CHIMERIC ANTIGEN RECEPTORS (CARs) HAVING MUTATIONS IN THE FC SPACER REGION AND METHODS FOR THEIR USE
Adoptive immunotherapy using T cells genetically redirected via expression of chimeric antigen receptors (CARs) is a promising approach for cancer treatment. However, this immunotherapy is dependent in part on the optimal molecular design of the CAR, which involves an extracellular ligand-binding domain connected to an intracellular signaling domain by spacer and/or transmembrane sequences.
This application claims priority to U.S. Provisional Patent Application No. 61/926,881, filed Jan. 13, 2014, which is incorporated herein in its entirety, including the drawings.
STATEMENT OF GOVERNMENT INTERESTThe present invention was made with government support under Grant Nos P50 CA107399 and P01 CA030206 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention.
BACKGROUNDAdoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells is a promising cancer treatment, because these cells can directly recognize and kill antigen-expressing tumor cells in a human leukocyte antigen (HLA)-independent manner. However, besides a careful choice of the target tumor associated antigen, this therapeutic approach is highly dependent on the optimal molecular design of the CAR.
Although CARs that contain a TAA-specific scFv that produces an intracellular signal via a cytoplasmic costimulatory (e.g., CD28 or 4-1BB) domain fused to CD3-zeta have been shown in various systems to exhibit significant anti-tumor potency (Brentjens et al. 2013; Brentjens et al. 2011; Grupp et al. 2013; Kalos et al. 2011; Kochenderfer et al. 2012), immunological rejection and clearance by the host remains a challenge to effective cancer treatment.
Certain modifications in CAR design have been used to prevent the FcR-mediated clearance of therapeutic cells. For example, hinge/spacer sequences that do not originate from Ig Fc domains may be used, such as those from CD8α or CD28 (Brentjens et al. 2007; Kalos et al. 2011; Imai et al. 2004; Kochenderfer et al. 2009). Although these spacer sequences may alleviate FcR binding, their length may not endow CAR T cells with optimal potency when targeting certain antigens. For instance, when targeting 5T4, NCAM and MUC1 using CAR T cells, longer linker regions (i.e., longer than those derived from CD8α or CD28) were required for optimal potency (Wilkie et al. 2008; Guest et al. 2005). Thus, it would be desirable to design a CAR that addresses these challenges, while maintaining its efficacy in killing cancer cells.
SUMMARYAccording to some embodiments, recombinant chimeric antigen receptors (CAR) having impaired binding to an Fc receptor (FcR) are provided. Such CARs may include, but are not limited to, an antigen recognition domain, a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR, and an intracellular signaling domain.
In another embodiment, a population of human immune cells transduced by a viral vector comprising an expression cassette that includes a CAR gene is provided. In some aspects, the CAR gene comprises a nucleotide sequence that encodes an antigen recognition domain, a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR, and an intracellular signaling domain, wherein the population of human immune cells expresses the CAR gene.
In another embodiment, a method of treating a cancer in a subject is provided. Such a method includes administering a population of human immune cells transduced with a CAR gene to the subject. In some aspects, the CAR gene comprises a nucleotide sequence that encodes an antigen recognition domain that targets a cancer associated antigen specific to the cancer, a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR, and an intracellular signaling domain.
Designing a CAR having a spacer domain that has decreased or impaired binding to FcRs (such as those described herein) helps prevent the FcR-expressing cells from recognizing and destroying, or unintentionally activating, the CAR-expressing immunotherapeutic cells in vivo. Therefore, such CARs help prevent immunological rejection and clearance of the cells meant to provide therapeutic benefit to patients
The following examples are intended to illustrate various embodiments of the invention. As such, the specific embodiments discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.
Chimeric Antigen ReceptorsAccording to the embodiments described herein, recombinant chimeric antigen receptors (CARs) to target cancer-related antigens and methods for their use are provided. As described by the embodiments below, a CAR may include a series of protein or peptide domains including, but not limited to, one or more of an antigen binding domain, a spacer domain, a transmembrane domain, an intracellular signaling domain and an intracellular costimulatory domain.
In some embodiments, a gene encoding the CAR is provided, wherein the gene includes a nucleotide or nucleic acid sequence which includes a series of regions which encode an amino acid sequence corresponding to the protein or peptide domains of the CAR described herein. Because the degeneracy of the genetic code is known, any amino acid sequences disclosed herein are also indicative of all degenerate nucleic acid codons corresponding to each amino acid in said sequences. As such, it is understood that the embodiments describing CARs and their domains may be provided as a gene comprising a nucleic acid sequence as well as the amino acid sequences encoded by said genes.
In one embodiment, a CAR may include, but is not limited to, an antigen binding domain, a spacer domain, optionally at least one intracellular signaling domain and optionally at least one intracellular costimulatory domain.
In other embodiments, a CAR may include, but is not limited to, an antigen binding domain, a spacer domain, and at least one intracellular signaling domain.
In other embodiments, a CAR may include, but is not limited to, an antigen binding domain, a spacer domain, at least one intracellular signaling domain and at least one intracellular costimulatory domain.
Antigen Binding Domain
A CAR antigen binding domain may include a nucleotide sequence that, when expressed as a peptide or polypeptide, binds an epitope of a cancer-related antigen. In some embodiments, a cancer-related antigen may be any antigen expressed by or overexpressed by a cancer cell (e.g., a tumor cell, a neoplastic cell, a malignant cell, or any other cancerous cell), and may be a protein, peptide, carbohydrate, glycoprotein, ganglioside, proteoglycan, or any combination or complex thereof. In some aspects, the cancer-related antigen is a tumor specific antigen (TSA) that may be expressed only on cancer or tumor cells, while in other aspects, the cancer-related antigen is a tumor-associated antigen (TAA) that may be expressed on both tumor cells and normal cells. In other aspects, the cancer-related antigen may be a product of a mutated oncogene or tumor suppressor gene, or a product of another mutated gene (e.g., overexpressed or aberrantly expressed cellular proteins, tumor antigens produced by oncogenic viruses, oncofetal antigens, altered cell surface glycolipids or glycoproteins, or cell type-specific differentiation antigens).
