Patent Application
20230312675
This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on Jan. 11, 2023, is named UTSCP1238USC1.xml and is 21,744 bytes in size.
The present invention relates generally to the fields of medicine, immunology, cell biology, and molecular biology. In certain aspects, the field of the invention concerns immunotherapy. More particularly, embodiments described herein concern the production of chimeric antigen receptors (CARs) and CAR-expressing T cells that can specifically target cells with elevated expression of a target antigen.
The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Such a combination of gene therapy with immunotherapy can redirect the specificity of T cells for B-lineage antigens and patients with advanced B-cell malignancies benefit from infusion of such tumor-specific T cells (Jena et al., 2010; Till et al., 2008; Porter et al., 2011; Brentjens et al., 2011; Cooper et al., 2012; Kalos et al., 2011; Kochenderfer et al., 2010; Kochenderfer et al., 2012; Brentjens et al., 2013). Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of chimeric antigen receptor (CAR) (Jena et al., 2010; Ertl et al., 2011; Kohn et al., 2011). This approach, although compliant with current good manufacturing practice (cGMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities.
One draw back of CAR T-cell based therapies is the potential for off-target effects when target antigens are also expressed in normal non-diseased tissues. Accordingly, new CAR T-cell therapies are needed that provide specific targeting of diseased cells whiles reducing the side effects on normal tissues.
Certain embodiments described herein are based on the finding that chimeric antigen receptor (CAR) T cells can be used to target cells that overexpress an antigen. Thus, in some aspects, cytotoxic activity of the CAR T cells can be focused only on intended target cells with a high level of antigen expression (e.g., cancer cells) while cytotoxic effects relative to cells having a lower level of antigen expression are minimized. In particular, it was found that by using CARs having an intermediate level of target affinity, CAR T cells could be produced that were selectively cytotoxic to cells with high antigen expression levels. Without being bound by any particular mechanism, the observed effect may be due to multivalent antigen binding by the CAR T cells to facilitate cell targeting. Alternatively or additionally, the expression level of a CAR may be adjusted in a selected CAR T cell so as reduce the off-target cytotoxicity of the cells.
Thus, in a first embodiment there are provided transgenic cells (e.g., an isolated transgenic cell) comprising an expressed CAR targeted to an antigen, said CAR having a Kd of between about 5 nM and about 500 nM relative to the antigen. In a further embodiment there is provided a transgenic T cell comprising an expressed CAR targeted to an antigen, said T cell exhibiting significant cytotoxic activity only upon multivalent binding of the antigen by the T cell. In an aspect, isolated cells of the embodiments are T cells or T-cell progenitors. In yet a further aspect, the cells are mammalian cells such as human cells.
In a further embodiment there are provided methods of selectively targeting cells expressing an antigen in a subject comprising (a) selecting a CAR T cell comprising an expressed CAR that binds to the antigen, said CAR T cells having: (i) cytotoxic activity only upon multivalent binding of the antigen by the T cells; and/or (ii) a CAR having a Kd of between about 5 nM and about 500 nM relative to the antigen; and (b) administering an effective amount of the selected CAR T cells to the subject to provide a T-cell response that selectively targets cells having elevated expression of the antigen. Thus, in certain aspects, a method of the embodiments is further defined as a method of treating a disease associated with an elevated level of antigen expression on diseased cells. For example, methods of the embodiments may be used for the treatment of a hyperproliferative disease, such as a cancer or autoimmune disease, or for the treatment of an infection, such as a viral, bacterial or parasitic infection.
In still a further embodiment there are provided methods of selectively targeting cells expressing an antigen in a mixed cell population comprising (a) selecting a CAR T cell comprising an expressed CAR that binds to the antigen, said CAR T cells having (i) cytotoxic activity only upon multivalent binding of the antigen by the T cells; and/or (ii) a CAR having a Kd of between about 5 nM and about 500 nM relative to the antigen; and (b) contacting a mixed cell population, said population including cells expressing different levels of the antigen, with the selected CAR T cells to selectively target cells having elevated expression of the antigen. For example, in certain aspects, a mixed cell population comprises non-cancer cells that express the antigen and cancer cells having elevated expression of the antigen. In some aspects, an elevated level of an antigen can refer to an expression level at least about: 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 times higher in a cell that is targeted by the CAR T cell.
In a further embodiment there are provided methods of selecting a CAR T cell comprising (a) obtaining a plurality of CAR T cells expressing CARs that bind to an antigen, said plurality of cells comprising (i) CARs with different affinities for the antigen (or having different on/off rates for the antigen) and/or (ii) CARs that are expressed at different levels in the cells (i.e., present at different levels on the cell surface); (b) assessing the cytotoxic activity of the cells on control cells expressing the antigen and on target cells expressing an elevated level of the antigen; and (c) selecting a CAR T cell that is selectively cytotoxic to target cells. In further aspects, methods of the embodiments further comprise expanding and/or banking a selected CAR T cell or population of selected T cells. In yet further aspects, methods of the embodiments comprise treating a subject with an effective amount of selected CAR T cells of the embodiments. In certain aspects, obtaining a plurality of CAR T cells can comprise generating a library of CAR T cells expressing CARs that bind to an antigen. For example, the library of CAR T cells may comprise random or engineered point mutations in the CAR (e.g., thereby modulating the affinity or on/off rates for the CARs). In a further aspect, a library of CAR T-cells comprises cells expressing CARs under the control of different promoter elements that provide varying levels of expression of the CARs.
In yet a further embodiment there are provided transgenic cells (e.g., an isolated transgenic cell) comprising an expressed CAR targeted to an EGFR antigen, said CAR having CDR sequences of nimotuzumab (see, e.g., SEQ ID NO: 1 and SEQ ID NO: 2) or the CDR sequences of cetuximab (see, e.g., SEQ ID NO: 3 and SEQ ID NO: 4). In some aspects, a cell of the embodiments is a human T cell comprising an expressed CAR sequence having the CDRs or the antigen binding portions of SEQ ID NO: 1 and SEQ ID NO: 2. In further aspects, a cell of the embodiments is a human T cell comprising an expressed CAR sequence having the CDRs or the antigen binding portions of SEQ ID NO: 3 and SEQ ID NO: 4.
Aspects of the embodiments concern antigens that are bound by a CAR. In some aspects, the antigen is an antigen that is elevated in cancer cells, in autoimmune cells or in cells that are infected by a virus, bacteria or parasite. In certain aspects, the antigen is CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII or VEGFR2. In some specific aspects the antigen is GP240, 5T4, HER1, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, IGF-1R, BAFF-R, TACI, APRIL, Fn14, ERBB2 or ERBB3. In some further aspects, the antigen is a growth factor receptor such as EGFR, ERBB2 or ERBB3.
Certain aspects of the embodiments concern a selected CAR (or a selected cell comprising a CAR) that binds to an antigen and has a Kd of between about 2 nM and about 500 nM relative to the antigen. For example, in some aspects, the CAR has a Kd of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nM or greater relative to the antigen. In still further aspects, the CAR has a Kd of between about 5 nM and about 450, 400, 350, 300, 250, 200, 150, 100 or 50 nM relative to the antigen. In still further aspects, the CAR has a Kd of between about 5 nM and 500 nM, 5 nM and 200 nM, 5 nM and 100 nM, or 5 nM and 50 nM relative to the antigen. As used herein reference to “Kd for a CAR” may refer to the Kd measured for a monoclonal antibody that is used to form the CAR.
In some aspects, a selected CAR of the embodiments can bind to 2, 3, 4 or more antigen molecules per CAR molecule. In some aspects, each to the antigen binding domains of a selected CAR has a Kd of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nM or greater relative to the antigen. In still further aspects, each to the antigen binding domains of a selected CAR has a Kd of between about 5 nM and about 450, 400, 350, 300, 250, 200, 150, 100 or 50 nM relative to the antigen. In still further aspects, each to the antigen binding domains of a selected CAR has a Kd of between about 5 nM and 500 nM, 5 nM and 200 nM, 5 nM and 100 nM, or 5 nM and 50 nM relative to the antigen.
In some aspects of the embodiments a selected CAR for use according to the embodiments is a CAR that binds to EGFR. For example, the CAR can comprise the CDR sequences of Nimotuzumab. For example, in some aspects a CAR of the embodiments comprises all six CDRs of Nimotuzumab (provided as SEQ ID NOs: 5-10). In some aspects a CAR comprises the antigen binding portions of SEQ ID NO: 1 and SEQ ID NO: 2. In some aspects, the CAR comprises a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 and/or SEQ ID NO: 2. In still further aspects, a CAR for use according the embodiments does not comprise the CDR sequences of Nimotuzumab.
In a further embodiment there are provided isolated cells comprising a selected CAR and at least a second expressed transgene, such as an expressed membrane-bound IL-15. For example, in some aspects, the membrane-bound IL-15 comprises a fusion protein between IL-15 and IL-15Rα. In some cases, such a second transgene is encoded by a RNA or a DNA (e.g., an extra chromosomal or episomal vector). In certain aspects, the cell comprises DNA encoding the membrane-bound IL-15 integrated into the genome of the cell (e.g., coding DNA flanked by transposon repeat sequences). In certain aspects, a cell of the embodiments (e.g., human CAR T cell expressing a membrane-bound cytokine) can be used to treat a subject (or provide an immune response in a subject) having a disease where disease cells express elevated levels of the antigen.
In some aspects, methods of the embodiments concern transfecting T cells with a DNA (or RNA) encoding a selected CAR and, in some cases, a transposase. Methods of transfecting cells are well known in the art, but in certain aspects, highly efficient transfection methods such as electroporation or viral transduction are employed. For example, nucleic acids may be introduced into cells using a nucleofection apparatus. Preferably, however, the transfection step does not involve infecting or transducing the cells with a virus, which can cause genotoxicity and/or lead to an immune response to cells containing viral sequences in a treated subject.
Certain aspects of the embodiments concern transfecting cells with an expression vector encoding a selected CAR. A wide range of expression vectors for CARs are known in the art and are further detailed herein. For example, in some aspects, the CAR expression vector is a DNA expression vector such as a plasmid, linear expression vector or an episome. In certain aspects, the vector comprises additional sequences, such as sequences that facilitate expression of the CAR, such as a promoter, enhancer, poly-A signal, and/or one or more introns. In preferred aspects, the CAR coding sequence is flanked by transposon sequences, such that the presence of a transposase allows the coding sequence to integrate into the genome of the transfected cell.
As detailed supra, in certain aspects, cells are further transfected with a transposase that facilitates integration of a CAR coding sequence into the genome of the transfected cells. In some aspects, the transposase is provided as a DNA expression vector. However, in preferred aspects, the transposase is provided as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells. Any transposase system may be used in accordance with the embodiments. However, in some aspects, the transposase is salmonid-type Tcl-like transposase (SB). For example, the transposase can be the “Sleeping beauty” transposase, see, e.g., U.S. Pat.6,489,458, incorporated herein by reference.
