitable fixed antigen concentration, 50% of a higher (i.e., stronger) affinity antibody, antigen-binding fragment, or CAR will bind antigen or ligand molecules at a lower antibody, antigen-binding fragment, or CAR concentration than would be required to achieve the same percent binding with a lower affinity antibody, antigen-binding fragment, or CAR. Thus a lower KD value indicates a higher (stronger) affinity. As used herein, “better” affinities are stronger affinities, and are of lower numeric value than their comparators, with a KD of 107M being of lower numeric value and therefore representing a better affinity than a KD of 106 M. Affinities better (i.e., with a lower KD value and therefore stronger) than 107 M, preferably better than 108 M, are generally preferred. Values intermediate to those set forth herein are also contemplated, and a preferred binding affinity can be indicated as a range of affinities.
As used herein, the term “T-cell” includes naive T cells, CD4+ T cells, CD8+ T cells, memory T cells, activated T cells, anergic T cells, tolerant T cells, chimeric B cells, and antigen-specific T cells.
As used herein, the term “vector” has the same meaning as commonly understood by one of ordinary skill in the art, and refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
In one aspect, the present disclosure provides membrane-inserting amphiphilic ligands. Membrane-inserting amphiphilic ligands can insert their lipid tails into the plasma membrane of cells (e.g., target cells, including, for example tumor cells) in vitro and in vivo with minimal to no toxicity, leading to cell surface display of membrane-inserting amphiphilic ligands. In some embodiments, membrane-inserting amphiphilic ligands redirect engineered immune cells expressing a CAR that specifically bind to the amphiphilic ligand, as described herein, to the cells comprising the amphiphilic ligands on their cell surface. Such membrane-inserting amphiphilic ligands on the cell surface enable recognition by engineered immune cells bearing amphiphilic ligand-specific chimeric antigen receptors, thereby triggering engineered immune cell activation, proliferation, and killing of the cells comprising the amphiphilic ligands on their cell surface.
In some embodiments, a membrane-inserting amphiphilic ligand is or comprises a fluorescein isothiocyanate lipid amphiphile ligand. In some embodiments, a membrane-inserting amphiphilic ligand comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG-FITC.
In some embodiments, membrane-inserting moieties (e.g., non-amphiphilic molecules) can be used. A plurality of membrane-inserting moieties are known in the art and one of ordinary skill would readily recognize and understand how to select and use such membrane-inserting moieties in accordance with the present disclosure.
In some embodiments, a membrane-inserting amphiphilic ligand comprises a amphiphilic poly(ethylene glycol) (PEG)-lipid. A plurality of amphiphilic PEG-lipids are known in the art and one of ordinary skill would readily recognize and understand how to select and use a known PEG-lipid in accordance with the present disclosure. PEG-lipids include, for example and without limitation, comprise dimyristoyl glycerol (DMG)-PEG 2000, Methoxypoly(ethylene glycol) (MPEG)2000C-DMG, α-[2-(ditetradecylamino)-2-oxoethyl]-ω-methoxy-poly(oxy-1,2-ethanediyl) (ALC-0159), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-PEG-Amine, molecular weight (MW) 1,000, DOPE-PEG-Amine, MW 2,000, DOPE-PEG-Amine, MW 3,400, DOPE-PEG-Amine, MW 5,000, DOPE-PEG-COOH, MW 1,000, DOPE-PEG-COOH, MW 2,000, DOPE-PEG-COOH, MW 3,400, DOPE-PEG-COOH, MW 5,000, DOPE-PEG-Mal, MW 1,000, DOPE-PEG-Mal, MW 2,000, DOPE-PEG-Mal, MW 3,400, DOPE-PEG-Mal, MW 5,000, DOPE-mPEG, MW 1,000, DOPE-mPEG, MW 2,000, DOPE-mPEG, MW 5,000, 1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine (DMPE)-mPEG, MW 2000, DSPE-PEG-NH2, MW 1,000, DSPE-PEG-Amine, MW 2,000, DSPE-PEG-Amine, MW 3,400, DSPE-PEG-Amine, MW 5,000, DSPE-PEG4-acid, DSPE-PEG-COOH, MW 1,000, DSPE-PEG-COOH, MW 3,400, DSPE-PEG-CH2COOH, MW 2,000, DSPE-PEG-CH2COOH, MW 5,000, DSPE-PEG-NHS, MW 600, DSPE-PEG-NHS, MW 1,000, DSPE-PEG-NHS, MW 2,000, DSPE-PEG-NHS, MW 3,400, DSPE-PEG-NHS, MW 5,000, DSPE-PEG13-TFP, DSPE-PEG5-azide, DSPE-PEG-Azide, MW 1,000, DSPE-PEG-Azide, MW 2,000, DSPE-PEG-Azide, MW 3,400, DSPE-PEG-Azide, MW 5,000, DSPE-PEG4-DBCO, DSPE-PEG-dibenzocycolctyne (DBCO), MW 2,000, DSPE-PEG-DBCO, MW 3,400, DSPE-PEG-DBCO, MW 5,000, DSPE-PEG5-propargyl, DSPE-PEG8-Mal, DSPE-PEG12-Mal, DSPE-PEG-Maleimide, MW 2,000, DSPE-PEG-Maleimide, MW 5,000, DSPE-PEG-Ald, MW 1,000, DSPE-PEG-Ald, MW 2,000, DSPE-PEG-Ald, MW 3,400, DSPE-PEG-Ald, MW 5,000, DSPE-PEG-Biotin, MW 2,000, DSPE-PEG-Biotin, MW 3,400, DSPE-PEG-Folate, MW 1,000, DSPE-PEG-Folate, MW 2,000, DSPE-PEG-Folate, MW 3,400, DSPE-PEG-Folate, MW 5,000, DSPE-PEG-OH, MW 1,000, DSPE-PEG-OH, MW 2,000, DSPE-PEG-OH, MW 3,400, DSPE-PEG-OH, MW 5,000, DSPE-PEG-SH, MW 1,000, DSPE-PEG-SH, MW 2,000, DSPE-PEG-SH, MW 3,400, DSPE-PEG-SH, MW 5,000, DSPE-PEG-SPDP, MW 1,000, DSPE-PEG-SPDP, MW 3,400, DSPE-PEG-SPDP, MW 5,000, DSPE-PEG-2-Aminoethyl-alpha-Mannopyranoside, MW 2,000, DSPE-PEG-IA, MW 2,000, DSPE-PEG-IA, MW 3,400, DSPE-PEG-IA, MW 5,000, DSPE-PEG-Boronate, MW 2,000, DSPE-PEG-Vinylsulfone, MW 1,000, DSPE-PEG-Vinylsulfone, MW 2,000, DSPE-PEG-Vinylsulfone, MW 3,400, DSPE-PEG-Vinylsulfone, MW 5,000, DSPE-PEG-Cy3, MW 2,000, DSPE-PEG-Cy3, MW 3,400, DSPE-PEG-Cy3, MW 5,000, DSPE-PEG-Cy5, MW 3,400, DSPE-PEG-Cy5, MW 5,000, DSPE-PEG-FITC, MW 1,000, DSPE-PEG-FITC, MW 2,000, DSPE-PEG-FITC, MW 3,400, DSPE-PEG-FITC, MW 5,000, DSPE-PEG-Rhodamine, MW 1,000, DSPE-PEG-Rhodamine, MW 2,000, DSPE-PEG-Rhodamine, MW 3,400, DSPE-PEG-Rhodamine, MW 5,000, m-PEG8-DSPE, m-PEG12-DSPE, m-PEG-DSPE, MW 550, m-PEG-DSPE, MW 1,000, m-PEG-DSPE, MW 2,000, m-PEG-DSPE, MW 3,000, m-PEG-DSPE, MW 5,000, Stearic acid-PEG-NHS, MW 1,000, Stearic acid-PEG-NHS, MW 2,000, Stearic acid-PEG-NHS, MW 3,400, Stearic acid-PEG-NHS, MW 5,000, Stearic acid-mPEG, MW 1,000, Stearic acid-mPEG, MW 2,000, Stearic acid-mPEG, MW 5,000, Stearic acid-PEG-Rhodamine, MW 1,000, Stearic acid-PEG-Rhodamine, MW 2,000, Stearic acid-PEG-Rhodamine, MW 3,400, Stearic acid-PEG-Rhodamine, MW 5,000, Stearic acid-PEG-FITC, MW 1,000, Stearic acid-PEG-FITC, MW 2,000, Stearic acid-PEG-FITC, MW 3,400, Stearic acid-PEG-FITC, MW 5,000, (R)-2,6-Bis-(m-PEG4)-amidohexanoic acid, (S)-2,6-Bis-(m-PEG8)-amidohexanoic acid, Pentacosadiynoic acid-MPEG, MW 1,000, Pentacosadiynoic acid-MPEG, MW 2,000, Pentacosadiynoic acid-MPEG, MW 5,000.
In some embodiments, a membrane-inserting amphiphilic ligand comprises a therapeutic compound conjugated to an amphiphilic poly(ethylene glycol)-lipid. In some embodiments, a therapeutic compound comprises one or more small molecules, polypeptides, polysaccharides and/or synthetic polymers. In some embodiments, a therapeutic compound is any small molecule, polypeptide, polysaccharide and/or synthetic polymer that can be conjugated to an amphiphilic poly(ethylene glycol)-lipid and targeted by an engineered immune cell described herein.
In some embodiments, a therapeutic compound is or comprises a small molecule, including, for example and without limitation, FITC, Texas Red, BODIPY, Cy3, Camptothecin, Doxorubicin, and Irinotecan, or derivatives thereof.