According to the embodiments described herein, cancer-related antigens that may be targeted by a CAR antigen binding domain described herein include, but are not limited to, 5T4, 8H9, αvβ6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcionoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-α, G250/CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor α2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated antigen (MAGE-A1), mesothelin, Murine CMV infected cells, mucin-1 (MUC1). mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors. In some embodiments, the antigen binding domain that is part of a CAR described herein targets CD19 or CD123.
An antigen binding domain may be any targeting moiety which targets an antigen associated with cancer. In some embodiments, the antigen binding domain is an antibody or functional fragment of an antibody. An antibody refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with an antigen or epitope, and includes both polyclonal and monoclonal antibodies, as well as functional antibody fragments, including but not limited to fragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain variable fragments (scFv) and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term “antibody or functional fragment thereof” also includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv). Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. In one embodiment, the antigen binding domain is an scFv having a heavy (VH) and light chain (VL). In other embodiments, the antigen binding domain is an scFv that targets CD19 or CD123. In such embodiments, the scFv that targets CD19 may have the following amino acid sequence:
And the scFv that targets CD123 may have one of the following amino acid sequences:
Spacer Domain
The spacer domain (also referred to as a “hinge region” or “spacer/hinge region”) may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain embodiments, the spacer domain includes at least a portion of an IgG1, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains. In some embodiments, the spacer domain may also include at least a portion of a corresponding immunoglobulin hinge region. In some embodiments, the spacer domain is derived from or includes at least a portion of a modified immunoglobulin Fc region, for example, a modified IgG1 Fc region, a modified IgG2 Fc region, a modified IgG3 Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region. The modified immunoglobulin Fc region may have one or more mutations (e.g., point mutations, insertions, deletions, duplications) resulting in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the spacer domain to an Fc receptor (FcR). In some aspects, the modified immunoglobulin Fc region may be designed with one or more mutations which result in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the spacer domain to one or more FcR including, but not limited to, FcγRI, FcγR2A, FcγR2B1, FcγR2B2, FcγR3A, FcγR3B, FcεRI, FcεR2, FcαRI, Fcα/μR, or FcRn.
Some amino acid sequences within the Fc CH2 domain have been identified as having involvement in antibody-FcR interaction (Strohl, 2009). FcRs, such as FcγRI, are integral membrane proteins located on immune cells including natural killer (NK) cells and macrophages, which then use this Fc-targeting ability to carry out various immune functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis.
Impairment of binding to FcRs by the spacer domain prevents the FcR-expressing cells from recognizing and destroying, or unintentionally activating, the CAR-expressing immunotherapeutic cells in vivo, thereby helping to prevent immunological rejection and clearance of the cells meant to provide therapeutic benefit to patients. The mutations described herein also contribute to reducing the CAR's off-target effects and, thereby increasing its specificity and efficacy.
An “amino acid modification” or an “amino acid substitution” or a “substitution,” as used herein, mean an amino acid substitution, insertion, and/or deletion in a protein or peptide sequence. An “amino acid substitution” or “substitution” as used herein, means a replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. For example, the substitution S228P refers to a variant protein or peptide, in which the serine at position 228 is replaced with proline.
Amino acid substitutions can be made by a mutation such that a particular codon in the nucleic acid sequence encoding the protein or peptide is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein.
The following are examples of various groupings of amino acids:
Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine.
Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine.
Amino acids with charged polar R groups (negatively charged at Ph 6.0): Aspartic acid, Glutamic acid.
Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0).
Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, Tyrosine.
Another grouping may be according to molecular weight (i.e., size of R groups) as shown below:
In certain embodiments, the spacer domain is derived from a modified IgG1, IgG2, IgG3, or IgG4 Fc region that includes one or more amino acid residues substituted with an amino acid residue different from that present in an unmodified hinge. The one or more substituted amino acid residues are selected from, but not limited to one or more amino acid residues at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336, 339, or a combination thereof.
In some embodiments, the spacer domain is derived from a modified IgG1, IgG2, IgG3, or IgG4 Fc region that includes, but is not limited to, one or more of the following amino acid residue substitutions: C220S, C226S, S228P, C229S, P230S, E233P, V234A, L234V, L234F, L234A, L235A, L235E, G236A, G237A, P238S, S239D, F243L, P2471, S267E, H268Q, S280H, K290S, K290E, K290N, R292P, N297A, N297Q, S298A, S298G, S298D, S298V, T299A, Y300L, V305I , V309L, E318A, K326A, K326W, K326E, L328F, A330L, A330S, A331S, P331S, 1332E, E333A, E333S, E333S, K334A, A339D, A339Q, P396L, or a combination thereof.
In some embodiments, the spacer domain is derived from an IgG Fc region having one or more modifications made to its CH2-CH3 region, wherein the unmodified IgG CH2-CH3 region corresponds to one of the following amino acid sequences:
In some embodiments, the spacer domain is derived from an IgG Fc region having one or more modifications made to its hinge region, wherein the unmodified IgG hinge region corresponds to one of the following amino acid sequences:
In some embodiments, the spacer domain is derived from an IgG4 Fc region having the following amino acid sequence:
In certain embodiments, the spacer domain is derived from a modified IgG4 Fc that includes one or more amino acid residues substituted with an amino acid residue different from that present in an unmodified IgG4 Fc region. The one or more substituted amino acid residues are selected from, but not limited to one or more amino acid residues at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336, 339, 396, or a combination thereof.
In some embodiments, the spacer domain is derived from a modified IgG4 Fc region that includes, but is not limited to, one or more of the following amino acid residue substitutions: 220S, 226S, 228P, 229S, 230S, 233P, 234A, 234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 2471, 267E, 268Q, 280H, 290S, 290E, 290N, 292P, 297A, 297Q, 298A, 298G, 298D, 298V, 299A, 300L, 305I, 309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S, 331S, 332E, 333A, 333S, 333S, 334A, 339D, 339Q, 396L, or a combination thereof, wherein the amino acid in the unmodified IgG4 Fc region is substituted with the above identified amino acids at the indicated position.