In still further aspects, a selected CAR T cell of the embodiments further comprises an expression vector for expression of a membrane-bound cytokine that stimulates proliferation of T cells. In particular, selected CAR T cells comprising such cytokines can proliferate with little or no ex vivo culture with antigen presenting cells due the simulation provided by the cytokine expression. Likewise, such CAR T cells can proliferate in vivo even when large amounts of antigen recognized by the CAR is not present (e.g., as in the case of a cancer patient in remission or a patient with minimal residual disease). In some aspects, the CAR T cells comprise a DNA or RNA expression vector for expression of a Cγ cytokine and elements (e.g., a transmembrane domain) to provide surface expression of the cytokine. For example, the CAR cells can comprise membrane-bound versions of IL-7, IL-15 or IL-21. In some aspects, the cytokine is tethered to the membrane by fusion of the cytokine coding sequence with the receptor for the cytokine. For example, a cell can comprise a vector for expression of an IL-15-IL-15Rα fusion protein. In still further aspects, a vector encoding a membrane-bound Cγ cytokine is a DNA expression vector, such as a vector integrated into the genome of the CAR cells or an extra-chromosomal vector (e.g., and episomal vector). In still further aspects, expression of the membrane-bound Cγ cytokine is under the control of an inducible promoter (e.g., a drug inducible promoter) such that the expression of the cytokine in the CAR cells (and thereby the proliferation of the CAR cells) can be controlled by inducing or suppressing promoter activity.
Aspects of the embodiments concern obtaining T cells or T-cell progenitors for expression of selected CARs. In some aspects, the cells are obtained from a third party, such as a tissue bank. In further aspects, cell samples from a patient comprising T cells or T-cell progenitors are used. For example, in some cases, the sample is an umbilical cord blood sample, a peripheral blood sample (e.g., a mononuclear cell fraction) or a sample from the subject comprising pluripotent cells. In some aspects, a sample from the subject can be cultured to generate induced pluripotent stem (iPS) cells and these cells used to produce T cells. Cell samples may be cultured directly from the subject or may be cryopreserved prior to use. In some aspects, obtaining a cell sample comprises collecting a cell sample. In other aspects, the sample is obtained by a third party. In still further aspects, a sample from a subject can be treated to purify or enrich the T cells or T-cell progenitors in the sample. For example, the sample can be subjected to gradient purification, cell culture selection and/or cell sorting (e.g., via fluorescence-activated cell sorting (FACS)).
In some aspects, a method of the embodiments further comprises obtaining, producing or using antigen presenting cells (APCs). For example, in some aspects, the antigen presenting cells comprise dendritic cells, such as dendritic cells that express or have been loaded with and an antigen of interest. In further aspects, the antigen presenting cell can comprise artificial antigen presenting cells that display an antigen of interest. For example, artificial antigen presenting cells can be inactivated (e.g., irradiated) artificial antigen presenting cells (aAPCs). Methods for producing such aAPCs are know in the art and further detailed herein.
Thus, in some aspects, transgenic CAR cells of the embodiments are co-cultured with antigen presenting cells (e.g., inactivated aAPCs) ex vivo for a limited period of time in order to expand the CAR cell population. The step of co-culturing CAR cells with antigen presenting cells can be done in a medium that comprises, for example, interleukin-21 (IL-21) and/or interleukin-2 (IL-2). In some aspects, the co-culturing is performed at a ratio of CAR cells to APCs of about 10:1 to about 1:10; about 3:1 to about 1:5; or about 1:1 to about 1:3. For example, the co-culture of CAR cells and APCs can be at a ratio of about 1:1, about 1:2 or about 1:3.
In some aspects, APCs for culture of selected CAR cells are engineered to express a specific polypeptide to enhance growth of the CAR cells. For example, the APCs can comprise (i) an antigen targeted by the CAR expressed on the transgenic CAR cells; (ii) CD64; (ii) CD86; (iii) CD137L; and/or (v) membrane-bound IL-15, expressed on the surface of the APCs. In some aspects, the APCs comprise a CAR-binding antibody or fragment thereof expressed on the surface of the APCs (see, e.g., International PCT pat. publication WO/2014/190273, incorporated herein by reference). Preferably, APCs for use in the instant methods are tested for, and confirmed to be free of, infectious material and/or are tested and confirmed to be inactivated and non-proliferating.
While expansion on APCs can increase the number or concentration of CAR cells in a culture, this proceed is labor intensive and expensive. Moreover, in some aspects, a subject in need of therapy should be re-infused with transgenic CAR cells in as short a time as possible. Thus, in some aspects, ex vivo culturing of selected CAR cells is for no more than 14 days, no more than 7 days or no more than 3 days. For example, the ex vivo culture (e.g., culture in the presence of APCs) can be performed for less than one population doubling of the transgenic CAR cells. In still further aspects, the transgenic cells are not cultured ex vivo in the presence of APCs.
In still further aspects, a method of the embodiments comprises a step for enriching the cell population for selected CAR-expressing T cells before administering or contacting the cells to a population (e.g., after transfection of the cells or after ex vivo expansion of the cells). For example, the enrichment step can comprise sorting of the cell (e.g., via Fluorescence-activated cell sorting (FACS)), for example, by using an antigen bound by the CAR or a CAR-binding antibody. In still further aspects, the enrichment step comprises depletion of the non-T cells or depletion of cells that lack CAR expression. For example, CD56+ cells can be depleted from a culture population. In yet further aspects, a sample of CAR cells is preserved (or maintained in culture) when the cells are administered to the subject. For example, a sample may be cryopreserved for later expansion or analysis.
In certain aspects, transgenic CAR cells of the embodiments are inactivated for expression of an endogenous T-cell receptor and/or endogenous HLA. For example, T cells can be engineered to eliminate expression of endogenous alpha/beta T-cell receptor (TCR). In specific embodiments, CAR+ T cells are genetically modified to eliminate expression of TCR. In some aspects, there is a disruption of the endogenous T-cell receptor in CAR-expressing T cells. For example, in some cases an endogenous TCR (e.g., a α/β or γ/δ TCR) is deleted or inactivated using a zinc finger nuclease (ZFN) or CRISPR/Cas9 system. In certain aspects, the T-cell receptor αβ-chain in CAR-expressing T cells is knocked-out, for example, by using zinc finger nucleases.
As further detailed herein, CAR cells of the embodiments can be used to treat a wide range of diseases and conditions. Essentially any disease that involves the enhanced expression of a particular antigen can be treated by targeting CAR cells to the antigen. For example, autoimmune diseases, infections, and cancers can be treated with methods and/or compositions of the embodiments. These include cancers, such as primary, metastatic, recurrent, sensitive-to-therapy, refractory-to-therapy cancers (e.g., chemo-refractory cancer). The cancer may be of the blood, lung, brain, colon, prostate, breast, liver, kidney, stomach, cervix, ovary, testes, pituitary gland, esophagus, spleen, skin, bone, and so forth (e.g., B-cell lymphomas or a melanomas). In certain aspects, a method of the embodiments is further defined as a method of treating a glioma, such as a diffuse intrinsic pontine glioma. In the case of cancer treatment, CAR cells typically target a cancer cell antigen (also known as a tumor-associated antigen (TAA)), such as EGFR.
The processes of the embodiments can be utilized to manufacture (e.g., for clinical trials) CAR+ T cells for various tumor antigens (e.g., CD19, ROR1, CD56, EGFR, CD123, c-met, GD2). CAR+ T cells generated using this technology can be used to treat patients with leukemias (AML, ALL, CML), infections and/or solid tumors. For example, methods of the embodiments can be used to treat cell proliferative diseases, fungal, viral, bacterial or parasitic infections. Pathogens that may be targeted include, without limitation, Plasmodium, trypanosome, Aspergillus, Candida, HSV, RSV, EBV, CMV, JC virus, BK virus, or Ebola pathogens. Further examples of antigens that can be targeted by CAR cells of the embodiments include, without limitation, CD19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23,CD30, CD56, c-Met, meothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2. In certain aspects, method of the embodiments concern targeting of CD19 or HERV-K-expressing cells. For example, a HERV-K targeted CAR cell can comprise a CAR including the scFv sequence of monoclonal antibody 6H5. In still further aspects, a CAR of the embodiments can be conjugated or fused with a cytokine, such as IL-2, IL-7, IL-15, IL-21 or a combination thereof.
In some embodiments, methods are provided for treating an individual with a medical condition comprising the step of providing an effective amount of cells from a population of CAR expressing T cells or T-cell progenitors (e.g., CAR expressing T-cells that selectively kill cells that have an elevated expression level of a target antigen) to the subject. In some aspects, the cells can be administered to an individual more than once (e.g., 2, 3, 4, 5 or more times). In further aspects, cells are administered to an individual at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days apart. In specific embodiments, the individual has a cancer, such a lymphoma, leukemia, non-Hodgkin’s lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, or B cell-associated autoimmune diseases.
In a further embodiment, there is provided an isolated transgenic cell (e.g., a T-cell or T-cell progenitor) comprising an expressed CAR targeted to EGFR. For example, the CAR can comprise the CDR sequences of Nimotuzumab. For example, in some aspects, a cell of the embodiments comprises a CAR comprising all six CDRs of Nimotuzumab (provided as SEQ ID NOs: 5-10). In some aspects, the CAR comprises the antigen binding portions of SEQ ID NO: 1 and SEQ ID NO: 2. In further aspects, the CAR comprises a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 and/or SEQ ID NO: 2. In still further aspects, a cell of the embodiments comprises a CAR that does not comprise the CDR sequences of Nimotuzumab. In some aspects, there is provided a pharmaceutical composition comprising an isolated transgenic cell of the embodiments. In a further related embodiment there is provided a method of treating a subject having an EGFR positive cancer comprising administering an effective amount of transgenic human T-cells to the subject said T-cells comprising an expressed CAR targeted to EGFR and comprising the CDR sequences of SEQ ID NOs: 5-10.
In a further embodiment, there is provided an isolated transgenic cell (e.g., a T-cell or T-cell progenitor) comprising an expressed CAR that comprises the CDR sequences of Cetuximab. For example, in some aspects, a cell of the embodiments comprises a CAR comprising all six CDRs of Cetuximab (provided as SEQ ID NOs: 11-16). In some aspects, the CAR comprises the antigen binding portions of SEQ ID NO: 3 and SEQ ID NO: 4. In further aspects, the CAR comprises a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 and/or SEQ ID NO: 4. In still further aspects, a cell of the embodiments comprises a CAR that does not comprise the CDR sequences of Cetuximab. In some aspects, there is provided a pharmaceutical composition comprising an isolated transgenic cell of the embodiments. In a further related embodiment there is provided a method of treating a subject having an EGFR positive cancer comprising administering an effective amount of transgenic human T-cells to the subject said T-cells comprising an expressed CAR targeted to EGFR and comprising the CDR sequences of SEQ ID NOs: 11-16.
As used herein in the specification and claims, “a” or “an” may mean one or more. As used herein in the specification and claims, when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein, in the specification and claim, “another” or “a further” may mean at least a second or more.
As used herein in the specification and claims, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Transient expression of CAR by RNA transfer has been proposed to reduce the potential for long-term, on-target, off-tissue toxicity of CAR T cell therapy directed against antigens with normal tissue expression. Numeric expansion of T cells prior to RNA transfer is appealing to obtain clinically relevant T cell numbers needed for patient infusion. The inventors explored numeric expansion of T cells independent of antigen-specificity by co-culturing on aAPC loaded with anti-CD3 antibody, OKT3. Altering the ratio of antigen presenting cells (e.g., aAPCs) to T cells in culture altered the phenotype of the resultant T cell population. T cells expanded with low density of aAPC (10 T cells to 1 aAPC) were associated with increased proportion of CD8+ T cells, increased presence of central memory phenotype T cells, reduced production of IFN-γ and TNF-α, but increased production of IL-2, and potentially less clonal loss of TCR diversity following expansion relative to T cells expanded with high density aAPC. T cells expanded with low density aAPC were more amenable to RNA electro-transfer, demonstrating higher expression of RNA transcripts and improved T-cell viability following electro-transfer than T cells expanded with high density aAPC.