In some embodiments, a therapeutic compound is or comprises a polypeptide, such as, for example, enzymes, monoclonal antibodies, and/or cytokines or fragments thereof. Non-limiting examples of polypeptides include, arginine deaminase, asparaginase, blue fluorescent protein, epidermal growth factor, granulocyte-colony stimulating factor, green fluorescent protein, interferons (e.g., interferon-α, interferon-β, etc.), mCherry, programmed cell death protein 1, programmed death ligand 1, red fluorescent protein, vascular endothelial growth factor receptors, yellow fluorescent protein, etc.
In one aspect, the present disclosure provides Chimeric Antigen Receptors (CARs) and nucleic acids encoding CARs. CARs are genetically-engineered, artificial membrane-bound proteins that, when expressed on an engineered immune cell (e.g., a T cell), direct such an engineered immune cell to a specific target molecule (e.g., ligand, antigen), and generally stimulates the engineered immune cell to kill the cell displaying the specific target molecule (e.g., a target cell, including for example, a tumor cell). Thus, CARs can be used to impart a desired target specificity to engineered immune cells, such as specificity to a tumor comprising a membrane-inserting amphiphilic ligand as described herein. In some embodiments, transfer of the coding sequence of the CAR is facilitated by nucleic acid vector, such as a retroviral vector. A plurality of appropriate technologies for transfer of coding sequences will be readily apparent to one of ordinary skill in the art.
In some embodiments, a CAR comprises three domains: (i) an extracellular domain typically comprising a signal peptide, a ligand, and/or an antigen recognition region (e.g., scFv), and a flexible spacer; (ii) a transmembrane domain; and (iii) an intracellular domain typically comprising one or more signaling domains. In some embodiments, a CAR can further comprise a linker.
In some embodiments, a subject CAR of the present disclosure may comprise any combination of any of the domains described herein (e.g., extracellular domains, transmembrane domains, costimulatory domains, intracellular domains, and/or hinge domains).
The target molecule specificity of a CAR is imparted by the extracellular domain of the CAR. In some embodiments, engagement of a CAR extracellular domain (e.g., a ligand and/or antigen recognition region, including, for example, a scFv) with its specific target molecule results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell. In some embodiments, CARs redirect engineered immune cells (e.g., T cells) to a particular target cell comprising a CAR-specific target molecule (e.g., a membrane-inserting amphiphilic ligand as described herein). Upon binding of the CAR to a specific target molecule, downstream signaling initiates, for example, production of molecules that can mediate cell death of a target cell (e.g., a tumor cell comprising a membrane-inserting amphiphilic ligand).
Extracellular Domains
A CAR extracellular domain (e.g., binding domain) comprises a ligand and/or antigen recognition region and is the region of a CAR that imparts specificity for binding a specific target molecule (e.g., an antigen, a ligand). In some embodiments, a specific target molecule is or comprises, for example, a polypeptide, a carbohydrate, a glycolipid, and/or a small molecule, including, for example, membrane-inserting amphiphilic ligands described herein. In some embodiments, a CAR comprises affinity to a specific target molecule (e.g., a membrane-inserting amphiphilic ligand described herein) on a target cell (e.g., a tumor cell). The specific target molecule may include, for example, any type of protein, small molecule (e.g., membrane-inserting amphiphilic ligands described herein), or epitope associated with the target cell. For example, in some embodiments, a CAR may comprise affinity to any membrane-inserting amphiphilic ligand described herein.
The extracellular domain can include any region that binds to a specific target molecule (e.g., a ligand, an antigen). In some embodiments, the extracellular domain comprises or is derived from natural binding proteins or receptors. In some embodiments, the extracellular domain comprises or is derived from an antigen binding fragment (Fab) or a single-chain variable fragment (scFv). In some embodiments, a scFv is specific to a membrane-inserting amphiphilic ligand as described herein. Non-limiting examples of a ScFv include those comprising, or derived from, 4m5.3 (see, e.g., Boder, E. T et al. Proceedings of the National Academy of Sciences of the United States of America 97, 10701-10705 (2000)), E2 (see, e.g., Vaughn, T. J. et al., Nature Medicine 2, 534-539 (1996)), or E2 with a His-H58-Ala mutation (“E2.7”) (see, e.g., Pedrazzi, G. et al., FEBS Letters 415, 289-293 (1997)).
In some embodiments, a CAR of the present disclosure may have affinity for one or more specific target molecules (e.g., ligands, antigens) on one or more target cells (e.g., tumor cells). In some embodiments, a CAR may have affinity for one or more specific target molecules on a single target cell. In some such embodiments, a CAR is a bispecific CAR or a multispecific CAR. In some embodiments, a CAR comprises one or more target molecule-specific recognition regions that confer affinity for one or more specific target molecules. In some embodiments, a CAR comprises one or more target-specific recognition regions that confer affinity for the same specific target molecule. For example, a CAR comprising one or more target-specific recognition regions having affinity for the same specific target molecule could bind distinct epitopes of the specific target molecule. When a plurality of target-specific recognition regions are present in a CAR, the recognition regions may be arranged in tandem and may be separated by linker peptides. For example, in a CAR comprising two target-specific recognition regions, the recognition regions are connected to each other covalently on a single polypeptide chain, through a polypeptide linker, an Fc hinge region, or a membrane hinge region.
In some embodiments, a polypeptide linker is rich in glycine. Without wishing to be bound by any one theory, a glycine-rich linker is utilized due to its enhanced solubility. In some embodiments, a polypeptide linker comprises serine and/or threonine. In some embodiments, a polypeptide linker links the heavy chain variable region and the light chain variable region of the extracellular domain. Non-limiting examples of linkers are disclosed in, for example, Shen et al, Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014/087010, the contents of which are hereby incorporated by reference in their entireties. Those of skill in the art would readily understand appropriate polypeptide linkers for use in the present invention.
In some embodiments, an extracellular domain comprises a signal peptide. A variety of signal peptides are known in the art and those of ordinary skill in the art would be able to select an appropriate signal peptide for use in accordance with the present disclosure. In some embodiments, a signal peptide comprises a CD8α signal peptide. In some embodiments, a CD8α signal peptide comprises the amino acid sequence: MALPVTALLLPLALLLHAARP. In some embodiments, a signal peptide comprises a CD8α signal peptide (e.g., MALPVTALLLPLALLLHAARP) followed by a Myc-tag (EQKLISEEDL).
In certain embodiments, the extracellular domain of a presently disclosed CAR has a high binding specificity and high binding affinity to a membrane-inserting amphiphilic ligand as described herein. For example, in some embodiments, the extracellular domain of the CAR (for example, a scFv) binds to a particular tumor antigen with a dissociation constant (Kd) of about 1×10−5 M or less. In certain embodiments, the Kd is about 5×10−6 M or less, about 1×10−6 M or less, about 5×10−7 M or less, about 1×10−7 M or less, about 5×10−8 M or less, about 1×10−8 M or less, about 5×10−9 M or less, about 1×10−9 M or less, about 5×10−10 M or less, about 1×10−10 M or less, about 5×10−11 M or less, about 1×10−11 M or less, about 5×10−12 M or less, about 1×10−12 M or less, about 5×10−13 M or less, about 1×10−13 M or less, about 5×10−14 M or less, about 1×10−14 M or less, about 5×10−15 M or less, about 4×10−15 or less, about 3×10−15 or less, about 2×10−15 or less, or about 1×10−15 M or less.
Binding of the extracellular domain of a presently disclosed CAR can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or a scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular domain of the tumor antigen-targeted CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).
In some embodiments, the extracellular domain of a CAR specifically binds to a membrane-inserting amphiphilic ligand described herein. In some embodiments, the extracellular domain of a CAR specifically binds to a membrane-inserted amphiphilic ligand present on the surface of a target cell (e.g., a tumor cell).
Extracellular domains described herein can be combined with any transmembrane domains described herein, any costimulatory domains described herein, any intracellular domains, and/or any hinge domains described herein.
Transmembrane Domains
A CAR transmembrane domain is a region that is capable of spanning at least a portion of the plasma membrane of a cell (e.g., an engineered immune cell). Different transmembrane domains can result in different receptor stability. In some embodiments, after specific target molecule recognition, receptors cluster and a signal is transmitted to the cell.
In some embodiments, a transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, a CAR transmembrane domain can be designed to connect the extracellular domain of the CAR to the intracellular domain of the CAR. In some embodiments, a transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
In some embodiments, a transmembrane domain is derived from a natural source. In some embodiments, a transmembrane domain is derived from a synthetic source. Where the source is natural, a transmembrane domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, a transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of transmembrane domains include, without limitation, transmembrane domains derived from (i.e., comprise at least the transmembrane region(s) or a fragment thereof of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8 (e.g., CD8α), CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.
In some embodiments, a transmembrane domain further comprises a hinge domain (also referred to as a spacer domain). A CAR hinge domain is a hydrophilic region located between the extracellular domain and the transmembrane domain. In some embodiments, a hinge domain facilitates proper protein folding of a CAR as described herein. In some embodiments, a hinge domain comprises a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. In some embodiments, the hinge domain is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge domain is a hinge domain polypeptide derived from a receptor (e.g., a CD8-derived hinge domain, including, for example CD8α). In some embodiments, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), and/or CHI and CH3 domains of IgGs (such as human IgG4).