In some embodiments, the spacer domain is derived from a modified IgG4 Fc region that includes, but is not limited to, two or more (i.e., “double mutated”), three or more (i.e., “triple mutated”), four or more, five or more, or more than five of the following amino acid residue substitutions: 220S, 226S, 228P, 229S, 230S, 233P, 234A, 234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 2471, 267E, 268Q, 280H, 290S, 290E, 290N, 292P, 297A, 297Q, 298A, 298G, 298D, 298V, 299A, 300L, 305I, 309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S, 331S, 332E, 333A, 333S, 333S, 334A, 339D, 339Q, 396L, or a combination thereof, wherein the amino acid in the unmodified IgG4 Fc region is substituted with the above identified amino acids at the indicated position.
In some embodiments, the spacer domain is derived from a modified IgG4 Fc region that includes, but is not limited to, a substitution of proline (P) in place of serine (S) at position 228 (S228P), a substitution of leucine (L) in place of glutamic acid (E) at position 235 (L235E), a substitution of asparagine (N) in place of glutamine (Q) at position 297 (N297Q), or a combination thereof. In certain embodiments, a modified IgG4 Fc region has a single mutation, as indicated in the following amino acid sequences (mutations are in bold and underlined):
In other embodiments, the spacer domain is derived from a modified IgG4 Fc region that is double mutated to include an L235E substitution and an N297Q substitution (“EQ”). In another embodiment, the modified IgG4 Fc region is triple mutated to include an S228P substitution, an L235E substitution, and an N297Q substitution (“S228P+L235E+N297Q”). In certain embodiments, a modified IgG4 Fc and/or hinge region may include a nucleotide sequence which encodes an amino acid sequence selected from the following (mutations are in bold and underlined):
In certain embodiments, the spacer domain is derived from a modified immunoglobulin Fc region that includes one or more deletions of all of a part of its CH2 domain. In one embodiment, the spacer domain is derived from a modified IgG4 Fc region that includes one or more deletions of all of a part of its CH2 domain (“ch2Δ”). In one aspect of such an embodiment, the spacer domain may include a nucleotide sequence which encodes the following amino acid sequence:
In some embodiments, the spacer domain may be modified to substitute the immunoglobulin Fc region for a spacer that does not have the ability to bind FcR, such as the hinge region of CD8a. Alternatively, the Fc spacer region of the hinge may be deleted. Such substitutions would reduce or eliminate Fc binding.
The term “position,” as used herein, is a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example a Kabat position or an EU position or EU index as in Kabat. For all positions discussed herein, numbering is according to the EU index or EU numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, hereby entirely incorporated by reference). The EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, which is hereby entirely incorporated by reference). Kabat positions, while also well known in the art, may vary from the EU position for a given position. For example, the S228P and L235E substitutions described above refer to the EU position. However, these substitutions may also correspond to Kabat positions 241 (5241 P) and 248 (L248E).
Transmembrane and Signaling DomainsThe intracellular signaling domain may include any suitable T cell receptor (TCR) complex signaling domain, or portion thereof. In some embodiments, the intracellular signaling domain is derived from a CD3 complex. In some embodiments, the intracellular signaling domain is a TCR zeta-chain (ζ-chain) signaling domain. In certain embodiments, a ζ-chain signaling domain may include a nucleotide sequence which encodes an amino acid sequence as follows:
The intracellular signaling domain may be associated with any suitable costimulatory domain including, but not limited to, a 4-1BB costimulatory domain, an OX-40 costimulatory domain, a CD27 costimulatory domain, a CD28 costimulatory domain, a DAP10 costimulatory domain, an inducible costiumulatory (ICOS) domain, or a 2B4 costimulatory domain. According to the embodiments described herein, a CAR may include at least one costimulatory signaling domain. In one aspect the CAR has a single costimulatory signaling domain, or it may include two or more costimulatory signaling domains such as those described above. In another aspect, the costimulatory domain may be made up of a single costimulatory domain such as those described above, or alternatively, may be made up of two or more portions of two or more costimulatory domains. Alternatively, in some embodiments, the CAR does not include a costimulatory signaling domain. In one embodiment, the CAR includes a costimulatory signaling domain which is a CD28 costimulatory domain. In this embodiment, such a modified CD28 costimulatory domain may have one or more amino acid substitutions or modifications including, but not limited to a substitution of leucine-leucine (LL) to glycine-glycine (GG). In certain embodiments, a modified costimulatory signaling domain region may include a nucleotide sequence which encodes an amino acid sequence selected from the following:
The signaling domain or domains may include a transmembrane domain selected from a CD28 transmembrane domain, a CD3 transmembrane domain, or any other suitable transmembrane domain known in the art. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In certain embodiments, a modified costimulatory signaling domain region may include a nucleotide sequence which encodes an amino acid sequence selected from the following:
In some embodiments, the CAR gene is part of an expression cassette. In some embodiments, the expression cassette may—in addition to the CAR gene—also include an accessory gene. When expressed by a T cell, the accessory gene may serve as a transduced T cell selection marker, an in vivo tracking marker, or a suicide gene for transduced T cells.
In some embodiments, the accessory gene is a truncated EGFR gene (EGFRt). An EGFRt may be used as a non-immunogenic selection tool (e.g., immunomagnetic selection using biotinylated cetuximab in combination with anti-biotin microbeads for enrichment of T cells that have been lentivirally transduced with EGFRt-containing constructs), tracking marker (e.g., flow cytometric analysis for tracking T cell engraftment), and suicide gene (e.g., via Cetuximab/Erbitux® mediated antibody dependent cellular cytotoxicity (ADCC) pathways). An example of a truncated EGFR (EGFRt) gene that may be used in accordance with the embodiments described herein is described in International Application No. PCT/US2010/055329, the subject matter of which is hereby incorporated by reference as if fully set forth herein. In other embodiments, the accessory gene is a truncated CD19 gene (CD19t).