A potential benefit of use of aAPC for T-cell expansion is the ability to form stable interactions with T cells by virtue of expression of adhesion molecules LFA-3 and ICAM-1 (Suhoski et al., 2007; Paulos et al., 2008). Additionally, aAPC can be modified with relative ease to express desired arrays of costimulatory molecules. Thus, aAPC for numeric T-cell expansion provides a platform to evaluate various combinations of costimulatory molecules for T-cell expansion to achieve an optimal T-cell phenotype for adoptive T-cell therapy. In addition to modification of aAPC, the inventors have described the impact of the density of aAPC in T cell culture on the phenotype of resulting T-cell populations. While CD8+ T cells, or cytotoxic T cells, are often thought of as the ideal T-cell population for anti-tumor immunotherapy, evidence suggests that CD8+ T cells require CD4+ T-cell help in vivo to achieve optimal anti-tumor response and memory formation (Kamphorts et al., 2013; Bourgeois et al., 2002; Sun et al., 20013). However, the ideal ratio of CD4+ to CD8+ T cells is unknown (Muranski et al., 2009). By altering density of aAPC in expansion cultures to skew CD4/CD8 ratio in T cells for adoptive immunotherapy, whether they be TIL isolated from patients or gene-modified T cells, these questions may be addressed in clinical trials. Finally, reducing density of aAPC in culture resulted in more T cells with a central memory phenotype (CCR7+CD45RAneg) than T cells expanded with higher density of aAPC. While the benefit of enhanced persistence of central memory phenotype T cells may not extend to RNA-modified T cells, which are only transiently redirected for tumor antigen, persistence of T cells has been shown to improve the anti-tumor efficacy of T-cell therapy (Kowolik et al., 2006; Robbins et al., 2004; Stephan et al., 2007; Wu et al., 2013). Therefore, ex vivo expansion with low density aAPC may be used to reprogram stably genetically modified T cells or TIL to a central memory phenotype for enhanced persistence.
Expression of CAR by RNA-modification in ex vivo expanded T cells was found to be more variable than expression of CAR by non-viral DNA-modification and expansion of T cells through CAR recognition of antigen. Expression of CAR at different densities did not impact the ability of the T cells to specifically lyse targets, although it is reasonable to expect that below a certain threshold, low CAR expression would have a negative impact on specific lysis of targets, as previously reported (Weijtens et al., 2000). Others have described tunable expression of CAR by RNA modification of T cells, such that the dose of RNA determines the level of transgene expression (Rabinovich et al., 2006; Yoon et al., 2009; Barrett et al., 2011). RNA modification of T cells in the present study was conducted using the same quantity of RNA, therefore, this does not account for variability of CAR expression by altering RNA dose. Instead, it is likely that variability between donors accounts for differences in CAR expression intensity following electro-transfer. The presently described protocol for T-cell expansion prior to RNA transfer may play a role in altering the sensitivity of T cells from certain donors to RNA uptake, and increasing the RNA quantity in electro-transfers may increase expression of CAR in these donors. High expression of CAR by transferring relatively high quantities of RNA can result in prolonged CAR expression and CAR-mediated activity over a prolonged period of time (Barrett et al., 2011). Prolonged CAR expression from RNA transfer may be beneficial to anti-tumor activity, particularly since stimulation of T cells seems to accelerate the loss of CAR expression. However, prolonging the expression of CAR may also increase T-cell activity in response to normal tissue antigen requiring the optimization of CAR expression to determine the optimal duration of expression to maximize anti-tumor activity while reducing normal tissue toxicity.
RNA-modification of T cells did not alter the proportion of effector memory and central memory T cells found in ex vivo expanded T cells prior to electro-transfer of RNA, similar to previous reports (Schaft et al., 2006). Only T cells expanded at relatively low aAPC density, 10 T cells to 1 aAPC, were capable of efficient RNA transcript uptake without significant toxicity, even with various electroporation conditions. This population of T cells also demonstrated a substantial proportion of T cells with a central memory phenotype (CCR7+CD45RAneg) that had reduced production of IFN-γ and TNF-α, and cytotoxic effector molecules granzyme B and perforin. As a result, RNA-modified T cells contained significantly more central memory phenotype T cells than DNA-modified T cells, demonstrated reduced production of IFN-γ and TNF-α in response to EGFR-expressing cells and slightly less specific lysis at low E:T ratios. Thus, the precursor T cell population for RNA-modification has a strong influence on CAR-mediated T cell function following RNA transfer and the reduced cytokine production and slightly less specific lysis of RNA-modified T cells may translate to reduced anti-tumor efficacy in an in vivo model where cytotoxic potential of T cells is short-lived and the enhanced persistence of a central memory T cell population may not be beneficial. RNA-modification of T cells expanded at 1 T cell to 2 aAPC, which demonstrated a more significant proportion of effector memory phenotype T cells, similar to DNA-modified CAR+ T cells, and consequently the capacity for higher production of IFN-γ and TNF-α is desirable. The addition of cytokines prior to RNA transfer may improve viability and additional electroporation programs may efficiently transfer RNA into these T cells.
Cetux-CAR introduced to T cells through RNA transfer was transiently expressed, and loss of expression was accelerated by stimulus to T cells, including addition of cytokines IL-2 and IL-21 and antigenic-stimulus through addition of EGFR-expressing cell lines. Concomitant with loss of CAR expression, RNA-modified T cells demonstrated reduced cytotoxicity against EGFR-expressing cell lines, including tumor cells and normal human renal cells. One concern for the use of RNA-modified T cells is that their inherently reduced capacity to target tumor over time will result in reduced anti-tumor efficacy relative to stably-modified T cells. Multiple injections of T cells modified to express a mesothelin-specific CAR by RNA transfer for the treatment of a murine model of mesothelioma demonstrated that biweekly, intratumoral injections demonstrated control of tumor growth, but following cessation of treatment, tumors relapsed (Zhao et al., 2010). Treatment of an in vivo disseminated leukemia murine model has demonstrated that while RNA-modified CAR+ T cells specific for CD19 have anti-tumor activity after a single injection, tumors often relapse after a time period consistent with CAR degradation (Barrett et al., 2011). In contrast, a single intratumoral injection of T cells stably expressing mesothelin-specific CAR mediated superior anti-tumor activity and was capable of curing most mice. Optimization of dosing of RNA-modified T cells demonstrated that a combination of cyclophosphamide to eliminate residual CARneg T cells before subsequent infusions and a weighted, split-dosing regimen was more effective in controlling disease burden, and was similar in anti-tumor efficacy to stably modified T cells (Barrett et al., 2013). Thus, optimizing a dosing regimen can improve the anti-tumor activity of RNA-modified T cells.
Cetux-CAR+ T cells can recognize normal tissue antigen, which could result in on-target, off-tissue toxicity. Thus, the inventors investigated expression of CAR as RNA species as a method to control on-target, off-tissue toxicity through transient expression of CAR. While CAR expression was transient and reduced potential for cytotoxicity against normal tissue EGFR after degradation of CAR, it did not address the potential for immediate T-cell effector function upon recognition of normal tissue EGFR before considerable degradation of CAR. Additionally, by limiting CAR expression, T cells are rendered non-responsive to EGFR-expressing tumor following CAR degradation, and the potential for lasting anti-tumor activity is compromised by this approach. Therefore, mechanisms to control CAR activity in the presence of normal tissue to limit deleterious on-target, off-tissue toxicity without compromising anti-tumor activity were investigated.
Endogenous T cell activation is dependent on both affinity of the TCR and density of peptide presented via MHC (Hemmer et al., 1998; Viola et al., 1996; Gottschalk et al., 2012; Gottschalk 2010). T cells are activated by a cumulative signal through the TCR that surpasses a certain threshold required for elicitation of effector functions Hemmer et al., 1998; Rosette et al., 2001; Viola et al., 1996). For high affinity TCRs, relatively low antigen density is sufficient to trigger T cell responses; however, low affinity TCRs required higher antigen density to achieve similar effector T cell responses (Gottschalk et al., 2012). Many tumors overexpress TAA at higher densities than their normal tissue expression (Barker et al., 2001; Lacunza et al., 2010; Hirsch et al., 2009). Amplification and overexpression of EGFR in glioma highlight this relationship as EGFR is overexpressed in glioma relative to normal tissue, and overexpression correlates with tumor grade, such that grade IV glioblastoma expresses the highest density of EGFR (Smith et al., 2001; Hu et al., 2013; Galanis et al., 1998). Therefore, the inventors determined if EGFR-specific CAR-modified T cells could distinguish malignant cells from normal cells based on EGFR density by reducing the binding affinity of the CAR.
The portion of Cetux-CAR that endows antigenic specificity is derived from the scFv portion of the monoclonal antibody cetuximab, which is characterized by a high affinity (Kd=1.9x10-9) (Talavera et al., 2009). Therefore, the inventors generated a CAR from the monoclonal antibody nimotuzumab, which shares a highly overlapping epitope with cetuximab and a 10-fold lower dissociation constant (Kd=2.1x10-8), characterized by a 59-fold reduced rate of association (Talavera et al., 2009; Garrido et al., 2011; Adams et al., Zuckier et al., 2000). The reduced association rate and subsequent reduction in overall affinity imposes a requirement for bivalent recognition of EGFR, which only occurs when EGFR is expressed at high density. Thus, a CAR derived from nimotuzumab may enable T cells to distinguish malignant tissue from normal tissue based on density of EGFR expression.
Recent clinical success in CLL and ALL note persistent B-cell aplasia in patients with complete tumor response to CD19-CAR+ T-cell therapy, but this toxicity is considered tolerable as CD19 is a lineage-restricted antigen and B cell aplasia is considered a tolerable toxicity in the setting of advanced lymphoma (Grupp et al., 2013; Porter et al., 2011). Serious adverse events in clinical trials targeting HER2 and CAIX with CAR-modified T cells highlights the need to control CAR T-cell activity against normal tissue antigen expression in order to broaden the range of safely targetable antigens beyond lineage and tumor restricted antigens (Lamers et al., 2013; Morgan et al., 2010). Aberrantly expressed TAAs are often overexpressed on tumor relative to normal tissue, such as EGFR expression in glioblastoma (Smith et al., 2001; Hu et al., 2013; Galanis et al., 1998). The inventors developed a CAR specific to EGFR with reduced capacity to respond to low antigen density to minimize the potential for normal tissue, while maintaining adequate effector function in response to high antigen density. This was accomplished by developing an EGFR-specific CAR from nimotuzumab, a monoclonal antibody with a highly-overlapping epitope, yet reduced binding kinetics compared to cetuximab (Talavera et al., 2009; Garrido et al., 2011). While Cetux-CAR+ T cells were capable of targeting low and high EGFR density, Nimo-CAR+ T cells were able to tune T-cell activity to antigen density and response was dependent on EGFR density expressed on target cells. While Nimo-CAR+ T cells demonstrate reduced activity relative to Cetux-CAR+ T cells in response to low EGFR density on tumor cells and normal renal cells, they were capable of equivalent redirected specificity and function in response to high EGFR density. CAR affinity influenced proliferation after antigen challenge and Cetux-CAR+ T cells demonstrated impaired proliferation when compared with Nimo-CAR+ T cells after antigen challenge, but not increased propensity for activation induced cell death (AICD). Additionally, CAR affinity influences downregulation of CAR from T-cell surface after interaction with antigen. Cetux-CAR exhibited more rapid and prolonged downregulation from the cell surface after interaction with high EGFR density than Nimo-CAR. Cetux-CAR+ T cells had impaired ability to respond to re-challenge with antigen, which could be a result of downregulated CAR or potentially functional exhaustion of Cetux-CAR+ T cells (James et al., 2010; Lim et al., 2002).