In some embodiments, a CAR of the present disclosure comprises a hinge domain that connects the extracellular domain with the transmembrane domain, which, in turn, connects to the intracellular domain. In some embodiments, a hinge domain is capable of supporting the extracellular domain recognize and bind to a specific target molecule on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, a hinge domain is a flexible domain, thus allowing the extracellular domain to have a structure to optimally recognize the specific structure and density of the specific target molecules on a target cell, such as tumor cell. Without wishing to be bound by any one theory, the flexibility of the hinge domain allows the hinge domain to adopt many different conformations.
In some embodiments, a transmembrane domain comprises a CD8α transmembrane domain. In some embodiments, a CAR comprises a CD8α transmembrane domain and a CD8α hinge domain.
Transmembrane domains described herein can be combined with any extracellular domains described herein, any costimulatory domains described herein, any intracellular domains, and/or any hinge domains described herein.
Intracellular Domains
A CAR intracellular domain is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., an engineered immune cell). The intracellular domain transduces effector function signal and directs the cell (e.g., an engineered immune cell) to perform its function (e.g., destruction of a target cell). Examples of an intracellular domain for use in accordance with the present disclosure include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and/or any molecule that acts in concert to initiate signal transduction in the engineered immune cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.
For example, in some embodiments, an intracellular signaling domain includes, without limitation, the z chain of the T cell receptor complex or any of its homologs, e.g., h chain, FcsRFy and b chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (D, d and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In some embodiments, an intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.
Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but are not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma R11a, DAP10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT gAD, CDlId, ITGAE, CD103, ITgAL, CDlla, LFA-1, ITGAM, CD lib, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, TRAN CE/R ANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMI, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combination thereof.
Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAPIO, and CD3z.
In some embodiments, the intracellular domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain. In certain embodiments, an intracellular domain comprises a costimulatory domain and an intracellular signaling domain. In certain embodiments, an intracellular domain comprises 4-1BB, CD28, and/or CD3ζ domains.
In some embodiments, an intracellular domain of a CAR comprises a costimulatory signaling domain which includes any portion of one or more costimulatory molecules, such as at least one signaling domain from CD2, CD3 (including, e.g., CD3ζ), CD8, CD27, CD28, 0X40, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.
Intracellular signaling domains suitable for use in a subject CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by specific target molecule).
While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
Intracellular domains described herein can be combined with any extracellular domains described herein, any costimulatory domains described herein, any transmembrane domains, and/or any hinge domains described herein.
In one aspect, the present disclosure provides engineered immune cells expressing any chimeric antigen receptor (CAR) described herein. In certain embodiments, provided herein are engineered immune cells transformed with a nucleic acid encoding any CAR described herein.
The engineered immune cells of the presently disclosed subject matter can be cells of the lymphoid lineage or myeloid lineage. The lymphoid lineage, comprising B, T, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of immune cells of the lymphoid lineage include T cells, Natural Killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells may be differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cells, including, but not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. In certain embodiments, the CAR-expressing T cells express Foxp3 to achieve and maintain a T regulatory phenotype.
Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.
T cells that can be for example, all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg) and gamma-delta T cells.
In some embodiments, engineered immune cells of the presently disclosed subject matter can be macrophages. Macrophages are effector cells of the innate immune system that phagocytose bacteria and secrete both pro-inflammatory and antimicrobial mediators as well as play an important role in eliminating diseased and/or damaged cells via programmed cell death.
In some embodiments, an engineered immune cell provided herein is autologous to a subject to whom they are to be administered (e.g., after their modification to express a CAR as described herein). In certain embodiments, an engineered immune cell provided herein is allogeneic to a subject to whom they are to be administered (e.g., after their modification to express a CAR as described herein). In some embodiments, wherein allogeneic immune cells are used to prepare an engineered immune cell expressing a CAR, engineered immune cells can be selected that will decrease the possibility of graft-versus-host disease in a subject in need of treatment. In some embodiments, wherein allogeneic immune cells are used to prepare an engineered immune cell expressing a CAR, engineered immune cells can be co-administered with one or more immunosuppressive agents. In some embodiments, an engineered immune cell is obtained from a subject, optionally expanded, and transformed with a polynucleotide comprising a nucleic acid sequence encoding a CAR described herein, and optionally further expanded.
In some embodiments, immune cells provided herein have been isolated from, or expanded from a buffy coat (e.g., a fraction of an anticoagulated blood sample). In certain embodiments, a buffy coat has been obtained and/or prepared from a donor (e.g., a human donor). In certain embodiments, the donor is a healthy human donor. In certain embodiments, the donor is a human donor that has cancer.
In some embodiments, immune cells provided herein have been isolated from, or expanded from whole blood. In certain embodiments, whole blood has been obtained and/or prepared from a donor (e.g., a human donor). In certain embodiments, the donor is a healthy human donor. In certain embodiments, the donor is a human donor that has cancer.
In some aspects, immune cells are obtained from a subject with a disease and/or a condition. In some embodiments, the disease and/or condition is a tumor. In some embodiments, the disease and/or condition is a cancer.
In some aspects, immune cells are obtained from a subject with a disease and/or condition and genetically modified (e.g., in vitro) to express at least one CAR with specificity for at least one membrane-inserting amphiphilic ligand as described herein (e.g., a fluorescein isothiocyanate lipid amphiphile ligand). In some embodiments, an immune cell is genetically modified to express a CAR with specificity for a membrane-inserting amphiphilic ligand described herein is then administered to treat a tumor and/or cancer in a subject in need of treatment. In some embodiments, an engineered immune cell performs at least one effector function (e.g., immune-mediated cell death) that is stimulated or induced by the specific binding of the CAR to the membrane-inserting amphiphilic ligand and that is useful for treatment of a subject in need of treatment. In some embodiments, an engineered immune cell exerts its effector function (e.g., a cytotoxic T cell response) on a target cell (e.g., a tumor cell) when brought in contact or proximity to the target and/or the target cell (see, e.g., Chang and Chen (2017) Trends Mol Med 23(5):430-450).
Stimulation of an engineered immune cell (e.g., by binding of the extracellular domain of the CAR to a membrane-inserting amphiphilic ligand as described herein) can result in the activation of one or more anti-cancer activities of the engineered immune cell. For example, in some embodiments, stimulation of an engineered immune cell can result in an increase in cytotoxic activity or helper activity of an engineered immune cell.
In some embodiments, a subject's immune cells are genetically modified with a CAR (see, e.g., Sadelain et al, Cancer Discov. 3:388-398, 2013). For example, in some embodiments, an immune cell obtained from a subject in need of treatment is provided a recombinant nucleic acid encoding a CAR to generate an engineered immune cell. In some embodiments, an immune cell not obtained from a subject in need of treatment is provided a recombinant nucleic acid encoding a CAR to generate an engineered immune cell. In some embodiments, immune cells are allogenic cells that have been engineered to be utilized as an “off the shelf” adoptive cell therapy.
A plurality of methods are known in the art to introduce nucleic acids into cells (e.g., immune cells). Any suitable method known in the art to introduce any nucleic acids encoding a CAR as described herein into an immune cell can be used. Non-limiting examples of methods for introducing nucleic acid into an immune cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection. Furthermore, the CRISPR/Cas9 genome editing technology known in the art can be used to introduce CAR nucleic acids into immune cells and/or to introduce other genetic modifications into immune cells to enhance engineered immune cell activity (for use of CRISPR/Cas9 technology in connection with engineered immune cells, see e.g., U.S. Pat. Nos. 9,890,393; 9,855,297; US 2017/0175128; US 2016/0184362; US 2016/0272999; WO 2015/161276; WO 2014/191128; CN 106755088; CN 106591363; CN 106480097; CN 106399375; CN 104894068).
In some aspects, a method for producing an engineered immune cell described herein comprises: (i) obtaining cells from peripheral blood, cord blood or lymph (e.g., from peripheral blood mononuclear cells (PMBC)), (ii) optionally, purifying the obtained cells, (iii) optionally, expanding the cells, (iv) optionally, activating the cells (e.g., with an anti-CD3 antibody or an antigen binding fragment thereof and/or an anti-CD28 antibody or an antigen-binding fragment thereof), (v) optionally, expanding the activated cells, (vi) transducing the cells with an expression vector comprising a CAR described herein, (vii) isolating the cells expressing the CAR, and (viii) optionally, expanding the isolated cells.
In some aspects, a method for producing an engineered immune cell described herein comprises: (i) obtaining a pluripotent stem cell (iPSC) (ii) inducing iPSC to differentiate into a particular immune cell (such as CD8+ T cell), (iii) optionally, expanding the cells, (iv) transducing the cells with an expression vector comprising a CAR described herein, (v) isolating the cells expressing the CAR, and (vi) optionally, expanding the isolated cells.
In some aspects, a method for producing an engineered immune cell described herein comprises: (i) obtaining whole blood or a buffy coat (e.g., from a healthy donor), (ii) isolating human primary immune cells (e.g., T cells) from the whole blood or buffy coat, (iii) optionally, expanding the cells, (iv) transducing the cells with an expression vector comprising a CAR described herein, (v) isolating the cells expressing the CAR, and (vi) optionally expanding the isolated cells.
In one aspect, the present disclosure provides compositions (e.g., pharmaceutical compositions) comprising a membrane-inserting amphiphilic ligand as described herein. In some embodiments, membrane-inserting amphiphilic ligands as described herein of pharmaceutical compositions can be purified (e.g., separated from one or more undesired components).
In one aspect, the present disclosure provides compositions (e.g., pharmaceutical compositions) comprising engineered immune cells as described herein. In some embodiments, engineered immune cells as described herein of pharmaceutical compositions can be purified (e.g., separated from one or more undesired components).
In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier. Appropriate pharmaceutically acceptable carriers, including but not limited to excipients and stabilizers, are known in the art (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA).