In another embodiment, the accessory gene is an inducible suicide gene. A suicide gene is a recombinant gene that will cause the cell that the gene is expressed in to undergo programmed cell death or antibody mediated clearance at a desired time. In one embodiment, an inducible suicide gene that may be used as an accessory gene is an inducible caspase 9 gene (see Straathof et al. (2005). An inducible caspase 9 safety switch for T-cell therapy. Blood. June 1; 105(11): 4247-4254, the subject matter of which is hereby incorporated by reference as if fully set forth herein).
In some embodiments, the expression cassette that include a CAR gene described above may be inserted into a vector for delivery—via transduction or transfection—of a target cell. Any suitable vector may be used, for example, a bacterial vector, a viral vector, or a plasmid. In some embodiments, the vector is a viral vector selected from a retroviral vector, a lentiviral vector, a poxvirus vector, an adenoviral vector, or an adeno-associated viral vector. In some embodiments, the vector may transduce a population of healthy immune cells, e.g., T cells. Successfully transduced or transfected target cells express the one or more genes that are part of the expression cassette.
As such, one or more populations of immune cells, such as T cells, may be transduced with a CAR gene such as those described above. The transduced T cells may be from a donor, or may be from a subject having a cancer and who is in need of a treatment for the cancer. In some embodiments, the transduced T cells are used in an adoptive immunotherapy treatment for the treatment of the cancer (residues in bold/underline indicate substitutions). In some embodiments, the transduced T cells express a CAR gene that encodes an amino acid sequence selected from SEQ ID NOS:24-27:
Further, the one or more populations of T cells may be part of a pharmaceutically acceptable composition for delivery for administration to a subject. In addition to the CAR-transduced T cells, the pharmaceutically effective composition may include one or more pharmaceutically effective carriers. A “pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a treatment of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. Such a carrier may comprise, for example, a liquid, solid, or semi-solid filler, solvent, surfactant, diluent, excipient, adjuvant, binder, buffer, dissolution aid, solvent, encapsulating material, sequestering agent, dispersing agent, preservative, lubricant, disintegrant, thickener, emulsifier, antimicrobial agent, antioxidant, stabilizing agent, coloring agent, or some combination thereof.
Each component of the carrier is “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the composition and must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) natural polymers such as gelatin, collagen, fibrin, fibrinogen, laminin, decorin, hyaluronan, alginate and chitosan; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as trimethylene carbonate, ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid (or alginate); (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) alcohol, such as ethyl alcohol and propane alcohol; (20) phosphate buffer solutions; (21) thermoplastics, such as polylactic acid, polyglycolic acid, (22) polyesters, such as polycaprolactone; (23) self-assembling peptides; and (24) other non-toxic compatible substances employed in pharmaceutical formulations such as acetone.
The pharmaceutical compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
In one embodiment, the pharmaceutically acceptable carrier is an aqueous carrier, e.g. buffered saline and the like. In certain embodiments, the pharmaceutically acceptable carrier is a polar solvent, e.g. acetone and alcohol.
The concentration of CAR-transduced T cells in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, organ size, body weight and the like in accordance with the particular mode of administration selected and the biological system's needs.
In certain embodiments, populations of T cells transduced with a CAR gene (i.e., CAR-transduced T cells) such as those described herein cells used in the methods for targeting and killing cancer or tumor cells may be grown in a cell culture. In certain aspects of this embodiment, the method may be used in an in vitro or research setting to investigate the role of a particular cancer-related antigen in the etiology of a cancer, or to evaluate the targeting abilities of new CAR constructs.
Treatment of Cancer with CAR-Transduced T Cells
According to some embodiments, CAR genes and populations of T cells that are transduced with CAR genes such as those described above may be used in methods for treating cancer in a subject. Such methods may include a step of administering a therapeutically effective amount of at least one population of T cells transduced with at least one CAR gene to the subject. In these embodiments, the population of CAR-transduced T-cells expresses one or more CAR genes, such as those described above. In certain embodiments, the T cells are transduced with and express a single mutant gene construct such as a CD19R(L235E) or CD19R(N297Q) construct as described herein, a double mutant gene construct which has both a L235E and N297Q mutation (e.g., CD19R(EQ)), as described herein, or a deletion gene construct (e.g., CD19Rch2Δ), as described herein. When such cells are administered via an adoptive immunotherapy treatment, the transduced T cells specifically target and lyse the cancer-related antigen expressing cells (i.e., cancer cells) in vivo, thereby delivering their therapeutic effect of eliminating cancer cells.
Cancers that may be treated using the population of transduced T cells may include, but are not limited to, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical, Carcinoma, AIDS-Related Cancers, Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Central Nervous System, Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma, Brain Stem Glioma, Brain Tumors, Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumors, Central Nervous System Cancers, Cervical Cancer, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Embryonal Tumors, Central Nervous System, Endometrial Cancer, Ependymoblastoma, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma Family of Tumors Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor Extrahepatic Bile Duct Cancer, Eye Cancer Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST)—see Soft Tissue Sarcoma, Germ Cell Tumor, Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis, Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors (Endocrine Pancreas), Kaposi Sarcoma, Kidney cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer, Lymphoma, Macroglobulinemia, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Medulloblastoma, Medulloepithelioma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia, Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma, Multiple, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer, Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Pancreatic Cancer, Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumors of Intermediate Differentiation, Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumors, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Sézary Syndrome, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer, Stomach (Gastric) Cancer, Supratentorial Primitive Neuroectodermal Tumors, T-Cell Lymphoma, Cutaneous, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Trophoblastic Tumor, Ureter and Renal Pelvis Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenström Macroglobulinemia, and Wilms Tumor.
The population or populations of T cells transduced with the CAR gene or genes that may be used in accordance with the methods described herein may be administered, by any suitable route of administration, alone or as part of a pharmaceutical composition. A route of administration may refer to any administration pathway known in the art, including but not limited to intracranial, parenteral, or transdermal. “Parenteral” refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intratumoral, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. In certain embodiments, transduced T cells are administered intravenously or intrathecally.