Complications in delineating the impact of scFv on CAR function stem from considerable debate surrounding the biochemical parameter of endogenous TCR binding that best predicts T-cell function. The kinetics of TCR binding can be described by the equation:
such that the dissociation constant, Kd, is equal to the ratio of the rate of dissociation (koff) and the rate of association (kon) (14). Both the dissociation constant (Kd) and the dissociation rate (koff) have been reported as important determinants of T-cell function following TCR recognition of pepMHC, however these two parameters are often strongly correlated, so it is difficult to separate their respective impact on T-cell function (Kersh et al., 1998; McKeithan T.W. 1995; Nauerth et al., 2013). The kinetic proofreading model of T-cell triggering states that koff impact T-cell function, such that sufficiently long dwell time is required to trigger T-cell signaling and activation. This has been amended to include a window of optimal dwell time, in which prolonged dwell time may be detrimental to T-cell activation by impairing the ability of serial triggering of multiple TCR by a single pepMHC complex (Kalergis et al., 2001). However, these models are contradicted by reports of very short dwell time interactions capable of producing functional T-cell responses (Govern et al., 2010; Tian et al., 2007; Aleksic et al., 2010; Gottschalk et al., 2012). Recent analysis aiming to reduce previous dataset bias by reducing the high degree of correlation between Kd and koff values and expanding dynamic range of kon values uncovered an important role in contribution of kon to T-cell activation, encompassed in a T-cell confinement model of T-cell triggering, in which T-cell function is directly correlated with the duration of T-cell confinement derived from a mathematical relationship between rate of association, rate of dissociation, and diffusion of TCR and pepMHC in their relative membranes (Tain et al., 2007; Aleksic et al., 2010). Interestingly, as kon becomes low, TCR and pepMHC are able to diffuse in their relative membranes before rebinding, thus the duration of interaction reduces to the koff value. In contrast, as kon becomes high, the TCR is capable of rapid rebinding to extend the dwell time, and the duration of interaction and resulting T-cell function is best predicted by Kd. This ongoing debate to define the role of TCR affinity components that control T-cell functional avidity cautions against universal models relying on one biochemical parameter of binding as a superior indicator of function over others. Instead, it is likely a combination of rates of association and dissociation as well as density of antigen freely moving through target cell membrane that defines functional response.
Endogenous TCR responses are generally described as much lower affinity than the binding of monoclonal antibodies, which are used to derive CAR specificity (Stone et al., 2009). However, SPR techniques used to measure TCR binding affinity are typically performed in three dimensions, and do not recapitulate the physiological interaction of a T cell with an antigen presenting cell, in which both binding partners are constrained in their respective membranes, increasing the probability of binding due to constrained intercellular space and proper molecule orientation (Huppa et al., 2010). Measurement of TCR binding kinetics in 2D suggests that TCR binding is of higher affinity than suggested by 3D measurements characterized by increased rates of association and decreased rates of dissociation (Huang et al., 2010; Robert et al., 2012). However, binding kinetics of other ligand/receptor pairs, such as ICAM-1 or LFA-1 did not show a difference between affinity measurements taken in 3D or 2D assays. Interestingly, ablation of cytoskeletal polymerization reduces measurements made in 2D to measurements made in 3D, highlighting the role of dynamic cellular and cytoskeletal processes in enhancing T cell binding to antigen (Robert et al., 2012). Whether similar cytoskeletal interactions or enhancement of binding affinity of CAR occurs is currently unknown, and therefore, it is unclear if assumptions made about binding affinity of the scFv domain of CAR can be directly made from measurements of monoclonal antibody affinity in 3D assays. In addition, several factors contribute to enhance overall T cell binding avidity, such as co-receptor binding to MHC and TCR nanocluster and microcluster formation on the T-cell surface prior to and following T cell activation (Holler et al., 2003; Schamel et al., 2005; Schamel et al., 2013; Kumar et al., 2011; Yokosuka et al., 2010). While it appears that CARs can be expressed in oligomeric form on the T cell surface, the degree of involvement of CAR with endogenous T cell signaling complexes is unclear. While reports of first generation CARs, signaling through only CD3-ζ demonstrate a requirement for association with endogenous CD3-ζ to achieve CAR-dependent T-cell activation, second generation CARs signaling through transmembrane CD28 and intracellular CD28 and CD3-ζ demonstrate no difference in CAR-dependent activation ability when endogenous TCR-CD3 complexes are restricted from the T cell surface (Bridgeman et al., 2010; Torikai et al., 2012). Therefore, the association of CAR with endogenous TCR signaling machinery may be dependent on CAR configuration.
Specific studies addressing the role of scFv affinity in CAR design are limited, and focus on contribution of the dissociation constant, Kd. Recent studies with ROR1-specific CAR compared a with 6-fold lower Kd, thus higher affinity, resulting from both increased kon and decreased koff and demonstrated that higher affinity ROR-1specific CAR increased T-cell function in vitro, including production of cytokines and specific lysis, without increased propensity for AICD (Hudecek et al., 2013). Additionally, high affinity ROR-1-specific CAR+ T cells mediated superior anti-tumor activity in vivo. Similarly, the higher affinity of Cetux-CAR+ T cells did not increase propensity for AICD, and had increased T-cell function, including production of cytokines and specific lysis, in response to reduced EGFR density. However, a previous study of a series of CARs derived from a panel of affinity-matured HER2-specific monoclonal antibodies with a wide range of Kd values, found that an affinity threshold existed, below which CAR-dependent T-cell activation was impaired; however, above this threshold, activation of T cells in response to various levels of HER2 did not improve with increased affinity (Chmielewski et al., 2004). In contrast, the present study identified different ability of high affinity CAR and low affinity CAR to target based on antigen density. Higher affinity Cetux-CAR+ T cells were associated with increased cytokine production and specific lysis in response to reduced EGFR density relative to Nimo-CAR+ T cells. While, Nimo-CAR is lower affinity relative to Cetux-CAR, the Kd value of Nimo-CAR was above the affinity threshold and within the range predicted to have effector function by the previous study. Similar to studies with endogenous TCRs, these results indicate that descriptions of CAR affinity should not be described solely by the dissociation constant, and support that relationship between individual dissociation and association rates be taken into consideration for CAR design.
The contradictions between the influence of affinity on CAR function between studies may be explained by the distinct relationships of the biochemical parameters koff and kon that constitute the dissociation constant Kd. The HER2-specific CARs were derived from antibodies that displayed a wide range of Kd values differing primarily in koff, with minimal correlation of kon values (Chmielewski et al., 2004). Thus, higher affinity interactions did not have increased rates of association, but increased duration of interaction with antigen. In contrast, the higher affinity of the ROR-1-specific CAR and Cetux-CAR were both influenced by increased association rates of binding. The higher affinity monoclonal antibody used to derive the ROR-1-specific CAR had a 6-fold lower Kd, from contributions of both increased kon and decreased koff, such that the higher affinity was characterized by both increased association rates and increased duration of interaction (Hudecek et al., 2013). The 10-fold difference in Kd between cetuximab and nimotuzumab is primarily impacted by a 59-fold increase in the kon and a 5.3x increase in the koff of cetuximab, such that cetuximab has greatly enhanced rate of association relative to nimotuzumab, but in contrast to most higher affinity interactions, a shorter duration of interaction (Talavera et al., 2009). Therefore, altering association rate rather than the dissociation rate of scFv domain in CAR design may have a greater impact on T-cell function.
Previous studies have established that a minimum CAR density is required for T-cell activation, below which T-cell activation is abrogated (James et al., 2010). However, sufficiently high antigen expression can mitigate this requirement and achieve CAR-dependent T-cell activation when CAR is expressed at low density (James et al., 2010). The interplay between CAR expression density, antigen density and CAR affinity and impact on CAR+ T cell function were evaluated in a study using high and low affinity HER-specific CARs. This study reported that reduced T-cell function of T cells with low CAR density in response to low antigen density was only apparent when T cells expressed a higher affinity HER2-specific CAR (Turatti et al., 2007). However, when CAR was expressed at higher density, CAR-mediated cytotoxicity was irrespective of affinity or antigen density. The authors attributed the reduced response of high affinity CAR when expressed low density to low HER2 density to a failure to induce serial triggering. Although it has been reported that CARs to do not serially trigger as endogenous TCRs (James et al., 2010), it is possible that this is CAR-specific, and that different transmembrane regions, endodomains, and scFv affinity may impact ability to serially trigger. The inventors did not observe any defect in Cetux-CAR+ T cells in initial response to low antigen density, however, the level of CAR expression culled out through repetitive stimulation on EGFR+ aAPC may select for an optimum CAR density, with T cells expressing suboptimal levels of CAR failing to expand and thus falling out of the repertoire. In contrast, the present findings suggest that the lower affinity Nimo-CAR+ T cells demonstrate reduced sensitivity to low antigen expression, but increasing density of Nimo-CAR did not restore Nimo-CAR+ T cell sensitivity to low antigen, thus it is likely controlled by a different mechanism.
Although expression of CAR at low density can reduce sensitivity to antigen, this is not likely to be an optimal strategy selectively target high antigen density in vivo, primarily because CAR expressed at low density demonstrate reduced sensitivity to all levels of antigen, and therefore the potential for reduced anti-tumor activity (James et al., 2010; Weijtens et al., 2000). Additionally, CAR downregulates from the T-cell surface at a constant number of CAR/antigen (James et al., 2010). Thus, T cells expressing CAR at lower density are more susceptible to downregulation below the minimum density to achieve T-cell activation.
In this study, Nimo-CAR, predicted to have lower affinity due to reduced association rate of binding relative to Cetux-CAR, mediated T-cell activation that directly correlated with EGFR expression density and reduced activity in response to normal renal cells with low EGFR density. Additionally, Nimo-CAR+ T cells showed enhanced proliferation and reduced CAR downregulation relative to Cetux-CAR+ T cells. Targeting EGFR on glioblastoma by Nimo-CAR+ T cells has the potential to mediate anti-tumor activity while reducing the potential for on-target, off-tissue toxicity.
Some tumors, such as glioblastoma, overexpress EGFR at a higher density relative to normal tissue expression and hypothesized that altering scFv domain of CAR to reduce binding affinity could preferentially activate T cells in the presence of high EGFR density but reduce T cell activity in the presence of low EGFR density. Cetux-CAR and Nimo-CAR bind overlapping epitopes on EGFR with distinct affinities and binding kinetics, such that Cetux-CAR has a 5.3-fold lower dissociation constant, and therefore higher affinity, characterized by a 59-fold higher rate of association. In vitro studies also demonstrated Cetux-CAR had reduced proliferation in response to antigen in the absence of exogenous cytokine, enhanced downregulation of CAR that was dependent on scFv domain of CAR binding EGFR and density of EGFR, and impaired cytokine production in response to re-challenge with antigen.