In some embodiments, a pharmaceutical composition is a sterile composition. In some such embodiments, a pharmaceutical composition (e.g., a sterile composition) can comprise cells, tethering means (e.g., lipid nanoparticles) and/or proteins or polypeptides, preferably in a pharmaceutically-acceptable carrier (e.g., one or more compatible solid or liquid filler, diluents or encapsulating substances that are suitable for administration to a human or other subject contemplated herein). In some embodiments, the carrier can be an organic or inorganic ingredient, natural or synthetic, with which the cells, tethering means (e.g., lipid nanoparticles) and/or proteins or peptides are combined to facilitate administration. The components of the pharmaceutical compositions are commingled in a manner that precludes interaction that would substantially impair their desired pharmaceutical efficiency.
Pharmaceutically acceptable carriers may include, for example and without limitation, a buffer, an emulsifying agent, a suspending agent, a dispersing agent, an isotonic agent, a wetting agent, a chelating agent, a sequestering agent, a pH buffering agent, a solubility enhance, an antimicrobial agent, an anesthetic, and/or an antioxidant.
Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, M D, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: saline, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, sucrose, dextrose, magnesium stearate, malt, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, glycerol, ethanol, polyvinyl pyrrolidone, povidone, starch (e.g., pregelatinized starch), propylene, propyl paraben, retinyl palmitate, shellac, silica gel, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium stearate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, talc, base cream, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.
In some embodiments, the pharmaceutical compositions disclosed herein may include at least one pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts that may be included in a composition of the disclosure include, but are not limited to, acid addition salts, alkali or alkaline earth metal salts, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like
In some embodiments, pharmaceutical compositions can be formulated such that they are suitable for administration to a subject (e.g., a human) in need of treatment. A pharmaceutical composition may be formulated for any route of administration.
In some embodiments, pharmaceutical compositions, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Such formulations can be prepared as liquid solutions, suspensions, emulsions or solid forms suitable making into a solution or suspension prior to injection. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers. In some embodiments, pharmaceutical parenteral formulations include aqueous solutions of the ingredients. Aqueous injection suspensions may contain, for example, substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively, suspensions of ingredients may be prepared as oil-based suspensions. Suitable lipophilic solvents or vehicles include, for example, fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. If administered parenterally, suitable pharmaceutically acceptable carriers may include, without limitation, physiological saline or phosphate buffered saline (PBS), or solutions containing, e.g., polyethylene glycol, polypropylene glycol or glucose.
A membrane-inserting amphiphilic ligand described herein and/or engineered immune cell described herein can be used or present in a therapeutically effective amount in the pharmaceutical composition disclosed herein. The therapeutically effective amount can be determined by standard clinical techniques.
The pharmaceutically acceptable compositions contemplated herein may include, in addition to a membrane-inserted amphiphilic ligand described herein and/or engineered immune cell described herein, an additional anti-cancer agent (e.g., any one, two, three or more anti-cancer agents described herein).
In one aspect, the present disclosure provides technologies for treating a tumor (e.g., inhibiting cancer proliferation, inhibiting cancer progression) in a subject in need of treatment comprising administering to the subject any membrane-inserting amphiphilic ligand as described herein, any engineered immune cell as described herein, or any pharmaceutical composition described herein. In some embodiments, the present disclosure provides technologies for treating a solid tumor. In some embodiments, the present disclosure provides technologies for treating a cancer, as described herein.
In some embodiments, the present disclosure provides methods for treating a tumor (e.g., a solid tumor, a tumor of a subject in need of treatment) and/or a cancer. In some such embodiments, a method of treating a tumor comprises introducing (e.g., administering) a membrane-inserting amphiphilic ligand as described herein to a subject in need of treatment followed by administering an engineered immune cell that specifically binds to the amphiphilic ligand as described herein, or a pharmaceutical composition described herein. In some embodiments, treatment locally at a primary tumor results in a systemic anti-tumor immune response, which can result in treatment of a secondary tumor (e.g., a tumor distal to the primary tumor).
In some embodiments, membrane-inserting amphiphilic ligands as described herein, engineered immune cells as described herein, and/or pharmaceutical compositions described herein can be used in the development of a targeted immunotherapy for treating a tumor (e.g., a solid tumor, a tumor of a subject in need of treatment) and/or a cancer.
In certain embodiments, the present disclosure provides technologies for preventing cancer in a subject in remission from cancer, comprising administering to the subject in need of treatment any membrane-inserting amphiphilic ligand described herein, any engineered immune cell described herein, or any pharmaceutical composition described herein.
In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer is a refractory cancer. In some embodiments, the cancer is an advanced stage cancer. In some embodiments, the cancer is resistant to one or more other therapies (e.g., chemotherapy, radiotherapy, stem cell transplantation, or another immunotherapy).
The effectiveness of any therapy described herein can be assessed by evaluating a parameter (e.g., tumor burden) before and after administration of the therapy (e.g., to the subject in need of treatment). Any assay known in the art can be used to evaluate the therapeutic effectiveness of the therapies described herein.
Technologies of the present disclosure (e.g., compositions, therapies) can be administered to a subject (e.g., a subject in need of treatment) by any suitable means which include, but are not limited to, parenteral route of administration. In some embodiments, compositions as described herein are administered via the same route of administration. In some embodiments, compositions described herein are administered via different routes of administration.
In some embodiments, composition(s) as described herein are administered to the patient parenterally. Non-limiting examples of parenteral administration include intravenous, intramuscular, intraarterial, subcutaneous, intratumoral, intrathecal and intraperitoneal administration. In one embodiments, a composition(s) described herein is administered intratumorally. In one embodiment, a composition(s) described herein is administered intravenously. In one embodiment, a composition(s) described herein is administered intraperitoneally. In one embodiment, a composition(s) described herein is administered intramuscularly. In one embodiment, a composition(s) described herein is administered subcutaneously. In certain embodiments, the administration is intravenous, intrathecal, intraosseous or into the spinal cord. In one embodiment, a composition(s) described herein is administered into the spinal cord or the spinal canal. In one embodiment, a composition(s) described herein is administered intrathecally. In one embodiment, a composition(s) described herein is administered intraosseously. In one embodiment, a composition(s) described herein is administered into the bone marrow.
In some embodiments, a membrane-inserting amphiphilic ligand as described herein is administered intratumorally. Intratumoral (i.t.) administration of a membrane-inserting amphiphilic ligand described herein can result in the membrane-inserting amphiphilic ligand remaining local after i.t. administration (e.g., in the tumor tissue in which it was administered without spreading or without significant spreading to neighboring tissue, such as non-tumor tissue). In some embodiments, a membrane-inserting amphiphilic ligand is administered intratumorally to a primary tumor. Without wishing to be bound by any one theory, it is understood that such local administration (e.g., i.t. administration) of a membrane-inserting amphiphilic ligand to a primary tumor results in redirection of an engineered immune cell to the primary tumor and can also lead to a systemic anti-tumor immune response, thus, resulting in the treatment of secondary tumors (e.g., a tumor at a site distal to the primary tumor).
In some embodiments, a membrane-inserting amphiphilic ligand as described herein is administered subcutaneously. In some such embodiments, the membrane-inserting amphiphilic ligand is introduced into dendritic cells in lymph nodes of the subject by subcutaneous injection. In some embodiments, a membrane-inserting amphiphilic ligand as described herein is administered intratumorally and subcutaneously (e.g., into dendritic cells in lymph nodes of the subject by subcutaneous injection).
In some embodiments, an engineered immune cell as described herein is administered by systemic infusion.
An appropriate dosage of membrane-inserting amphiphilic ligand and/or engineered immune cells as described herein will vary with the particular condition and/or disease (e.g., a tumor, a cancer) being treated, the age, weight, and physical condition of the subject in need of treatment, the severity of the cancer, the route of administration, the duration of the treatment, the responsiveness of the subject being treated, the nature of the concurrent or combination therapy (if any), the specific route of administration and like factors within the knowledge and expertise of a health practitioner. In certain embodiments, a maximal tolerable dose of a membrane-inserting amphiphilic ligand as described herein and/or an engineered immune cell as described herein is to be used, that is, the highest safe dose according to sound medical judgement. In preferred embodiments, a membrane-inserting amphiphilic ligand and/or an engineered immune cell as described herein is to be administered in effective amounts. An effective amount is, for example, a dose of the composition(s) sufficient to provide a medically desirable result. A medically desirable result, for example, for a subject in need of treatment with a tumor, may be an amount that decreases tumor volume or load. Effective amounts may also be assessed by the presence and/or frequency of tumor cells in the blood or other body fluid or tissue. If a tumor impacts the standard functioning of a tissue or organ, the effective amount may be assessed by measuring standard functioning of the tissue or organ.
In certain embodiments, a membrane-inserting amphiphilic ligand as described herein is administered into a tumor of a subject in an amount of about or at least 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 20 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 250 nM, 500 nM, 750 nM, or 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 750 μM or 1 mM (or any value or range in between). In certain embodiments, for example, a membrane-inserting amphiphilic ligand as described herein is administered into a tumor of a subject in an amount of about or at least 1 nM to 1 mM, 10 nM to 1 mM, 100 nM to 1 mM, 250 nM to 1 mM, 500 nM to 1 mM, 750 nM to 1 mM, 1 nM to 750 μM, 1 nM to 500 μM, 1 nM to 250 μM, 1 nM to 100 μM, or 1 nM to 10 μM.
In certain embodiments, an engineered immune cell as described herein are administered in an amount of about or at least 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 1×109, 1×1010, 5×1010, 1×1011, or 5×1011, 1×1012, or 5×1012 cells (or any value or range in between).