The term “effective amount” as used herein refers to an amount of an agent, compound, treatment or therapy that produces a desired effect. For example, a population of cells may be contacted with an effective amount of an agent, compound, treatment or therapy to study its effect in vitro (e.g., cell culture) or to produce a desired therapeutic effect ex vivo or in vitro. An effective amount of an agent, compound, treatment or therapy may be used to produce a therapeutic effect in a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, or producing a desired physiological effect. In such a case, the effective amount of a compound is a “therapeutically effective amount,” “therapeutically effective concentration” or “therapeutically effective dose.” The precise effective amount or therapeutically effective amount is an amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject or population of cells. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. Further an effective or therapeutically effective amount may vary depending on whether the compound is administered alone or in combination with another compound, drug, therapy or other therapeutic method or modality. One skilled in the clinical and pharmacological arts will be able to determine an effective amount or therapeutically effective amount through routine experimentation, namely by monitoring a cell's or subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005, which is hereby incorporated by reference as if fully set forth herein. Agents, compounds treatments or therapies that may be used in an effective amount or therapeutically effective amount to produce a desired effect in accordance with the embodiments described herein may include, but are not limited to, a CAR gene, an expression cassette that includes a CAR gene, a vector that delivers an expression cassette that includes a CAR gene to a target cell such as a T cell, and a population of T cells that are transduced with a CAR gene.
The terms “treating” or “treatment” of a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof. Treatment may also mean a prophylactic or preventative treatment of a condition.
The term “subject” as used herein refers to a human or animal, including all mammals such as primates (particularly higher primates), sheep, dog, rodents (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbit, and cow. In some embodiments, the subject is a human.
In certain embodiments, the methods for treating cancer may include a step of administering a therapeutically effective amount of a first population of T cells transduced with a first CAR gene in combination with a therapeutically effective amount of a second population of T cells transduced with a second CAR gene.
In other embodiments, CAR-transduced T cells may be administered in combination with one or more additional anti-cancer therapies. “In combination” or “in combination with,” as used herein, means in the course of treating the same cancer in the same subject using two or more agents, drugs, therapeutics, procedures, treatment regimens, treatment modalities or a combination thereof, in any order. This includes simultaneous administration, as well as in a temporally spaced order of up to several days apart. Such combination treatment may also include more than a single administration of any one or more of the agents, drugs, therapeutics, procedures, treatment regimens, and treatment modalities. Further, the administration of the two or more agents, drugs, therapeutics, procedures, treatment regimens, treatment modalities or a combination thereof may be by the same or different routes of administration.
Additional anti-cancer therapies that may be used in accordance with the methods described herein may include one or more anti-cancer procedures, treatment modalities, anti-cancer therapeutics or a combination thereof. In some embodiments, the CAR-transduced T cells may be administered in combination with one or more anti-cancer procedures or treatment modalities including, but not limited to, stem cell transplantation (e.g., bone marrow transplant or peripheral blood stem cell transplant using allogenic stem cells, autologous stem cells; or a non-myeloablative transplant), radiation therapy, or surgical resection. In other embodiments, the CAR-transduced T cells may be administered in combination with one or more anti-cancer therapeutics or drugs that may be used to treat cancer including, but not limited to, chemotherapeutics and other anti-cancer drugs, immunotherapeutics, targeted therapeutics, or a combination thereof.
Chemotherapeutics and other anti-cancer drugs that may be administered in combination with the CAR-transduced T cells in accordance with the embodiments described herein include, but are not limited to, all-trans-retinoic acid (ATRA), arsenic trioxide, anthracycline antibiotics and pharmaceutically acceptable salts thereof (e.g., doxorubicin hydrochloride, daunorubicin hydrochloride, idarubicin, mitoxantrone), alkylating agents (e.g., cyclophosphamide, laromustine), antimetabolite analogs (cytarabine, 6-thioguanine, 6-mercaptopurine, methotrexate), demethylating agents (e.g., decitabine, 5-azacytidine), nucleic acid synthesis inhibitors (e.g., hydroxyurea), topoisomerase inhibitors (e.g., etoposide), vinca alkaloids (e.g., vincristine sulfate), or a combination thereof (e.g., “ADE,” which is a combination treatment that includes a combination of Cytarabine (Ara-C), Daunorubicin Hydrochloride and Etoposide).
Immunotherapeutics that may be administered in combination with the CAR-transduced T cells in accordance with the embodiments described herein include, but are not limited to, immune modulatory reagents (e.g., STAT3 inhibitors, Lenalidomide) and therapeutic monoclonal antibodies. The therapeutic monoclonal antibodies may be designed to target one or more additional cancer-related antigens
Targeted therapeutics that may be administered in combination with the CAR-transduced T cells in accordance with the embodiments described herein include, but are not limited to, tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, sunitinib), farnesyl transferase inhibitors (e.g., tipifarnib), FLT inhibitors, and c-Kit (or CD117) inhibitors (imatinib, dasatinib, nilotinib).
The following examples are intended to illustrate various embodiments of the invention. As such, the specific embodiments discussed are not to be construed as limitations on the scope of the invention. For example, although the example below relates to an embodiment for a CAR that targets CD19, it is appreciated that a CAR may be generated to target any antigen. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.
EXAMPLES Example 1 Chimeric Antigen Receptors (CARs) Incorporating Mutations in the IgG4 Fc Spacer Region Avoid Fc Receptor Mediated Recognition and Clearance of CAR T Cells, Resulting in Improved T Cell Persistence and Anti-Tumor EfficacyTo determine whether cellular FcR-mediated interactions play a role in immunological rejection and clearance, or even unintentional activation of adoptively transferred CAR-expressing T cells, a CD19-specific CAR that has been mutated at one or two sites within the CH2 region (L235E and/or N297Q) of its IgG4 Fc spacer—referred to herein as CD19R(L235E), CD19R(N297Q) or CD19R(EQ)—as well as a CD19-specific CAR that has a CH2 deletion in its IgG4 Fc spacer—referred to herein as CD19Rch2Δ. T cells expressing these mutated CAR were then compared to T cells expressing a non-mutated CAR (CD19R) or only a truncated EGFR molecule (EGFRt) as a tracking marker (Wang et al. 2011), for in vitro FcγR binding and CAR-mediated cytolytic activity, as well as in vivo engraftment and therapeutic efficacy. The results provide evidence that elimination of cellular FcγR interactions improves the persistence and anti-tumor responses of adoptively transferred CAR-expressing T cells.