Evaluation of efficacy of Cetux-CAR+ and Nimo-CAR+ T cells in treatment of intracranial glioma xenografts supported in vitro conclusions by demonstrating that both Cetux-CAR+ T cells and Nimo-CAR+ T cells can mediate anti-tumor activity against U87med, expressing intermediate EGFR density, but only Cetux-CAR+ T cells demonstrated anti-tumor activity against U87 with endogenously low EGFR density.
Some studies have demonstrated that higher affinity TCR interactions can result in superior in vivo activity (Nauerth et al., 2013; Zhong et al., 2013); however, it has been demonstrated that in vitro T-cell activity does not always mirror in vivo efficacy (Chervin et al., 2013; Janicki et al., 2008). High affinity T cells with high potency in vitro have been shown to have attenuated responses in vivo, characterized by decreased signaling, expansion and T-cell mediated function (Corse et al., 2010). Similarly, low affinity interaction have been demonstrated to have curtailed T-cell expansion in vivo, resulting in fewer T cells present at each stage of the immune response (Zehn et al., 2009). Models assessing the role of TCR affinity in anti-tumor efficacy have demonstrated that high affinity TCR interactions have impaired anti-tumor function, characterized by decreased presence in tumor and impaired cytolytic function (Chervin et al., 2013; Engels et al., 2012; Janicki et al., 2008). Thus, it has been suggested that T cells with intermediate affinity may better control tumor growth relative to high affinity T cells (Corse et al., 2010; Janicki et al., 2008). Combining these observation with in vitro observations that Cetux-CAR+ T cells have decreased proliferative capacity when stimulated in the absence of exogenous cytokine, enhanced CAR downregulation following engagement with antigen, and reduced ability to respond to re-challenge with antigen, Cetux-CAR+ T cells may have reduced anti-tumor efficacy in vivo. The inventors did not observe impaired anti-tumor efficacy relative to Nimo-CAR+ T cells; however, the fate of CAR+ after intratumoral injection was not followed, and therefore, differences in vivo expansion were not assessed. Intratumoral injection of CAR+ T cells was chosen to avoid the confounding variable of disparate abilities of CAR+ T cells to home to tumor when evaluating anti-tumor activity; however, it is possible that Cetux-CAR+ T cells may have reduced tumor infiltration due to retention in tumor periphery.
Nimo-CAR+ T cell treatment did not significantly reduce tumor burden or improve the survival of mice relative to untreated mice in response to low EGFR density on U87, which is about 2-fold higher than EGFR density measured on normal renal epithelial cells (
Unexpectedly, Cetux-CAR+ T cells showed significant toxicity within 7 days of T cell treatment, with 6/14 mice dying within 7 days of a T-cell injection. Previously, an EGFR-specific CAR has been reported to have no detectable in vivo toxicity by measurement of liver enzymes 48 hours after T-cell infusion in mice bearing no tumor (Zhou et al., 2013). Because this CAR was derived from a murine antibody, it is unlikely that the EGFR-specific CAR would recognize murine EGFR on normal tissue. Additionally, measurement of toxicity in the absence of antigen does not replicate physiologic CAR+ T-cell activation in patients expressing antigen on tumors, as these cells will activate, proliferate, and produce cytokine in response to tumor lysis, which could all contribute to measureable toxicity (Barrett et al., 2014). In fact, in the present study, treatment of mice with Cetux-CAR+ T cells bearing low antigen tumor or no tumor did not result in detectable toxicity (
Because cetuximab does not recognize murine EGFR, on-target, off-tissue toxicity is not likely a cause of Cetux-CAR+ T cell-related toxicity (Mutsaers et al., 2009). Possible mechanisms for Cetux-CAR mediated toxicity in this model include cytokine-related toxicity resulting from T cell activation or possibly enhanced avidity of Cetux-CAR due to clustering, immune synapse formation or association with T-cell cytoskeleton that reduces antigenic-specificity, as has been described in the contribution of CD8 coreceptor binding to enhance avidity of high affinity TCRs, resulting in loss of specificity (Stone et al., 2013).
In summary, Nimo-CAR+ T cells demonstrate anti-tumor activity and improved survival comparable to higher affinity Cetux-CAR+ T cells in an intracranial orthotopic xenograft model, without T-cell related toxicity associated with Cetux-CAR+ T cells. In contrast, Cetux-CAR+ T cells, but not Nimo-CAR+ T cells demonstrate anti-tumor activity against tumors with low EGFR density. These findings are consistent with in vitro observations that Nimo-CAR+ T cells have reduced activity in response to low EGFR density.
Methods developed to achieve safety of CAR+ T cells can be categorized into three main strategies: (i) restricting CAR+ T cells to tumor tissue, (ii) limiting CAR expression/T cell persistence, and (iii) restricting CAR-mediated T cell activation to tumor (
Strategies to temporally limit CAR+ T cell presence include suicide gene modification of T cells, such as expression of CAR as a transient RNA species, and introduction of iCaspase9 suicide switch, which is specifically activated by a chemical inducer of dimerization (CID) to result in T-cell death (Zhao et al., 2010; DiStassi et al., 2011; Budde et al., 2013; Barrett et al., 2011; Barrett et al., 2013). Both methods have high penetrance and result in almost complete abrogation of CAR+ T cells, either after induction of apoptosis by drug delivery or loss of RNA transgene expression over time. Because both strategies permanently ablate CAR+ T cells, they also limit therapeutic efficacy against tumor while protecting normal tissue. One limitation of these strategies is that before CAR reduction or T cell ablation, potent activity against normal cells exists, and there is no short-term limitation of toxicity. Serious adverse events from T-cell therapy can progress rapidly from onset of clinical symptoms, therefore, it is desirable to have a strategy to protect normal tissue from the moment of CAR+ T-cell infusion (Grupp et al., 2013; Porter et al., 2011).
Dual-specific, complementary CARs have achieved selective activation in response to co-expression of two antigens mutually expressed only on tumor by dissociating signaling domains and expressing two chimeric receptors with two specificities. In this strategy, one specificity is fused to CD3ζ to express a first generation CAR and a different, complementary specificity is fused to costimulation endodomains, termed a chimeric costimulation receptor (CCR), such that full activation and T-cell function is only attained with simultaneous engagement of CAR and CCR by co-expression of by antigens (Wilkie et al., 2014; Lanitis et al., 2013; Kloss et al., 2013). This approach has been piloted with different pairs of CAR and CCR with redirected specificities towards HER2 and MUC1 for breast cancer, PSMA and PSCA for prostate cancer and mesothelin and α-folate receptor for ovarian cancer treatment. Early studies have demonstrated that T-cell activation and lytic function can occur against single antigen expressing targets via first generation CAR expression in the absence of CCR activation. Although this cytotoxicity is lower than that observed with second generation CARs, there is still some residual risk of CAR targeting normal tissue expressing single antigen (Wilkie et al., 2014; Lanitis et al., 2013). One strategy to overcome this limitation is to develop a first generation CAR with suboptimal affinity, such that it barely renders T cell function when activated by single antigen and toxicity is only rescued by ligation of CCR (Kloss et al., 2013). However, this strategy functions by blunting T cell sensitivity to tumor antigen. While this strategy prevents recognition and targeting of single antigen expression tissue, thereby potentially reduced normal tissue toxicity, it also reduces anti-tumor activity. Additionally, the requirement for two antigens to be expressed for efficient T-cell activation and tumor elimination reduces the fraction of tumor capable of CAR activation and increases the potential for the development of tumor escape variants.
An inhibitory CAR (iCAR) fusing specificity for antigen found only on normal tissue, and not on tumor to PD-1 signaling endodomains is capable of significantly inhibiting T-cell-mediated killing and cytokine production in response to binding normal tissue antigen (Fedorov et al., 2013). Impressively, iCAR inhibition of T-cell function is reversible, and T cells are capable of subsequent functionally productive responses upon encounter with tumor antigen. The success of this strategy is dependent of stoichiometry of CAR, iCAR and both antigens. Therefore, it is reasonable to predict that normal tissue toxicity could occur if iCAR expression or antigen is insufficient in the presence of overwhelming CAR/tumor antigen expression. This stoichiometric parameter must evaluated and tightly control for each set of antigens for this strategy to be successful.
Described herein is a method to control T-cell activation to the site of tumor based on the affinity of the scFv used in CAR design to mitigate activation of CAR+ T cells in response to low density of EGFR on normal tissue while mediating T-cell cytotoxicity in response to high EGFR density on tumor tissue. Advantages of this method are that (i) reduction of normal tissue toxicity is not associated with mitigated activity in response to tumor and (ii) activation/inhibition of T cells does not require recognition of multiple antigens, for which the stoichiometry of expression and binding to relative receptors must be tightly controlled. Additionally, requiring multiple antigens for T cell activation further reduces the proportion of a tumor that will be efficiently targeted. None of the methods to restrict T-cell on-target, off-tissue tissue toxicity are mutually exclusive, and combinations of multiple strategies may provide improve avoidance of normal tissue destruction.
Glioblastoma patients may be an ideal patient population for initial evaluation of safety of T cells specific for EGFR for cancer immunotherapy. EGFR is overexpressed in 40-50% of patients with globlastoma (Parsons et al., 2008; Hu et al., 2013). Additionally, EGFR expression is not reported in normal brain tissue (Yano et al., 2003). Because EGFR is widespread on normal epithelial surfaces, intracavitary delivery of T cells following tumor resection can maximize anti-tumor potential while minimizing the potential for interaction with epithelial surfaces outside of the CNS. Following initial safety evaluation in patients with glioblastoma, it may be possible to extend EGFR-specific CAR+ T cell therapy to other EGFR-expressing malignancies, which include breast, ovarian, lung, head and neck, colorectal, and renal cell carcinoma (Hynes et al., 2005).
Although transient expression of CAR through RNA modification of T cells may result in reduced anti-tumor efficacy due to limited presence of CAR+ T cells, multiple infusions of RNA-modified T cells, particularly with a weighted initial dose, may overcome these potential limitations, as previously demonstrated with CD19 CAR+ T cells modified by RNA transfer in an advanced leukemia murine model (Barrett et al., 2013). While clinical trials with mesothelin-specific CAR transferred by RNA expression have demonstrated the potential for anaphylaxis attributed to the development of IgE antibody responses specific for CAR moieties in response to repeated CAR infusions, a dosing strategy with no more than 10 days between CAR+ T cell infusions and treatment to be completed over a course of 21 days has been proposed to avoid isotype switching of IgG antibodies to IgE antibodies and is currently being evaluated (Maus et al., 2013). Despite these challenges, there are many attractive advantage of RNA modification to express CAR in clinical application. First, RNA-modification of T cells does not involve genomic integration of transgenes, and thus have the potential for less cumbersome processes for regulatory approval, which may shorten the preclinical development period for CAR+ T cell therapy. In addition, generation of CAR-modified T cells by RNA transfer is much quicker than DNA-modification using the Sleeping Beauty transposon/transposase system, resulting in >90% CAR+ T cells in about half of the ex vivo culture time as is required for DNA-modification of T cells. Improving the speed of regulatory approval processes and ex vivo manufacture time could result in getting new CAR+ T cell therapies to the clinic faster, quicken the communication time from bench-to-bedside and back to mediate improved efficiency in fine-tuning these therapies for clinical application.