Various dosing schedules of the compositions described herein are contemplated including single administration or multiple administrations over a period of time. The methods of administration include, without limitation, bolus administration and infusions (e.g., continues or pulse infusions).
The therapeutic regimen for use in the methods described herein may include administration of a composition as described herein once a day, once every two days, once every three days, once every four days, once every five days, once every six days, twice a week, once every week, once every two weeks, once every three weeks, once every month or 4 weeks, once every six weeks, once every two months or eight weeks, or once every three months or twelve weeks. In certain embodiments, the subject receives a single dose of any therapy described herein. In certain embodiments, the subject receives from at least two, at least three, at least four, at least five, at least six, at least eight, or at least ten doses of any therapy described herein. In certain embodiments, a therapy described herein is administered daily, every other day, or two times a week. In certain embodiments, a therapy described herein is administered for a period of time, such as one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, or one year.
In some embodiments, the initial treatment period (where the therapy is administered, e.g., a single time, twice a week, once a week, twice in two weeks, or once a month) is followed by a withdrawal period in which the antibody is not administered (for, e.g., a week, two weeks, three weeks, 1 month or four weeks, six weeks, two months or 8 weeks, three months, four months, five months, six months, or 1 year), and then followed by a second treatment period (where the therapy is administered, e.g., a single time, twice a week, once a week, twice in two weeks, or once a month). Such initial treatment and such second treatment periods can last, for example, two weeks, three weeks, four weeks, six weeks, two months, or three months (where the initial treatment period can be the same or different from the second treatment period).
In some embodiments, a subject in need of treatment in accordance with the technologies disclosed herein include, but are not limited to, humans and non-human vertebrates. In some embodiments, a subject in need of treatment in accordance with the technologies disclosed herein comprise, for example, a mammal. In some such embodiments, a mammal includes, for example and without limitation, a household pet (e.g., a dog, a cat, a rabbit, a ferret, a hamster, etc.), a livestock or farm animal (e.g., a cow, a pig, a sheep, a goat, a pig, a chicken or another poultry), a horse (e.g., a thoroughbred horse), a monkey, a laboratory animal (e.g., a mouse, a rat, a rabbit, etc.), and the like. Subjects also include fish and other aquatic species. In a preferred embodiment, the subject being treated in accordance with the methods described herein is a human. In some embodiments, technologies of the present disclosure can be practiced in any subject in need of treatment that is likely to benefit from targeted immunotherapy for the treatment of a tumor, including, for example, a solid tumor.
In some aspects, the technologies disclosed herein can be practiced in any subject that has (e.g., has been diagnosed with) a cancer that may (or is likely to) benefit from any immunotherapy described herein. A subject having a tumor (e.g., a solid tumor) is a subject that has detectable tumor cells. The disclosure contemplates, among other things, any membrane-inserting amphiphilic ligand described herein and/or any engineered immune cell described here to a subject in need of treatment (e.g., a subject with a tumor).
In some aspects, the therapeutic methods and uses of the present disclosure can be practiced in a subject that has cancer. A subject that has cancer is a subject that has detectable cancer cells. Certain exemplary cancers that may, in some embodiments, be treated in accordance with the present disclosure include, for example, adrenocortical carcinoma, astrocytoma, basal cell carcinoma, carcinoid, cardiac, cholangiocarcinoma, chordoma, chronic myeloproliferative neoplasms, craniopharyngioma, ductal carcinoma in situ, ependymoma, intraocular melanoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, glioma, histiocytosis, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, myelogenous leukemia, and myeloid leukemia), lymphoma (e.g., Burkitt lymphoma (non-Hodgkin lymphoma), cutaneous T-cell lymphoma, Hodgkin lymphoma, mycosis fungoides, Sezary syndrome, AIDS-related lymphoma, follicular lymphoma, diffuse large B-cell lymphoma), melanoma, merkel cell carcinoma, mesothelioma, myeloma (e.g., multiple myeloma), myelodysplastic syndrome, papillomatosis, paraganglioma, pheochromocytoma, pleuropulmonary blastoma, retinoblastoma, sarcoma (e.g., Ewing sarcoma, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, uterine sarcoma, vascular sarcoma), Wilms' tumor, and/or cancer of the adrenal cortex, anus, appendix, bile duct, bladder, bone, brain, breast, bronchus, central nervous system, cervix, colon, endometrium, esophagus, eye, fallopian tube, gall bladder, gastrointestinal tract, germ cell, head and neck, heart, intestine, kidney (e.g., Wilms' tumor), larynx, liver, lung (e.g., non-small cell lung cancer, small cell lung cancer), mouth, nasal cavity, oral cavity, ovary, pancreas, rectum, skin, stomach, testes, throat, thyroid, penis, pharynx, peritoneum, pituitary, prostate, rectum, salivary gland, ureter, urethra, uterus, vagina, or vulva.
In some embodiments, a cancer may involve one or more tumors. In some embodiments, a tumor comprises a solid tumor. In some embodiments, solid tumors include but are not limited to tumors of the bladder, breast, central nervous system, cervix, colon, esophagus, endometrium, head and neck, kidney, liver, lung, ovary, pancreas, skin, stomach, uterus, or upper respiratory tract.
Tests for diagnosing the cancers to be treated by the methods described herein are known in the art and will be familiar to the ordinary medical practitioner. These laboratory tests include, without limitation, microscopic analyses, cultivation dependent tests (such as cultures), and nucleic acid detection tests. These include wet mounts, stain-enhanced microscopy, immune microscopy (e.g., FISH), hybridization microscopy, particle agglutination, enzyme-linked immunosorbent assays, urine screening tests, DNA probe hybridization, serologic tests, etc. The medical practitioner generally takes a full history and conducts a complete physical examination in addition to running the laboratory tests listed above.
In some aspects, the subject being treated has previously undergone one or more other cancer therapies (e.g., chemotherapy, radiotherapy, or stem cell transplantation). In certain embodiments, the subject being treated has previously undergone one or more other cancer therapies (e.g., chemotherapy, radiotherapy, or stem cell transplantation), and the subject's cancer has relapsed. In certain embodiments, the subject being treated has previously undergone one or more other cancer therapies (e.g., chemotherapy, radiotherapy, or stem cell transplantation), and the subject has developed resistance to the one or more other cancer therapies. In certain embodiments, the subject being treated is in remission (e.g., in partial remission or in complete remission of cancer). In certain embodiments, the subject being treated is refractory to one or more other cancer therapies (e.g., chemotherapy, radiotherapy, or stem cell transplantation).
In other embodiments, contemplated herein is treating a subject that is at risk of developing cancer that may (or is likely to) benefit from any immunotherapy described herein in accordance with the therapeutic methods and uses of the disclosure. A subject at risk of developing a cancer is a subject that has a higher than normal probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality that has been demonstrated to be associated with a higher likelihood of developing a cancer, subjects having a familial disposition to cancer, subjects exposed to cancer causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, and subjects previously treated for cancer and in apparent remission. The disclosure contemplates administration of membrane-inserting amphiphilic ligands described herein and engineered immune cells as described herein, or compositions comprising the same, to subjects at risk of developing a cancer.
In on embodiment, the subject in need of treatment is an adult. In one embodiment, the subject is a human subject over 18 years of age. In one embodiment, the subject is a human subject over 21 years of age. In one embodiment, the subject is a human subject over 45 years of age. In one embodiment, the subject is a human subject over 65 years of age. In one embodiment, the subject is a human subject under 18 years of age. In one embodiment, the subject is a human subject under 45 years of age (or between 18 and 45 years of age, or between 21 and 45 years of age). In one embodiment, the subject is a human subject under 65 years of age (or between 18 and 65 years of age, between 21 and 65 years of age, or between 45 and 65 years of age).
In some aspects, therapeutic methods and uses described herein further include methods of treating a tumor (e.g., a tumor of a subject in need of treatment) with additional agents that enhance therapeutic responses, such as, for example, enhance an anti-tumor response in a subject in need of treatment and/or that are cytotoxic to the tumor (e.g., chemotherapeutic agents).
In some aspects, a method of treatment described herein is administered to a subject in combination with one or more anti-cancer therapy, e.g., a chemotherapy, a radiation therapy, stem cell transplantation, a small molecule with an anti-cancer activity, another anti-cancer immunotherapy (e.g., another anti-cancer antibody or fragment thereof, or another T cell therapy), or any other anti-cancer therapy known in the art.
In some aspects, one or more of the membrane-inserting amphiphilic ligands, or compositions comprising the same, as described herein, are administered to a subject in need of treatment in combination with one or more anti-cancer therapy, e.g., a chemotherapy, a radiation therapy, stem cell transplantation, a small molecule with an anti-cancer activity, another anti-cancer immunotherapy (e.g., an anti-cancer antibody or fragment thereof, or another T cell therapy), or any other anti-cancer therapy known in the art.
In some aspects, one or more of the engineered immune cells, or compositions comprising the same, as described herein are administered to a subject in need of treatment in combination with stem cell transplantation or another anti-cancer immunotherapy (e.g., an anti-cancer antibody or fragment thereof, or another cell therapy). For administration with engineered immune cells, any combination of therapies that would not negatively affect the viability of the engineered immune cells are contemplated herein.
In some aspects, one or more of the membrane-inserting amphiphilic ligands and engineered immune cells, or compositions comprising the same, as described herein are administered to a subject in need of treatment in combination with stem cell transplantation or another anti-cancer immunotherapy (e.g., an anti-cancer antibody or fragment thereof, or another cell therapy). For administration with engineered immune cells, any combination of therapies that would not negatively affect the viability of the engineered immune cells are contemplated herein.