Materials and Methods
DNA Constructs and Lentiviral Vectors. The CD19R28Z-T2A-EGFRt_epHIV7 lentiviral construct contains a) the chimeric antigen receptor (CAR) sequence consisting of the VH and VL gene segments of the CD19-specific FMC63 mAb, an IgG4 hinge-CH2—CH3, the transmembrane and cytoplasmic signaling domains of the costimulatory molecule CD28 that contains gg mutations that enhance chimeric receptor expression and function (Nguyen et al. 2003), and the cytoplasmic domain of the CD3λ chain (Kowolik et al. 2006); b) ribosomal skip T2A sequence (Szymczak et al. 2004) and c) the truncated EGFR sequence (Wang et al. 2011a). The EGFRt-T2A-DHFRFS-T2A-IMPDH2IY_epHIV7 lentiviral vector was generated as previously described (Jonnalagadda et al. 2013). The CD19R(L235E)28Z-T2A-EGFRt_epHIV7, CD19R(N297Q)28Z-T2A-EGFRt_epHIV7 and CD19R(EQ)28Z-T2A-EGFRt_epHIV7 vectors were generated by site directed mutagenesis using the QuikChange II XL kit (Agilent Technologies, Santa Clara, Calif.) of a codon optimized CD19R28Z_pGA plasmid that had been synthesized by Geneart, digested with NheI/RsrII and ligated with a similarly digested CD19R28Z-T2A-EGFRt_epHIV7. The CD19Rch2Δ28Z-T2A-EGFRt_epHIV7 vector was generated from a codon optimized CD19R-HL-CH3(CO)_pMK-RQ plasmid that had been synthesized by Geneart, digested with NheI/RsrII and ligated with a similarly digested CD19R28Z-T2A-EGFRt_epHIV7.
Cell Lines and Maintenance.
Human peripheral blood mononuclear cells (PBMCs) were isolated as described (Wang, 2011b) from heparinized peripheral blood obtained from discard kits containing residual blood components of healthy donors that had undergone apheresis at the City of Hope National Medical Center (COHNMC). Because this was de-identified discard blood material, informed consent was waived with the approval of the COHNMC Internal Review Board (IRB protocol #09025), and the COHNMC Office of Human Subjects Protection. TCM isolation (using CD14- and CD45RA-depletion followed by CD62L-selection), anti-CD3/CD28 bead stimulation and lentiviral-mediated transduction was then done as previously described (Wang et al. 2012). In some cases, transduced T cells were immunomagnetically enriched for EGFRt expression as previously described (Wang et al. 2011a).
EBV-transformed lymphoblastoid cell lines (LCL) and LCL that expressed OKT3 (LCL-OKT3) (Wang et al. 2011 b) or ffLuc+ LCL cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana, Calif.) supplemented with 10% heat-inactivated fetal calf serum (FCS, Hyclone, Logan, Utah) 2 mM L-glutamine (Irvine Scientific), and 25 mM HEPES (Irvine Scientific). ffLuc+ LCL were generated by transduction with lentiviral vector eGFP-ffluc_epHIV7 at an MOI of 20 in the presence of 5 μg/mL polybrene in 500 uL medium, and subsequent purification by sorting GFP+ cells.
Mouse myeloma cells secreting human homeostatic IL-15 cytokine (NSO-IL15) were generated as previously described (Wang et al. 2011b).
SupB15 and K562 leukemia cell lines (ATCC) were grown in the corresponding ATCC recommended media.
Antibodies and Flow Cytometry.
Fluorochrome-conjugated isotype controls, anti-CD3, anti-CD4, anti-CD8, anti-CD45 and streptavidin were obtained from BD Biosciences (San Jose, Calif.). Biotinylated anti-Fc was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa.). Generation of biotinylated-cetuximab was previously described (Wang et al. 2011a). Biotinylated huFcγR1, muFcγR1, huFcγR2a, huFcγR2b, and muFcγR2b were obtained from Sino Biological, Inc. (Beijing, P.R. China). The percentage of immunofluorescent cells were analyzed by a FACScalibur system (BD Biosciences), and the percentage of cells in a region of analysis were calculated using FCS Express V3 (De Novo Software, CA, USA).
In Vivo T Cell Engraftment and Therapy.
All mouse experiments were approved by the COHNMC Institute Animal Care and Use Committee. For engraftment studies, 6-10 week old NOD/Scid IL-2RγCnull (NSG) mice were injected intravenously (i.v.) on day 0 with 107 of the indicated TCM-derived cells, and intraperitoneal (i.p.) injections three times a week of 2×107 irradiated NS0-IL15 to provide a systemic supply of human IL-15 in vivo. Peripheral blood was harvested from retro-orbital bleeds, red blood cells were lysed and cell suspensions were analyzed by flow cytometry. For the therapeutic study, 1.5×106 ffLuc+ LCL cells were administered i.v. into 6-8 week old NSG mice on day 0, and then 5×106 of the indicated CAR+ TCM-derived cells were administered i.v. on day 3. Luciferase activity was measured by Xenogen imaging as previously described (Kahlon et al. 2004).
Chromium-Release Assays.
4-hour 51Cr-release assays were performed as previously described (Stastny et al. 2007) using the indicated effector/target cell ratios.
Results
CD19R+ T Cells Fail to Engraft in NSG Mice.