RNA-modification may also provide a platform to test transiently modified T cells specific to widely expressed normal tissue antigens, such as EGFR, in patients to determine safety profiles of CAR structures prior to evaluating permanently integrated CARs as an additional measure of safety. Because Cetux-CAR demonstrates T-cell activation and lytic activity in response to low EGFR density, DNA-modification of T cells to permanently express Cetux-CAR is not likely to be a viable clinical strategy due to the high risk of normal tissue toxicity. However, initial clinical evaluation of Nimo-CAR+ T cells modified by RNA transfer may determine the capacity of Nimo-CAR+ T cells to mediate normal tissue toxicity with the additional safety feature of transient CAR expression to alleviate concerns of long-term normal tissue toxicity.
While the reduced capacity of Nimo-CAR+ T cells to mediate cytotoxicity against low density EGFR functions to reduce normal tissue toxicity, it also may reduce effectiveness against tumors that express low density EGFR, increasing the potential for outgrowth of tumor escape variants expressing EGFR at low density. In contrast, specific lytic activity of Cetux-CAR+ T cells against all levels of EGFR expression may reduce the risk of outgrowth of low EGFR expressing tumor escape variants, but does so at the expense of potential toxicity against normal tissue with low EGFR expression. In addition, Cetux-CAR+ T cells appear to mediate some degree of T-cell related toxicity independent of targeting normal tissue expressing EGFR, as demonstrated in treatment of intracranial U87 expressing moderate density of EGFR, perhaps due to enhanced cytokine production or induction of local inflammation. The relationship between Cetux-CAR+ and Nimo-CAR+ T cells highlight the balance that must be achieved between safety and efficacy of gene-modified T cell therapies. Choosing which strategy might have better clinical outcome, Cetux-CAR+ T cells with increased risk of toxicity but potential for greater tumor control or Nimo-CAR+ T cells with reduced risk of toxicity, but greater potential for development of tumor escape variants, does not have a simple solution. One potential clinical strategy for coping with this balance may be infusing Nimo-CAR+ T cell modified by DNA for stable control of high EGFR-expressing tumor variants combined with multiple infusions of Cetux-CAR+ T cells modified by RNA to eliminate low EGFR-expressing tumor cells.
The term “chimeric antigen receptors (CARs),” as used herein, may refer to artificial T-cell receptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a tumor associated antigen, for example. In some embodiments, CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins. In some embodiments, one can target malignant B cells by redirecting the specificity of T cells by using a CAR specific for the B-lineage molecule, CD19. In certain embodiments, the spacing of the antigen-recognition domain can be modified to reduce activation-induced cell death. In certain embodiments, CARs can comprise domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10, and/or OX40. In some embodiments, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.
The term “T-cell receptor (TCR)” as used herein refers to a protein receptor on T cells that is composed of a heterodimer of an alpha (α) and beta ( β ) chain, although in some cells the TCR consists of gamma and delta ( γ / δ ) chains. In some embodiments, the TCR may be modified on any cell comprising a TCR, including a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell, for example.
As used herein, the term “antigen” is a molecule capable of being bound by an antibody or T-cell receptor. An antigen may generally be used to induce a humoral immune response and/or a cellular immune response leading to the production of B and/or T lymphocytes.
The terms “tumor-associated antigen” and “cancer cell antigen” are used interchangeably herein. In each case, the terms refer to proteins, glycoproteins or carbohydrates that are specifically or preferentially expressed by cancer cells.
As used herein the phrase “in need thereof” with reference to treating a subject or selectively targeting cells in a subject refers to a subject having a disease condition that could benefit from selective killing of cells expressing a target antigen (or an elevated level of a target antigen). In some aspects, the disease condition may be a cancer that expresses an elevated level of a target antigen relative to non-cancerous cells in the subject. For example, the cancer can be a glioma that expresses an elevated level of EGFR relative to non-cancerous cells in the subject.
As used herein the phrase “effective amount” relative to CAR T-cells, or pharmaceutical compositions comprising CAR T-cells, refers to an amount of CAR T-cells that is sufficient, when administered to a subject, to kill cells that express (or express an elevated level of) a target antigen bound by the CAR.
Embodiments described herein involve generation and identification of nucleic acids encoding an antigen-specific chimeric antigen receptor (CAR) polypeptide. In some embodiments, the CAR is humanized to reduce immunogenicity (hCAR).
In some embodiments, the CAR may recognize an epitope comprised of the shared space between one or more antigens. Pattern recognition receptors, such as Dectin-1, may be used to derive specificity to a carbohydrate antigen. In certain embodiments, the binding region may comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and/or antigen binding fragments thereof. In some embodiments the binding region is an scFv. In another embodiment, a peptide (e.g., a cytokine) that binds to a receptor or cellular target may be included as a possibility or substituted for a scFv region in the binding region of a CAR. Thus, in some embodiments, a CAR may be generated from a plurality of vectors encoding multiple scFv regions and/or other targeting proteins. A complementarity determining region (CDR) is a short amino acid sequence found in the variable domains of antigen receptor (e.g., immunoglobulin and T-cell receptor) proteins that complements an antigen and therefore provides the receptor with its specificity for that particular antigen. Each polypeptide chain of an antigen receptor contains three CDRs (CDR1, CDR2, and CDR3). Since the antigen receptors are typically composed of two polypeptide chains, there are six CDRs for each antigen receptor that can come into contact with the antigen -- each heavy and light chain contains three CDRs. Because most sequence variation associated with immunoglobulins and T-cell receptor selectivity are generally found in the CDRs, these regions are sometimes referred to as hypervariable domains. Among these, CDR3 shows the greatest variability as it is encoded by a recombination of the VJ (VDJ in the case of heavy chain and TCR αβ chain) regions.
A CAR-encoding nucleic acid generated via the embodiments may comprise one or more human genes or gene fragments to enhance cellular immunotherapy for human patients. In some embodiments, a full length CAR cDNA or coding region may be generated via the methods described herein. The antigen binding regions or domain may comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Pat. 7,109,304, incorporated herein by reference. In some embodiments, the scFv comprises an antigen binding domains of a human antigen-specific antibody. In some embodiments, the scFv region is an antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.
The arrangement of the antigen-binding domain of a CAR may be multimeric, such as a diabody or multimers. The multimers can be formed by cross pairing of the variable portions of the light and heavy chains into what may be referred to as a diabody. The hinge portion of the CAR may in some embodiments be shortened or excluded (i.e., generating a CAR that only includes an antigen binding domain, a transmembrane region and an intracellular signaling domain). A multiplicity of hinges may be used with the present embodiments, e.g., as shown in Table 1. In some embodiments, the hinge region may have the first cysteine maintained, or mutated by a proline or a serine substitution, or be truncated up to the first cysteine. The Fc portion may be deleted from scFv used to as an antigen-binding region to generate CARs according to the embodiments. In some embodiments, an antigen-binding region may encode just one of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin. One may also include the hinge, CH2, and CH3 region of a human immunoglobulin that has been modified to improve dimerization and oligermerization. In some embodiments, the hinge portion of may comprise or consist of an 8-14 amino acid peptide (e.g., a 12 AA peptide), a portion of CD8α, or the IgG4 Fc. In some embodiments, the antigen binding domain may be suspended from cell surface using a domain that promotes oligomerization, such as CD8 alpha. In some embodiments, the antigen binding domain may be suspended from cell surface using a domain that is recognized by monoclonal antibody (mAb) clone 2D3 (mAb clone 2D3 described, e.g., in Singh et al., 2008).
The endodomain or intracellular signaling domain of a CAR can generally cause or promote the activation of at least one of the normal effector functions of an immune cell comprising the CAR. For example, the endodomain may promote an effector function of a T cell such as, e.g., cytolytic activity or helper activity including the secretion of cytokines. The effector function in a naive, memory, or memory-type T cell may include antigen-dependent proliferation. The terms “intracellular signaling domain” or “endodomain” refers to the portion of a CAR that can transduce the effector function signal and/or direct the cell to perform a specialized function. While the entire intracellular signaling domain may be included in a CAR, in some cases a truncated portion of an endodomain may be included. Generally, endodomains include truncated endodomains, wherein the truncated endodomain retains the ability to transduce an effector function signal in a cell.
In some embodiments, an endodomain comprises the zeta chain of the T-cell receptor or any of its homologs (e.g., eta, delta, gamma, or epsilon), MB1 chain, B29, Fc RIII, Fc RI, and combinations of signaling molecules, such as CD3ζ and CD28, CD27, 4-1BB, DAP-10, OX40, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins can be used, such as FcγRIII and FcεRI. Examples of these alternative transmembrane and intracellular domains can be found, e.g., Gross et al. (1992), Stancovski et al. (1993), Moritz et al. (1994), Hwu et al. (1995), Weijtens et al. (1996), and Hekele et al. (1996), which are incorporated herein by reference in their entireties. In some embodiments, an endodomain may comprise the human CD3ζ intracellular domain.
The antigen-specific extracellular domain and the intracellular signaling-domain are preferably linked by a transmembrane domain. Transmembrane domains that may be included in a CAR include, e.g., the human IgG4 Fc hinge and Fc regions, the human CD4 transmembrane domain, the human CD28 transmembrane domain, the transmembrane human CD3ζ domain, or a cysteine mutated human CD3ζ domain, or a transmembrane domains from a human transmembrane signaling protein such as, e.g., the CD16 and CD8 and erythropoietin receptor. Examples of transmembrane domains are provided, e.g., in Table 1.
In some embodiments, the endodomain comprises a sequence encoding a costimulatory receptor such as, e.g., a modified CD28 intracellular signaling domain, or a CD28, CD27, OX-40 (CD134), DAP10, or 4-1BB (CD137) costimulatory receptor. In some embodiments, both a primary signal initiated by CD3 ζ, an additional signal provided by a human costimulatory receptor may be included in a CAR to more effectively activate transformed T cells, which may help improve in vivo persistence and the therapeutic success of the adoptive immunotherapy. As noted in Table 1, the endodomain or intracellular receptor signaling domain may comprise the zeta chain of CD3 alone or in combination with an Fc γ RIII costimulatory signaling domains such as, e.g., CD28, CD27, DAP10, CD137, OX40, CD2, 4-1BB. In some embodiments, the endodomain comprises part or all of one or more of TCR zeta chain, CD28, CD27, OX40/CD134, 4-1BB/CD137, Fc ε RI γ, ICOS/CD278, IL-2Rbeta/CD122, IL-2Ralpha/CD132, DAP10, DAP12, and CD40. In some embodiments, 1, 2, 3, 4 or more cytoplasmic domains may be included in an endodomain. For example, in some CARs it has been observed that at least two or three signaling domains fused together can result in an additive or synergistic effect.
In some aspects, an isolated nucleic acid segment and expression cassette including DNA sequences that encode a CAR may be generated. A variety of vectors may be used. In some preferred embodiments, the vector may allow for delivery of the DNA encoding a CAR to immune such as T cells. CAR expression may be under the control of regulated eukaryotic promoter such as, e.g., the MNDU3 promoter, CMV promoter, EFlalpha promoter, or Ubiquitin promoter. Also, the vector may contain a selectable marker, if for no other reason, to facilitate their manipulation in vitro. In some embodiments, the CAR can be expressed from mRNA in vitro transcribed from a DNA template.