Suitable therapeutic agents for use in combination therapy include small molecule chemotherapeutic agents, including protein tyrosine kinase inhibitors, as well as biological anti-cancer agents, such as anti-cancer antibodies, including but not limited to those discussed further below.
In some aspects, combination therapy includes administering to the subject an immune checkpoint inhibitor to enhance anti-tumor immunity, such as a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, or a CTLA-4 inhibitor. Other modulators of immune checkpoints may target TIM-3, OX-40, OX-40L or ICOS. In one embodiment, an agent that modulates an immune checkpoint is an antibody (e.g., an antagonistic antibody to PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, or OX-40). In another embodiment, an agent that modulates an immune checkpoint is a protein or small molecule modulator. In another embodiment, the agent (such as an mRNA) encodes an antibody modulator of an immune checkpoint. In one embodiment, any therapy described herein is administered in combination with a TIM-3 inhibitor. In one embodiment, any therapy described herein is administered in combination with a PD-1 inhibitor. In one embodiment, any therapy described herein is administered in combination with a PD-L1 inhibitor. In one embodiment, any therapy described herein is administered in combination with a CTLA-4 inhibitor. Non-limiting examples of immune checkpoint inhibitors that can be used in combination therapy include pembrolizumab, alemtuzumab, nivolumab, pidilizumab, ofatumumab, rituximab, MEDIO680, PDR001, AMP-224, PF-06801591, BGB-A317, REGN2810, SHR-1210, TSR-042, affimer, avelumab (MSB0010718C), atezolizumab (MPDL3280A), durvalumab (MEDI4736), BMS936559, ipilimumab, tremelimumab, AGEN1884, MEDI6469 and MOXR0916.
In a specific embodiment, a therapy described herein is administered to a subject in combination with chemotherapy. Examples of types of chemotherapeutic agents that can be used in the combination therapy described herein include, without limitation, an alkylating agent, a nitrosourea agent, an antimetabolite, a platinum complex derivative, a topoisomerase inhibitor, an aromatase inhibitor, an alkaloid derived from a plant, a hormone antagonist, an antitumor antibiotic, and a P-glycoprotein inhibitor. Specific examples of chemotherapeutic drugs that can be used in the combination therapy described herein include, without limitation, taxol, paclitaxel, nab-paclitaxel, 5-fluoro uracil (5-FU), gemcitabine, doxorubicin, daunorubicin, colchicin, mitoxantrone, tamoxifen, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, busulfan, uramustine, mustargen, ifosamide, bendamustine, carmustine, lomustine, semustine, fotemustine, streptozocin, thiotepa, mitomycin, diaziquone, tetrazine, altretamine, dacarbazine, mitozolomide, temozolomide, procarbazine, hexamethylmelamine, altretamine, hexalen, trofosfamide, estramustine, treosulfan, mannosulfan, triaziquone, carboquone, nimustine, ranimustine, azathioprine, sulfanilamide, fluoropyrimidine, thiopurine, thioguanine, mercaptopurine, cladribine, capecitabine, pemetrexed, fludarabine, methotrexate, hydroxyurea, nelarabine or clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, thioquanine, azacitidine, cladribine, pentostatin, mercaptopurine, imatinib, dactinomycin, cerubidine, bleomycin, actinomycin, luteomycin, epirubicin, idarubicin, plicamycin, vincristin, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, etoposide, teniposide, periwinkle, vinca, taxane, irinotecan, topotecan, camptothecin, teniposide, pirarubicin, novobiocin, merbarone, aclarubicin, amsacrine, antiandrogen, anti-estrogen, bicalutamide, medroxyprogesterone, fluoxymesterone, diethylstilbestrol, estrace, octreotide, megestrol, raloxifene, toremifene, fulvestrant, prednisone, flutamide, leuprolide, goserelin, aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, androstene, resveratrol, myosmine, catechin, apigenin eriodictyol isoliquiritigenin, mangostin, amiodarone, azithromycin, captopril, clarithromycin, cyclosporine, piperine, quercetine, quinidine, quinine, reserpine, ritonavir, tariquidar, verapamil, cisplatin, carboplatin, oxaliplatin, transplatin, nedaplatin, satraplatin, triplatin and carboplatin.
In a specific embodiment, a therapy described herein is administered to a subject in combination with radiation therapy.
In a specific embodiment, any therapy described herein is administered to a subject in combination with stem cell transplantation.
In certain embodiments, any therapy described herein can be administered before, during (i.e., concurrently) or after one or more additional anti-cancer therapy. In one embodiment, the subject being treated in accordance with the methods described herein has not previously received an anti-cancer therapy. In one embodiment, the subject being treated in accordance with the methods described herein has previously received an anti-cancer therapy (e.g., a chemotherapy, a radiation therapy, or a stem cell transplant).
In one aspect, provided herein are kits comprising one or more containers comprising (i) a membrane-inserting amphiphilic ligand as described herein, a CAR polypeptide described herein, engineered immune cell (e.g., a T cell) comprising a CAR polypeptide described herein, or a pharmaceutical composition described herein; (ii) optionally, one or more additional anti-cancer agents (e.g., a chemotherapeutic agent), and (iii) optionally, instructions for use in treating a tumor.
In certain embodiments, the disclosure pertains to kits comprising a membrane-inserting amphiphilic ligand as disclosed herein, and instructions for use. In certain embodiments, the disclosure pertains to kits comprising an engineered immune cell (e.g., a T cell) comprising a CAR polypeptide described herein, and instructions for use. In certain embodiments, the disclosure pertains to kits comprising an engineered immune cell as disclosed herein, and instructions for use.
In some embodiments, a kit may comprise, in the same or separate suitable containers a membrane-inserting amphiphilic ligand as disclosed herein and a pharmaceutically acceptable carrier (e.g., a buffer). In some embodiments, a kit may comprise in the same or separate suitable containers, an engineered immune cell as disclosed herein and a pharmaceutically acceptable container (e.g., a buffer). In some embodiments, a kit may comprise, in the same or separate suitable containers, a membrane-inserting amphiphilic ligand as disclosed herein, an engineered immune cell as disclosed herein, and a pharmaceutically acceptable carrier (e.g., a buffer).
A suitable container may include, without limitation, a vial, well, test tube, flask, bottle, syringe, infusion bag, or other container means, into which the membrane-inserting amphiphilic ligand described herein or engineered immune cell as described herein, may be placed (and in some instances, suitably aliquoted). Where an additional component is provided, the kit can contain additional containers into which this component may be placed. The containers may further include injection or blow-molded plastic containers in which the desired vials are retained. Containers and/or kits can include labeling with instructions for use and/or warnings.
The following materials and methods were used for the following Examples.
Materials: DSPE-PEG-FITC (PEG MW 1, 2, 3.4, 5 kDa) was purchased from Creative PEGworks (cat #PLS-9926 to 9929). DSPE-FITC and DSPE-PEG10k-FITC were purchased from Nanosoft Polymers (cat #10805) and Nanocs (cat #PG2-DSFC-10k), respectively. Cyclic di-GMP was purchased from Invivogen.
Cell lines: Phoenix-ECO, B16F10, and MSTO-211H cells were purchased from ATCC. 293T cells were purchased from Clontech. MC38 and CT-2A-Luc cells were kindly gifted. CT-2A and MSTO-211H cells were lentivirally transduced to stably express murine EGFRvIII and tdTomato or human mesothelin, respectively, with selection using puromycin and sorting on a BD FACSAria™ III Cell Sorter. B16F10, CT-2A, MC38, 293T, and Phoenix-ECO cells were cultured in complete Dulbecco's modified Eagle's medium (DMEM, Cytiva; supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin). MSTO-211H cells were cultured in complete RPMI medium (Cytiva).
Construct design: All murine CARs contained the CD8α signal peptide (MALPVTALLLPLALLLHAARP) followed by a Myc-tag (EQKLISEEDL), for analysis of cell surface expression) and a FITC-specific scFv derived from monoclonal antibody clone 4m5.3, E2, or E2 with a His-H58-Ala mutation (“E2.7”) (see, e.g., Boder, E. T et al. Proceedings of the National Academy of Sciences of the United States of America 97, 10701-10705 (2000); Vaughn, T. J. et al., Nature Medicine 2, 534-539 (1996)); Pedrazzi, G. et al., FEBS Letters 415, 289-293 (1997)). Extracellular domains were fused to the CD8α hinge and transmembrane domains, CD28 or 4-1BB costimulatory domains, and a CD3ζ intracellular domain, and were cloned into a pMIG retroviral vector. For BLI studies, T cells were co-transduced with MI-FLuc-IRES-mCherry (Addgene plasmid #75020; http://n2t.net/addgene:75020; RRID:Addgene_75020). All humanized CARs were similarly cloned as above using humanized domains, with the exception that CARs containing CD28 costimulatory domains were paired with a CD28 transmembrane domain. Humanized CARs were cloned into pLenti6.3 lentiviral vector (ThermoFisher) under the control of a cytomegalovirus (CMV) promoter. For BLI studies, T cells were co-transduced with pCDH-EF1-Luc2-P2A-tdTomato (Addgene plasmid #72486; http://n2t.net/addgene:72486; RRID:Addgene_72486). Expression of all CARs was determined by flow cytometry staining for the Myc-tag using an anti-Myc tag antibody (9B11, PE conjugate, Cell Signaling) with analysis on a BD LSR-II flow cytometer.