Central memory T cells (TCM) as a T cell subpopulation, have been characterized as having superior engraftment potential, and thus therapeutic efficacy, after adoptive transfer (Wang et al. 2011 b). Further evidence has shown that CAR expression on the TCM-derived cells seem to correlate with decreased in vivo persistence in an in vivo xenograft model using NSG mice. As the studies described herein indicate, this decrease in persistence was shown in an experiment comparing the engraftment of non-transduced TCM-derived cells to (i) TCM-derived cells that were lentivirally transduced to express both a CD19-specific CAR (CD19R) and a truncated EGFR (EGFRt) as a tracking marker, and (ii) TCM-derived cells that were lentivirally transduced to express just the EGFRt tracking marker on the cell surface (
HuFcγR Binds CD19R+ T Cells.
The CD19R construct includes a CD19-specific scFv derived from mouse monoclonal antibody FMC63, a human IgG4 Fc linker, human CD28 transmembrane and cytoplasmic domains, and a human CD3-zeta cytoplasmic domain. Because the CAR construct includes a portion of a human IgG4 Fc region, the propensity of FcR-mediated innate immune responses to selectively clear the CD19R/EGFRt+ cells—but not the EGFRt+ cells—was investigated. Indeed, a binding assay using soluble human FcγR1 revealed that, in contrast to TCM-derived cells that were non-transduced or expressed only the EGFRt, those that expressed CD19R exhibited binding of the FcγR1 molecules that could be titrated down with higher dilutions (
Generation of CD19R Mutants.
To further test the significance of potential FcR-mediated effects on the CAR-expressing TCM population, the CD19-specific CAR was mutated at amino acids within the IgG4 CH2 domain that may be involved with FcR binding—L235E and/or N297Q (
huFcγR Binding to CARs with Mutated IgG4 Spacer is Impaired.
To determine the efficacy of the different mutations/deletion in the CAR to affect FcR binding, flow cytometric analysis was performed using various human and murine biotinylated soluble FcγRs, and PE-streptavidin (SA-PE) to detect the binding of the FcγRs to the different cell populations. T cells that expressed the non-mutated CD19R were bound by human FcγR1, FcγR2a and FcγR2b, as well as murine FcγR1 and FcγR2b (
T Cells with CD19R Mutants Exhibit Improved In Vivo Engraftment and Persistence.
To determine whether the CD19R mutations or deletion which helped prevent FcγR binding would translate to an increased in vivo persistence upon adoptive transfer, 107 T cells expressing either the parental CD19R, the EGFRt marker alone, the CD19R(L235E), the CD19R(N297Q), the CD19R(EQ), or the CD19Rch2Δ were infused i.v. into NSG mice. One and two weeks later, peripheral blood was assayed for CD45+ EGFRt+ cell engraftment (
T Cells with CD19R Mutants Exhibit Improved Therapeutic Efficacy.
Based on the engraftment findings, the effects of the CD19R(EQ) or CD19Rch2Δ on the anti-tumor efficacy of the TCM-derived cells were compared. LCL is a CD19-expressing tumor cell line that was transduced to express firefly luciferase (ffLuc) to allow for bioluminescent monitoring of in vivo tumor growth. Three days after the ffLuc+ LCL were administered to NSG mice i.v., the mice were treated i.v. with either PBS as a control or 5×106 T cells expressing either the non-mutated CD19R, the EGFRt marker alone, the double point-mutated CD19R(EQ), or the CH2-deleted CD19Rch2Δ. Expression of either the CD19R(EQ) or the CD19Rch2Δ on the TCM-derived cells resulted in significant control of tumor growth (
Clinically, the in vivo therapeutic efficacy of adoptive T cell strategies directly correlates with engraftment and persistence upon adoptive transfer (Heslop et al. 2003; Brenner & Heslop 2010). Various approaches have been suggested to improve transferred T cell persistence, including lymphodepletion of the host prior to cell transfer (Gattinoni et al. 2005), cytokine support after cell transfer (most recently reviewed in (Overwijk & Schluns 2009), and use of the optimal T cell population(s) for transfer (Berger et al. 2008; Hinrichs et al. 2011; Yang et al. 2013; Gattinoni et al. 2011; Cieri et al. 2013). The studies described above provide further evidence that chimeric antigen receptor (CAR) design plays a significant role in directing the engraftment and persistence of therapeutic cells. Previously, CAR design has been exploited to benefit engraftment and persistence of therapeutic cells is by including costimulatory signaling domains in second and third generation CARs (see Cartellieri et al. 2010). However, as the data above also suggests, sequences that are used to connect the ligand-binding domain to the signaling domain(s) of the CAR (known as either the spacer, hinge and/or linker) are of previously unappreciated importance for in vivo therapeutic outcome in murine models of malignant disease. Specifically, it was found that the use of an Ig Fc spacer can potentially inhibit the engraftment and/or persistence of CAR-expressing cells in NSG mouse models in a manner that correlates with FcγR binding. Prevention of FcγR binding by either point mutation or deletion of the relevant sequences within the CAR Fc domain can then restore the in vivo persistence of the adoptively transferred cells to that of cells which do not express a CAR. The increased in vivo persistence that is mediated by the spacer-optimized CAR then translates, into significantly improved CAR-directed anti-tumor therapy in an in vivo mouse model.
The immunological clearance of adoptively transferred T cells is not a new issue. For example, cellular immune rejection responses against the HyTK and NeoR selection genes have been shown to be coordinately expressed with the CAR (Berger et al. 2006; Jensen et al. 2010). However, the studies described above highlights the importance of FcR-mediated responses against CAR-expressing T cells for in vivo T cell persistence and anti-tumor efficacy. Consequently, the studies also show that there is a ‘fix’ to avoid this form of immunogenicity—namely, the incorporation of mutations in the CAR design to prevent FcγR-recognition.