Chimeric antigen receptor molecules are recombinant and are distinguished by their ability to both bind antigen and transduce activation signals via immunoreceptor activation motifs (ITAM’s) present in their cytoplasmic tails. Receptor constructs utilizing an antigen-binding moiety (for example, generated from single chain antibodies (scFv)) afford the additional advantage of being “universal” in that they can bind native antigen on the target cell surface in an HLA-independent fashion. For example, a scFv constructs may be fused to sequences coding for the intracellular portion of the CD3 complex’s zeta chain (ζ), the Fc receptor gamma chain, and sky tyrosine kinase (Eshhar et al., 1993; Fitzer-Attas et al., 1998). Re-directed T cell effector mechanisms including tumor recognition and lysis by CTL have been documented in several murine and human antigen-scFv: ζ systems (Eshhar et al., 1997; Altenschmidt et al., 1997; Brocker et al., 1998).
The antigen binding region may, e.g., be from a human or non-human scFv. One possible problem with using non-human antigen binding regions, such as murine monoclonal antibodies, is reduced human effector functionality and a reduced ability to penetrate into tumor masses. Furthermore, non-human monoclonal antibodies can be recognized by the human host as a foreign protein, and therefore, repeated injections of such foreign antibodies might lead to the induction of immune responses leading to harmful hypersensitivity reactions. For murine-based monoclonal antibodies, this effect has been referred to as a Human AntiMouse Antibody (HAMA) response. In some embodiments, inclusion of human antibody or scFv sequences in a CAR may result in little or no HAMA response as compared to some murine antibodies. Similarly, the inclusion of human sequences in a CAR may be used to reduce or avoid the risk of immune-mediated recognition or elimination by endogenous T cells that reside in the recipient and might recognize processed antigen based on HLA.
In some embodiments, the CAR comprises: a) an intracellular signaling domain, b) a transmembrane domain, c) a hinge region, and d) an extracellular domain comprising an antigen binding region. In some embodiments, the intracellular signaling domain and the transmembrane domain are encoded with the endodomain by a single vector that can be fused (e.g., via transposon-directed homologous recombination) with a vector encoding a hinge region and a vector encoding an antigen binding region. In other embodiments, the intracellular signaling region and the transmembrane region may be encoded by two separate vectors that are fused (e.g., via transposon-directed homologous recombination).
In some embodiments, the antigen-specific portion of a CAR, also referred to as an extracellular domain comprising an antigen binding region, selectively targets a tumor associated antigen. A tumor associated antigen may be of any kind so long as it is expressed on the cell surface of tumor cells. Examples of tumor associated antigens that may be targeted with CARs generated via the embodiments include, e.g., CD19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, Dectin-1, and so forth. In some embodiments that antigen specific portion of the CAR is a scFv. Examples of tumor-targeting scFv are provided in Table 1. In some embodiments, a CAR may be co-expressed with a membrane-bound cytokine, e.g., to improve persistence when there is a low amount of tumor-associated antigen. For example, a CAR can be co-expressed with membrane-bound IL-15.
In some embodiments, an intracellular tumor associated antigen such as, e.g., HA-1, survivin, WT1, and p53 may be targeted with a CAR. This may be achieved by a CAR expressed on a universal T cell that recognizes the processed peptide described from the intracellular tumor associated antigen in the context of HLA. In addition, the universal T cell may be genetically modified to express a T-cell receptor pairing that recognizes the intracellular processed tumor associated antigen in the context of HLA.
Additional examples of target antigens for use according to the embodiments include, without limitation CD19, CD20, ROR1, CD22carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, meothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, GP240, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, IGF-1R, BAFF-R, TACI, APRIL, Fn14, ERBB2 or ERBB35T4, MUC-1, and EGFR. In certain specific aspects, a selected CAR of the embodiments comprises CDRs or the antigen binding portions of nimotuzumab, such as set forth in SEQ ID NOs: 1-2. For example, the CAR can comprise VL CDR1 RSSQNIVHSNGNTYLD (SEQ ID NO: 5); VL CDR2 KVSNRFS (SEQ ID NO: 6); VL CDR3 FQYSHVPWT (SEQ ID NO: 7); VH CDR1 NYYIY (SEQ ID NO: 8); VH CDR2 GINPTSGGSNFNEKFKT (SEQ ID NO: 9) and VH CDR3 QGLWFDSDGRGFDF (SEQ ID NO: 10), see e.g., Mateo et al., 1997, incorporated herein by reference. In further specific aspects, a CAR of the embodiments comprises CDRs or the antigen binding portions of cetuximab, such as set forth in SEQ ID NOs: 3-4. For example, the CAR can comprise VL CDR1 RASQSIGTNIH (SEQ ID NO: 11); VL CDR2 ASEIS (SEQ ID NO: 12); VL CDR3 QQNNNWPTT (SEQ ID NO: 13); VH CDR1 NYGVH (SEQ ID NO: 14); VH CDR2 VIWSGGNTDYNTPFTS (SEQ ID NO: 15) and VH CDR3 ALTYYDYEFAY (SEQ ID NO: 16), see e.g., International (PCT) Pat. Publn. WO2012100346, incorporated herein by reference.
As discussed supra, in some aspects, a selected CAR that binds to an antigen and has a Kd of between about 2 nM and about 500 nM relative to the antigen, wherein a T-cell comprising the selected CAR exhibits cytotoxicity to a target cell (e.g., a cancer cell) expressing the antigen. For example, in some aspects, the CAR has a Kd of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nM or greater relative to the antigen and a T-cell comprising the selected CAR exhibits cytotoxicity to a target cell expressing the antigen. In still further aspects, the CAR has a Kd of between about 5 nM and about 450, 400, 350, 300, 250, 200, 150, 100 or 50 nM relative to the antigen. In still further aspects, the CAR has a Kd of between about 5 nM and 500 nM, 5 nM and 200 nM, 5 nM and 100 nM, or 5 nM and 50 nM relative to the antigen and a T-cell comprising the selected CAR exhibits cytotoxicity to a target cell expressing the antigen.
In some aspects, a selected CAR of the embodiments can bind to 2, 3, 4 or more antigen molecules per CAR molecule and a T-cell comprising the selected CAR exhibits cytotoxicity to a target cell (e.g., a cancer cell) expressing the antigen. In some aspects, each to the antigen binding domains of a selected CAR has a Kd of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nM or greater relative to the antigen and a T-cell comprising the selected CAR exhibits cytotoxicity to a target cell expressing the antigen. In still further aspects, each to the antigen binding domains of a selected CAR has a Kd of between about 5 nM and about 450, 400, 350, 300, 250, 200, 150, 100 or 50 nM relative to the antigen and a T-cell comprising the selected CAR exhibits cytotoxicity to a target cell expressing the antigen. In still further aspects, each to the antigen binding domains of a selected CAR has a Kd of between about 5 nM and 500 nM, 5 nM and 200 nM, 5 nM and 100 nM, or 5 nM and 50 nM relative to the antigen and a T-cell comprising the selected CAR exhibits cytotoxicity to a target cell expressing the antigen.
The pathogen recognized by a CAR may be essentially any kind of pathogen, but in some embodiments the pathogen is a fungus, bacteria, or virus. Exemplary viral pathogens include those of the families of Adenoviridae, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV), JC virus, BK virus, HSV, HHV family of viruses, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, chickenpox, ebola, and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella. In some embodiments the pathogen receptor Dectin-1 may be used to generate a CAR that recognizes the carbohydrate structure on the cell wall of fungi such as Aspergillus. In another embodiment, CARs can be made based on an antibody recognizing viral determinants (e.g., the glycoproteins from CMV and Ebola) to interrupt viral infections and pathology.
In some embodiments, naked DNA or a suitable vector encoding a CAR can be introduced into a subject’s T cells (e.g., T cells obtained from a human patient with cancer or other disease). Methods of stably transfecting T cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor of the embodiments contained in a plasmid expression vector in proper orientation for expression. In some embodiments, the use of naked DNA may reduce the time required to produce T cells expressing a CAR generated via methods of the embodiments.
Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into T cells. Generally, a vector encoding a CAR that is used for transfecting a T cell from a subject should generally be non-replicating in the subject’s T cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain viability of the cell. Illustrative vectors include the pFB-neo vectors (STRATAGENE®) as well as vectors based on HIV, SV40, EBV, HSV, or BPV.
Once it is established that the transfected or transduced T cell is capable of expressing a CAR as a surface membrane protein with the desired regulation and at a desired level, it can be determined whether the chimeric receptor is functional in the host cell to provide for the desired signal induction. Subsequently, the transduced T cells may be reintroduced or administered to the subject to activate anti-tumor responses in the subject. To facilitate administration, the transduced T cells may be made into a pharmaceutical composition or made into an implant appropriate for administration in vivo, with appropriate carriers or diluents, which are preferably pharmaceutically acceptable. The means of making such a composition or an implant have been described in the art (see, for instance, Remington’s Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980)). Where appropriate, transduced T cells expressing a CAR can be formulated into a preparation in semisolid or liquid form, such as a capsule, solution, injection, inhalant, or aerosol, in the usual ways for their respective route of administration. Means known in the art can be utilized to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed-release of the composition. Generally, a pharmaceutically acceptable form is preferably employed that does not ineffectuate the cells expressing the chimeric receptor. Thus, desirably the transduced T cells can be made into a pharmaceutical composition containing a balanced salt solution such as Hanks’ balanced salt solution, or normal saline.
In certain aspects, the embodiments described herein include a method of making and/or expanding the antigen-specific redirected T cells that comprises transfecting T cells with an expression vector containing a DNA construct encoding the hCAR, then, optionally, stimulating the cells with antigen positive cells, recombinant antigen, or an antibody to the receptor to cause the cells to proliferate.
In another aspect, a method is provided of stably transfecting and redirecting T cells by electroporation, or other non-viral gene transfer (such as, but not limited to sonoporation) using naked DNA. Most investigators have used viral vectors to carry heterologous genes into T cells. By using naked DNA, the time required to produce redirected T cells can be reduced. “Naked DNA” means DNA encoding a chimeric T-cell receptor (cTCR) contained in an expression cassette or vector in proper orientation for expression. An electroporation method according to the embodiments produces stable transfectants that express and carry on their surfaces the chimeric TCR (cTCR).
In certain aspects, the T cells are primary human T cells, such as T cells derived from human peripheral blood mononuclear cells (PBMC), PBMC collected after stimulation with G-CSF, bone marrow, or umbilical cord blood. Conditions include the use of mRNA and DNA and electroporation. Following transfection the cells may be immediately infused or may be stored. In certain aspects, following transfection, the cells may be propagated for days, weeks, or months ex vivo as a bulk population within about 1, 2, 3, 4, 5 days or more following gene transfer into cells. In a further aspect, following transfection, the transfectants are cloned and a clone demonstrating presence of a single integrated or episomally maintained expression cassette or plasmid, and expression of the chimeric receptor is expanded ex vivo. The clone selected for expansion demonstrates the capacity to specifically recognize and lyse CD19 expressing target cells. The recombinant T cells may be expanded by stimulation with IL-2, or other cytokines that bind the common gamma-chain (e.g., IL-7, IL-12, IL-15, IL-21, and others). The recombinant T cells may be expanded by stimulation with artificial antigen presenting cells. The recombinant T cells may be expanded on artificial antigen presenting cell or with an antibody, such as OKT3, which cross links CD3 on the T cell surface. Subsets of the recombinant T cells may be deleted on artificial antigen presenting cell or with an antibody, such as Campath, which binds CD52 on the T cell surface. In a further aspect, the genetically modified cells may be cryopreserved.