Retrovirus production and engineering of murine CAR T cells: To produce ecotropic retrovirus for murine T cell transduction, Phoenix-ECO cells were transfected with a 1:3 ratio of pCL-Eco and transfer plasmid using a CalPhos Mammalian Transfection Kit (Takara Bio). Primary murine T cells were isolated from spleens of WT or CD45.1+ C57BL/6 mice using the EasySep Mouse CD8+ T Cell Isolation Kit or EasySep Mouse T cell Isolation Kit (StemCell Technologies) and activated for 48 hours on 6-well non-TC-treated plates precoated with 0.5 μg/mL anti-CD3 (BioXCell, Clone 2C11) and 5 μg/mL anti-CD28 (BioXCell, Clone 37.51) at 106 cells/mL with 3-5 mL/well. T cells were cultured in complete RPMI with 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol, and 1× minimal essential medium (MEM) non-essential amino acids (ThermoFisher) supplemented with 10 ng/mL murine IL-2 (BioLegend). Following activation, T cells were transduced by spinfection at 3×106 cells/well in complete T cell media for 90 min at 1100×g and 32° C. with 10 ng/mL polybrene (Sigma) on non-TC-treated plates precoated with 15 μg/mL RetroNectin (Takara Bio). CAR expression was assayed via flow cytometry 24 hr post-transduction; T cells were maintained at a cell density of 106 cells/mL, then used for adoptive transfer or in vitro assays at 48 hr post-transduction.
Lentivirus production and engineering of human CAR T cells: For humanized CARs, vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus was produced via transfection of 293T cells with psPAX2, pMD2.G, and transfer plasmid in a 2:1:2 ratio using Effectene Transfection Reagent (QIAGEN). Primary human T cells were isolated from buffy coats from healthy donors (Massachusetts General Hospital Blood Donor Center) using the EasySep Human CD8+ T Cell Isolation Kit or EasySep Human T Cell Isolation Kit (StemCell Technologies) and activated for 48 hours using Dynabeads Human T-Activator CD3/CD28 (ThermoFisher) at a 3:1 bead-to-cell ratio. Human T cells were cultured in complete RPMI supplemented with 30 U/mL human IL-2 (PeproTech). Following activation, T cells were transduced and stained as described above, and used for adoptive transfer or in vitro studies at 48-72 hr post-transduction.
Amph-FITC labeling and cytotoxicity studies: For in vitro labeling of tumor cells with amph-FITC, tumor cells were washed with 1×PBS and incubated at a cell density of 106 cells/mL for 30 minutes at 37° C. with DSPE-PEGx-FITC, where x=0, 1, 2, 3.4, 5, or 10 kDa (Creative PEGworks) in PBS. For cocultures, target cells were labeled simultaneously with 100 nM amph-FITC and CellTrace Violet (ThermoFisher); 20,000 target cells were seeded per well in a 96-well flat-bottom plate with CAR T cells at the indicated effector-to-target ratios. Following a 16 hr incubation, cells were stained with SYTOX red (ThermoFisher) for flow cytometry, and IFN-γ in the supernatant was quantified using a Mouse IFN gamma Uncoated ELISA Kit (Invitrogen).
Biodistribution analysis: To assess biodistribution of amph-FITC following intratumoral injection, C57BL/6 mice (6-8 weeks, Jackson Laboratory, n=5) were inoculated subcutaneously with 106 B16F10 tumor cells. Once tumors were ˜25 mm2 in area, 10 nmol DSPE-PEG2k-FITC (Creative PEGworks) was injected intratumorally. After 24 hr, tissues (tumor, liver, kidney, spleen, bone marrow, and both axillary and inguinal LNs) were homogenized in ethanol (pH 8.0-8.5) and supernatant fluorescence was quantified on a FlexStation 3 plate reader (Molecular Devices, excitation 495 nm, emission 519 nm).
For cellular localization studies, MC38 tumor-bearing mice were injected intratumorally with 10 nmol DSPE-PEG2k-FITC, and 24 hr later tumors were enzymatically digested at 37° C. for 30 min with 1 mg/mL collagenase D and 0.2 mg/mL DNase I in complete RPMI media before mechanical dissociation through a 70 μm cell strainer. Samples were stained using the following antibodies: PE-Cy7 anti-CD45 (clone 30-F11, BioLegend), BV605 anti-CD11b (clone M1/70, BioLegend), PE anti-CD11c (clone N418, BioLegend), BV711 anti-mouse CD3 (clone 17A2, BioLegend), BV421 anti-CD8α (clone 53-6.7, BioLegend), and Alexa Fluor 647 anti-FITC (Jackson ImmunoResearch) and were analyzed on a BD LSRFortessa flow cytometer.
In vivo therapy studies: All animal work was conducted under a Massachusetts Institute of Technology (MIT) Division of Comparative Medicine institute animal care and use committee-approved protocol in accordance with federal, state and local guidelines.
C57BL/6 WT, CD45.1+, Batf3 KO, and Rag1 KO mice (Jackson Laboratory, 6 to 8 weeks old) were inoculated with 106 B16F10, 106 MC38, 106 TC-1, or 3×106 CT-2A tumor cells subcutaneously at the flank. Once tumors grew to ˜25 mm2 in area, mice underwent a non-myeloablative 5 Gy dose of TBI administered by a [137Cs] gamma radiation source, 24 hr prior to adoptive transfer of 107 CD45.1+ CAR T cells IV via the tail vein. One day later, mice were vaccinated subcutaneously at the tail base with 10 nmol DSPE-PEG2k-FITC (Creative PEGworks) and 25 μg cyclic di-GMP (Invivogen), and vaccinated for 3 total doses, once weekly. Intratumoral injection of 10 nmol amph-FITC was administered beginning the day after the first vaccination, and continuing every 3 days.
For xenograft studies, 6- to 12-week-old NOD.Cg-PrkdcscidIL2rgtm1Wj1/SzJ (NSG) mice (Jackson Laboratory) were inoculated with 3×106 MSTO-211H cells subcutaneously at the flank. Once tumors grew to ˜25 mm2, mice were adoptively transferred with 107 CAR T cells IV via the tail vein. Intratumoral injection of 10 nmol amph-FITC was administered beginning the day after adoptive transfer, and continuing every 3 days.
For all studies, tumor progression was monitored by caliper measurements. Survival was evaluated over time until tumors exceeded 200 mm2 in area, were severely ulcerated >1 week, or experienced >20% weight loss. To monitor trafficking of Fluc-expressing CAR T cells, mice were injected subcutaneously at the scruff with 150 mg/kg D-luciferin K+ Salt (Perkin-Elmer) and imaged on a Xenogen IVIS Spectrum. Prior to imaging, C57BL/6 mice were depilated on their back to improve signal sensitivity.
Flow cytometry: For quantification of murine CAR T and endogenous T cells in the peripheral blood, tumor, and spleen, tissues were homogenized through a 70 μm cell strainer (with the exception of MC38 tumors, which required enzymatic digestion as described above). Blood and spleen samples were incubated with ACK lysing buffer (Gibco) to isolate splenocytes and PBMCs, respectively. Samples were then stained with PE anti-CD45.1 (clone A20, BioLegend), BUV805 anti-CD8α (clone 53-6.7, BioLegend), Alexa Fluor 647 anti-CD4 (clone GK1.5, BioLegend), APC-Cy7 anti-CD45.2 (clone 104, BioLegend), and LIVE/DEAD Fixable Aqua (ThermoFisher) and analyzed on a BD LSRFortessa flow cytometer.
For antigen uptake studies, tumors and LNs were stained with Zombie Aqua Fixable Viability Kit (BioLegend), BUV395 anti-CD45 (clone 30-F11, BD Biosciences), BV421 anti-CD103 (clone 2E7, BioLegend), BV605 anti-Ly6C (clone HK1.4, BioLegend), BV711 anti-F4/80 (clone BMB, BioLegend), BV785 anti-CD11b (clone M1/70, BioLegend), Alexa Fluor 700 anti-CD86 (clone GL1, BioLegend), PE-Cy7 anti-I-A/I-E (clone M5/114.15.2, BioLegend), APC anti-CD24 (clone 30-F1, BioLegend), APC-Cy7 anti-CD11c (clone N418, BioLegend), PE-Cy5 anti-CCR7 (clone 4B12, BioLegend), PerCP-Cy5.5 anti-CD169 (clone 3D6.112, BioLegend), and BUV737 anti-CD8α (clone 53-6.7, BD Biosciences).
For quantification of human CAR T cells in the peripheral blood, spleen, and tumor, tissues were processed as described above, then stained with LIVE/DEAD Aqua, BUV496 anti-CD4 (clone SK3/Leu3a, BD Biosciences), BUV805 anti-CD8α (clone SK1, BD Biosciences), PE anti-myc tag (clone 9B11, Cell Signaling), and APC-Cy7 anti-CD45 (clone 2D1, BioLegend).
ELISPOT: Spleens were processed as described above. CT-2A and B16F10 target cells were pretreated with 100 U/mL and 500 U/mL murine IFN-γ (Abcam), respectively for 16 hr, then irradiated at 120 Gy with a [137Cs] gamma radiation source. Splenocytes (several dilutions starting with 106 cells/well) were cocultured with 25,000 target cells/well for 16 hr and ELISPOT was performed using a Mouse IFN-γ ELISPOT Set (BD Biosciences) according to manufacturer's instructions.