Based on these results, the mutations described herein may be extrapolated to humans and should therefore augment the persistence and therapeutic efficacy of T cells expressing IgG-spacer containing CAR in humans. Any discrepancy in CAR T cell engraftment and in vivo anti-tumor efficacy is likely impacted by the nature of the murine NSG model system. Human IgG4 has been shown to efficiently bind murine FcRs to mediate potent antibody dependent cell-mediated cytotoxicity (Isaacs et al. Steplewski et al. 1988). In contrast, human FcRs have the strongest affinity toward IgG1 and IgG3, and reduced affinity for IgG4 (Schroeder & Cavacini 2010; Nirula et al. 2011). Additionally, given that NSG mice lack serum antibodies, FcRs expressed by their innate immune cells are unoccupied and thus have a greater potential to bind the IgG-Fc spacer within the CAR. With the exception of hypoglobulinemia cases, immunocompetent humans have high serum IgG levels of approximately 10 mg/mL (Stoop et al., 1969), which could potentially compete for recognition of IgG-containing CARs. Indeed, several groups have administered IgG-Fc bearing CAR T cells to humans, and in some cases low levels of CAR T cells were detectable by quantitative PCR up to 6 weeks (Savoldo et al. 2011) and even one year (Till et al. 2012) after administration. Incorporation of the mutations described herein would likely further improve this CAR T cell persistence in humans.
Overall, the studies reported here provide evidence that CARs containing components of an Ig Fc spacer should incorporate modifications that prevent the FcR-mediated recognition of the cells in vivo. Such modifications can involve either point mutations to change the amino acid sequence, or sequence deletions such as that seen with the CD19R(EQ) and CD19Rch2Δ constructs described herein. Not only will such modifications prevent the ability of FcR-expressing cells to recognize the CAR-expressing immunotherapeutic cellular product in vivo, but they might also prevent the unintentional activation of the transferred T cells and/or the host immune responses (Hombach et al. 2010), which could contribute to various unwanted side-effects of this immunotherapeutic strategy.
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Claims
1. A recombinant chimeric antigen receptor (CAR) having impaired binding to an Fc receptor (FcR) comprising:
- an antigen recognition domain;
- a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR; and
- an intracellular signaling domain.
2. The method of claim 1, wherein the antigen recognition domain is an scFv.
3. The method of claim 1, wherein the antigen recognition domain targets a cancer associated antigen selected from the group consisting of 5T4, 8H9, αvβ6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcionoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-α, G250/CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor α2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated antigen (MAGE-A1), mesothelin, Murine CMV infected cella, mucin-1 (MUC1). mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors.
4. The method of claim 1, wherein the modified immunoglobulin Fc region is a modified IgG1, IgG2, IgG3, or IgG4 Fc region.
5. The method of claim 1, wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more amino acid substitutions selected from an S228P amino acid substitution, an L235E amino acid substitution, an N297Q amino acid substitution, or a combination thereof.
6. The method of claim 1, wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more deletions.
7. The method of claim 1, further comprising a transmembrane domain.
8. The method of claim 1, wherein the intracellular signaling domain is a T cell receptor (TCR) zeta chain signaling domain.
9. The method of claim 8, further comprising one or more costimulatory intracellular signaling domain derived from CD28, inducible costimulatory (ICOS), OX40, CD27, DAP10, 4-1BB, p56lck, or 2B4.
10. The method of claim 1, wherein the CAR is encoded by a nucleic acid sequence which that is inserted within a viral vector.
11. A population of human immune cells transduced by a viral vector comprising an expression cassette that includes a CAR gene, the gene comprising a nucleotide sequence that encodes:
- an antigen recognition domain;
- a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR; and
- an intracellular signaling domain;
- wherein the population of human immune cells expresses the CAR gene.
12. The method of claim 11, wherein the antigen recognition domain targets a cancer associated antigen selected from the group consisting of 5T4, 8H9, αvβ6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcionoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-α, G250/CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor α2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated antigen (MAGE-A1), mesothelin, Murine CMV infected cella, mucin-1 (MUC1). mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors.
13. The method of claim 11, wherein the modified immunoglobulin Fc region is a modified IgG1, IgG2, IgG3, or IgG4 Fc region.
14. The method of claim 11, wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more amino acid substitutions selected from an S228P amino acid substitution, an L235E amino acid substitution, an N297Q amino acid substitution, or a combination thereof.
15. The method of claim 11, wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more deletions.
16. The method of claim 11, wherein the intracellular signaling domain is a T cell receptor (TCR) zeta chain signaling domain.
17. The method of claim 16, further comprising one or more costimulatory intracellular signaling domain derived from CD28, inducible costimulatory (ICOS), OX40, CD27, DAP10, 4-1BB, p56lck, or 2B4.
18. A method of treating a cancer in a subject comprising administering a population of human immune cells transduced with a CAR gene to the subject, wherein the CAR gene comprises a nucleotide sequence that encodes:
- an antigen recognition domain that targets a cancer associated antigen specific to the cancer;
- a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR; and
- an intracellular signaling domain.
19. The method of claim 18, wherein the impaired binding to the FcR results in improved persistence of the human immune cells as compared to human immune cells transduced with a CAR gene comprising a nucleotide sequence that encodes a spacer domain derived from an unmodified immunoglobulin Fc region.
20. The method of claim 18, further comprising administering the population of human immune cells transduced with the CAR gene in combination with one or more anti-cancer therapy selected from stem cell transplantation, radiation therapy, surgical resection, chemotherapeutics, immunotherapeutics, targeted therapeutics or a combination thereof.
21. A recombinant chimeric antigen receptor (CAR) having impaired binding to an Fc receptor (FcR) comprising:
- an antigen recognition domain comprising an scFv;
- a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR, wherein the one or more mutations are selected from an S228P amino acid substitution, an L235E amino acid substitution, an N297Q amino acid substitution, or a combination thereof; and
- an intracellular signaling domain.
Type: Application
Filed: Mar 14, 2014
Publication Date: Nov 17, 2016
Inventors: Stephen J. Forman (Duarte, CA), Christine E. Brown (Duarte, CA), Umamaheswararao Jonnalagadda (Troy, MI), Armen Mardiros (Glendale, CA)
Application Number: 15/111,384