Immunohistochemistry and immunofluorescence: Tissues (tumor, lungs, liver, and kidney) were fixed in 4% paraformaldehyde (PFA) in PBS for 24 hr at 4° C. For immunofluorescence studies, tumors were embedded in 3% low gelling temperature agarose (Sigma-Aldrich) before processing into 100 μm sections on a Leica VT1000S vibratome. Tissue sections were permeabilized with 2% BSA, 0.2% Triton-X 100 in PBS for 1 hr at 37° C. prior to staining with Alexa Fluor 647- or DyLight 405-conjugated anti-FITC (Jackson ImmunoResearch) for 16 hr at 37° C., followed by several PBS washes. Confocal microscopy was performed on an Olympus FV1200 Confocal Laser Scanning Microscope. For immunohistochemistry, PFA-fixed samples were paraffin-embedded and 10 μm sections were obtained. Sections were stained with anti-myc tag (9B11, Cell Signaling) or anti-CD45.1 (A20, BioLegend). For immunohistochemistry studies, tumors were frozen in O.C.T. and processed into 14 m thick sections on a Leica CM1950 cryostat. Tissue sections were permeabilized with 2% BSA, 0.2% Triton-X 100 in PBS for 2 hours at room temperature prior to staining with biotinylated anti-Trp1 [TA99] (kindly gifted), BV421-conjugated streptavidin (Biolegend), Alexa Fluor 647-conjugated anti-FITC (clone SPM395, Novus Biologicals), and CellMask™ Orange Plasma Membrane Stain (Invitrogen). Imaging was performed on a Leica SP8 Spectral Confocal Microscope.
Statistical analysis: Statistical analyses were performed using GraphPad Prism 9. All data was presented as mean±SD. To assess statistical significance, the following tests were used: between two groups, unpaired two-tailed Student's t test; for multiple comparisons, two-way ANOVA with Tukey's multiple comparisons; for repeated measures over a time course, two-way ANOVA with repeated measures; for Kaplan-Meier survival curves, log-rank (Mantel-Cox) test. All P values are provided in the figures or in their legends.
The present example demonstrates Amph-FITC can label diverse cancer cells for effective CAR T cell activation and target cell killing. Exemplary CAR T cell ligand, fluorescein isothiocyanate (FITC), was utilized. FITC is a safe, non-immunogenic compound that can redirect CAR T cells against tumors. It was hypothesized, without wishing to be bound by any one theory, that intratumoral administration of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-FITC (DSPE-PEG-FITC, or amph-FITC) would enable recognition and killing of tumor cells by FITC-specific CAR T cells (
Labeling of cancer cells in vitro was evaluated by incubating amph-FITC with B16F10 murine melanoma cells for 30 min at 37° C. Flow cytometry analysis of the melanoma cells revealed dose-dependent association of amph-FITC with the cells (
Next, CAR T cell recognition of B16 cells decorated with amph-FITC molecules was tested. FITC-specific CAR T cells were generated by transducing primary murine CD8+ T cells with a CAR comprised of the FITC-specific scFvs 4m5.3 (see, e.g., Boder, E. T. et al. Proceedings of the National Academy of Sciences of the United States of America 97, 10701-10705 (2000)) or E2 (see, e.g., Vaughn, T. J. et al. Nature Medicine 2, 534-539 (1996)) fused to a CD8 hinge, CD8 transmembrane domain, CD28 costimulatory domain, and CD3C signaling domain (abbreviated hereafter as the 4m5.3-28z and E2-28z CARs,
The present example demonstrates intratumoral (i.t.) administration of exemplary membrane-inserting amphiphilic ligand, amph-FITC, labels tumor cells. In vivo tumor labeling following intratumoral injection of amph-FITC was assessed using an amph-FITC dose of 10 nmol. Following a single injection into established B16F10 tumors, use of histology demonstrated that amph-FITC labeled large regions of the tumor (
Flow cytometry analysis demonstrated that FITC was taken up by a large proportion of tumor cells 24 hour after injection, and remained accessible at cell surfaces as detected by staining with an anti-FITC antibody (
Lymphodepletion (LD) can be used to promote CAR T cell engraftment and could have affected amph-FITC uptake. Thus, analysis of FITC cellular distribution was conducted in animals that received LD by 5 Gy total body irradiation (TBI) 24 hours prior to amph-FITC injection. LD pre-treatment significantly depleted immune cells and favored labeling of cancer cells in an even greater proportion (
The present example demonstrates that exemplary FITC-specific CAR T cells home to and expand in tumors. The ability of FITC-specific CAR T cells to recognize tumors injected with amph-FITC was characterized and CAR T cell expansion and tumor homing via bioluminescence imaging was evaluated. Mice bearing B16F10 tumors were lymphodepleted by total body irradiation (TBI), followed by adoptive transfer of firefly luciferase-expressing E2-28z CAR T cells. Beginning two days later, amph-FITC was administered intratumorally and every 3 days thereafter (e.g., to re-tag any tumor cells that had not yet been killed by CAR T cells) (
The present example demonstrates anti-tumor activity of exemplary amph-FITC/FITC-specific CAR T cell therapy. The therapeutic impact of FITC-targeting CAR T cell transfer combined with intratumoral amph-FITC and amph-FITC vaccination in the B16F10 and CT-2A tumor models was evaluated following the experimental timelines outline in
The CT-2A tumor model is a slowly progressing tumor more amenable to immunotherapy treatment. To determine factors governing the therapeutic efficacy of FITC CAR T treatment, the effect of the CD4/CD8 composition in the CAR T cell product was tested. Adoptive transfer of exclusively CD8+ E2-28z CAR T cells combined with intratumoral amph-FITC and vaccination halted progression of CT-2A tumors for 3 weeks (
The impact of CAR design parameters (e.g., CAR affinity and costimulatory domain selection) on the efficacy of amph-FITC-redirected CAR T treatment was evaluated. To modulate CAR affinity, the efficacy of CAR T cells bearing the E2 FITC-specific scFv (KD=2.4 nM) with CARs prepared from a very high affinity FITC scFv, 4m5.3 (KD=300 fM), or the lower affinity scFv E2.7 (KD=8.9 nM), which differs from E2 WT by a single amino acid substitution were compared. Each of these three scFv variant CARs was prepared with either a CD28 or 4-1BB costimulatory domain to further interrogate the role of costimulatory signaling. Murine CD8+ T cells showed efficient transduction and expression of each of these CARs, though the CD28-based CARs expressed at about 10-fold higher levels than the 4-1BB CARs (
To determine the dosing regimen of amph-FITC and preparation of maximally-effective CAR T cells in this treatment paradigm, the importance of dosing frequency was assessed. Interestingly, reducing injection frequency to once every 6 days did not reduce the therapeutic effect (
The present example demonstrates exemplary Amph-FITC and FITC CAR T therapy is well-tolerated with minimal toxicity. One concern with the chemical delivery of a CAR T ligand is the potential for dissemination of amph-FITC to trigger CAR T cell attack on healthy tissues. Studies herein demonstrated that local i.t. administration of amph-FITC minimally labeled normal (e.g., non-cancerous) tissues (
The present example demonstrates CAR T cell-mediated cytotoxicity stimulates an endogenous anti-tumor T cell response. TBI was utilized to promote CAR T cell engraftment at the start of treatment. Infiltration of host CD45.2+ CD4+ and CD8+ T cells into CT-2A tumors treated with intratumoral amph-FITC and CD45.1+ CAR T cells was evaluated. While untreated tumors showed very low levels of tumor-infiltrating lymphocytes (TILs), amph-FITC/CAR T treatment elicited robust infiltration by CD45.2+ host T cells (
An important step for priming of endogenous T cell responses is acquisition of tumor antigen by dendritic cells (DCs). To determine if amph-FITC/CAR T therapy promoted DC uptake of tumor cell debris, tdTomato+ CT-2A tumors were treated and found that immune cells, most notably dendritic cells in both the tumor and tumor-draining lymph node, became tdTomato+, suggesting uptake of tumor antigens (
The present example demonstrates that the endogenous T cell response initiated by local redirection of CAR T cells with amph-FITC injection leads to immune attack of distal untreated tumors. In order to assess whether this localized therapy could trigger systemic anti-tumor immune response, CT-2A tumors were implanted bilaterally in the flanks of mice, and only the right flank tumor was treated by administration of i.t. amph-FITC (in combination with amph-FITC vaccination,
The present example demonstrates Amph-FITC tagging enables human CAR T cells to target xenograft tumors. Studies were conducted in immunocompetent mouse models using murine CAR T cells to enable analysis of antigen spreading and endogenous T cell immunity. The ability of amph-FITC tagging to effectively redirect human CAR T cells against human cancer cells was evaluated using an engineered FITC-specific CARs that were highly expressed in human T cells (
In most syngeneic murine tumor treatment studies, intratumoral amph-FITC alongside subcutaneously administered amph-FITC (with adjuvant) was administered was efficiently taken up in draining lymph nodes as a vaccine booster. However, because lymphatic vessels and peripheral lymph nodes are defective in the immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wj1/SzJ (NSG) mice used for human CAR T cell evaluation (see, e.g., Puchalapalli, M. et al. PLoS ONE 11, 1-15 (2016)). FITC-specific CAR T cells were stimulated through intratumoral amph-FITC. To test the in vivo activity of CAR T cells responding to amph-FITC-coated tumors, MSTO-211H were inoculated tumors in NSG mice and adoptively transferred luciferase-expressing 4m5.3 or E2 CAR T cells with CD28 or 4-1BB costimulatory domains 7 days later, and then treated animals with repeated doses of intratumoral amph-FITC (
By treating tumors as shown in
This application claims priority to U.S. Provisional Application No. 63/343,804, filed May 19, 2022, the entire contents of which are incorporated herein by reference.
This invention was made with government support under CA247632 and GM007753 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63343804 | May 2022 | US |