T-cell propagation (survival) after infusion may be assessed by: (i) q-PCR using primers specific for the CAR; (ii) flow cytometry using an antibody specific for the CAR; and/or (iii) soluble TAA.
Embodiments described hereinalso concern the targeting of a B-cell malignancy or disorder including B cells, with the cell-surface epitope being CD19-specific using a redirected immune T cell. Malignant B cells are an excellent target for redirected T cells, as B cells can serve as immunostimulatory antigen-presenting cells for T cells. Preclinical studies that support the anti-tumor activity of adoptive therapy with donor-derived CD19-specific T-cells bearing a human or humanized CAR include (i) redirected killing of CD19+ targets, (ii) redirected secretion/expression of cytokines after incubation with CD19+ targets/stimulator cells, and (iii) sustained proliferation after incubation with CD19+ targets/stimulator cells.
In certain embodiments, the CAR cells are delivered to an individual in need thereof, such as an individual that has cancer or an infection. The cells then enhance the individual’s immune system to attack the respective cancer or pathogenic cells. In some cases, the individual is provided with one or more doses of the antigen-specific CAR T-cells. In cases where the individual is provided with two or more doses of the antigen-specific CAR T-cells, the duration between the administrations should be sufficient to allow time for propagation in the individual, and in specific embodiments the duration between doses is 1, 2, 3, 4, 5, 6, 7, or more days.
The source of the allogeneic T cells that are modified to include both a chimeric antigen receptor and that lack functional TCR may be of any kind, but in specific embodiments the cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells, for example. Suitable doses for a therapeutic effect would be at least 105 or between about 105 and about 1010 cells per dose, for example, preferably in a series of dosing cycles. An exemplary dosing regimen consists of four one-week dosing cycles of escalating doses, starting at least at about 105 cells on Day 0, for example increasing incrementally up to a target dose of about 1010 cells within several weeks of initiating an intra-patient dose escalation scheme. Suitable modes of administration include intravenous, subcutaneous, intracavitary (for example by reservoir-access device), intraperitoneal, and direct injection into a tumor mass.
A pharmaceutical composition of the embodiments can be used alone or in combination with other well-established agents useful for treating cancer. Whether delivered alone or in combination with other agents, a pharmaceutical composition of the embodiments can be delivered via various routes and to various sites in a mammalian, particularly human, body to achieve a particular effect. One skilled in the art will recognize that, although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route.
A composition of the embodiments can be provided in unit dosage form wherein each dosage unit, e.g., an injection, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition of the embodiments, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the novel unit dosage forms of the embodiments depend on the particular pharmacodynamics associated with the pharmaceutical composition in the particular subject.
Desirably an effective amount or sufficient number of the isolated transduced T cells is present in the composition and introduced into the subject such that long-term, specific, anti-tumor responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment. Desirably, the amount of transduced T cells reintroduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the transduced T cells are not present.
Accordingly, the amount of transduced T cells administered should take into account the route of administration and should be such that a sufficient number of the transduced T cells will be introduced so as to achieve the desired therapeutic response. Furthermore, the amounts of each active agent included in the compositions described herein (e.g., the amount per each cell to be contacted or the amount per certain body weight) can vary in different applications. In general, the concentration of transduced T cells desirably should be sufficient to provide in the subject being treated at least from about 1 × 106 to about 1 × 109 transduced T cells, even more desirably, from about 1 × 107 to about 5 × 108 transduced T cells, although any suitable amount can be utilized either above, e.g., greater than 5 × 108 cells, or below, e.g., less than 1 × 107 cells. The dosing schedule can be based on well-established cell-based therapies (see, e.g., Topalian and Rosenberg, 1987; U.S. Pat. No. 4,690,915), or an alternate continuous infusion strategy can be employed.
These values provide general guidance of the range of transduced T cells to be utilized by the practitioner upon optimizing the methods of the embodiments. The recitation herein of such ranges by no means precludes the use of a higher or lower amount of a component, as might be warranted in a particular application. For example, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in CAR-expressing cells (e.g., CAR binding affinity to a target antigen). One skilled in the art readily can make any necessary adjustments in accordance with the exigencies of the particular situation.
In some cases, APCs are useful in preparing CAR-based therapeutic compositions and cell therapy products. APCs for use according to the embodiments include but arte not milted to dendritic cells, macrophages and artificial antigen presenting cells. For general guidance regarding the preparation and use of antigen-presenting systems, see, e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Pat. Application Publication Nos. 2009/0017000 and 2009/0004142; and International Publication No. WO2007/103009).
APCs may be used to expand T Cells expressing a CAR. During encounter with tumor antigen, the signals delivered to T cells by antigen-presenting cells can affect T-cell programming and their subsequent therapeutic efficacy. This has stimulated efforts to develop artificial antigen-presenting cells that allow optimal control over the signals provided to T cells (Turtle et al., 2010). In addition to antibody or antigen of interest, the APC systems may also comprise at least one exogenous assisting molecule. Any suitable number and combination of assisting molecules may be employed. The assisting molecule may be selected from assisting molecules such as co-stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD70 and B7.1 (also called B7 or CD80), which can bind to CD28 and/or CTLA-4 molecules on the surface of T cells, thereby affecting, e.g., T-cell expansion, Thl differentiation, short-term T-cell survival, and cytokine secretion such as interleukin (IL)-2 (see Kim et al., 2004). Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), that promote, for example, cell-to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for selection, cloning, preparation, and expression of exemplary assisting molecules, including co-stimulatory molecules and adhesion molecules, are exemplified in, e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001.
Cells selected to become aAPCs, preferably have deficiencies in intracellular antigen-processing, intracellular peptide trafficking, and/or intracellular MHC Class I or Class II molecule-peptide loading, or are poikilothermic (i.e., less sensitive to temperature challenge than mammalian cell lines), or possess both deficiencies and poikilothermic properties. Preferably, cells selected to become aAPCs also lack the ability to express at least one endogenous counterpart (e.g., endogenous MHC Class I or Class II molecule and/or endogenous assisting molecules as described above) to the exogenous MHC Class I or Class II molecule and assisting molecule components that are introduced into the cells. Furthermore, aAPCs preferably retain the deficiencies and poikilothermic properties that were possessed by the cells prior to their modification to generate the aAPCs. Exemplary aAPCs either constitute or are derived from a transporter associated with antigen processing (TAP)-deficient cell line, such as an insect cell line. An exemplary poikilothermic insect cells line is a Drosophila cell line, such as a Schneider 2 cell line (e.g., Schneider, J.m 1972). Illustrative methods for the preparation, growth, and culture of Schneider 2 cells, are provided in U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001.
APCs may be subjected to a freeze-thaw cycle. For example, APCs may be frozen by contacting a suitable receptacle containing the APCs with an appropriate amount of liquid nitrogen, solid carbon dioxide (dry ice), or similar low-temperature material, such that freezing occurs rapidly. The frozen APCs are then thawed, either by removal of the APCs from the low-temperature material and exposure to ambient room temperature conditions, or by a facilitated thawing process in which a lukewarm water bath or warm hand is employed to facilitate a shorter thawing time. Additionally, APCs may be frozen and stored for an extended period of time prior to thawing. Frozen APCs may also be thawed and then lyophilized before further use. Preservatives that might detrimentally impact the freeze-thaw procedures, such as dimethyl sulfoxide (DMSO), polyethylene glycols (PEGs), and other preservatives, may be advantageously absent from media containing APCs that undergo the freeze-thaw cycle, or are essentially removed, such as by transfer of APCs to media that is essentially devoid of such preservatives.
In other embodiments, xenogenic nucleic acid and nucleic acid endogenous to the aAPCs may be inactivated by crosslinking, so that essentially no cell growth, replication or expression of nucleic acid occurs after the inactivation. For example, aAPCs may be inactivated at a point subsequent to the expression of exogenous MHC and assisting molecules, presentation of such molecules on the surface of the aAPCs, and loading of presented MHC molecules with selected peptide or peptides. Accordingly, such inactivated and selected peptide loaded aAPCs, while rendered essentially incapable of proliferating or replicating, may retain selected peptide presentation function. The crosslinking can also result in aAPCS that are essentially free of contaminating microorganisms, such as bacteria and viruses, without substantially decreasing the antigen-presenting cell function of the aAPCs. Thus crosslinking can be used to maintain the important APC functions of aAPCs while helping to alleviate concerns about safety of a cell therapy product developed using the aAPCs. For methods related to crosslinking and aAPCs, see for example, U.S. Pat. Application Publication No. 20090017000, which is incorporated herein by reference.
Any of the compositions described herein may be comprised in a kit. In some embodiments, allogeneic CAR T-cells are provided in the kit, which also may include reagents suitable for expanding the cells, such as media, antigen presenting cells (e.g., aAPCs), growth factors, antibodies (e.g., for sorting or characterizing CAR T-cells) and/or plasmids encoding CARs or transposase.
In a non-limiting example, a chimeric receptor expression construct, one or more reagents to generate a chimeric receptor expression construct, cells for transfection of the expression construct, and/or one or more instruments to obtain allogeneic cells for transfection of the expression construct (such an instrument may be a syringe, pipette, forceps, and/or any such medically approved apparatus).
In some embodiments, an expression construct for eliminating endogenous TCR α/β expression, one or more reagents to generate the construct, and/or CAR+ T cells are provided in the kit. In some embodiments, there includes expression constructs that encode zinc finger nuclease(s).
In some aspects, the kit comprises reagents or apparatuses for electroporation of cells.
The kits may comprise one or more suitably aliquoted compositions of the embodiments or reagents to generate compositions of the embodiments. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits may include at least one vial, test tube, flask, bottle, syringe, or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third, or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the embodiments also will typically include a means for containing the chimeric receptor construct and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained, for example.
The following specific and non-limiting examples are to be construed as merely illustrative, and do not limit the present disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
Cetuximab-derived CAR transposon. Cetuximab-derived CAR is composed of the following: a signal peptide from human GMCSFR2 signal peptide (amino acid 1-22; NP_758452.1), variable light chain of cetuximab (PDB:1YY9_C) whitlow linker (AAE37780.1), variable heavy chain of cetuximab (PDB:1YY9_D), human IgG4 (amino acids 161-389, AAG00912.1), human CD28 transmembrane and signaling domains (amino acids 153-220, NP_006130), and human CD3-ζ intracellular domain (amino acids 52 through 164, NP_932170.1). Sequence of GMCSFR2, variable light chain, whitlow linker, variable heavy chain and partial IgG4 were human codon optimized and generated by GeneART (Regensburg, Germany) as 0700310/pMK. Previously described CD19CD28mZ(CoOp)/pSBSO under control of human elongation factor 1-alpha (HEF1α) promoter was selected as backbone for SB transposon. 0700310/pMK and previously described CD19CD28mZ/pSBSO (93, 94) underwent double digestion with NheI and XmnI restriction enzymes. CAR insert and transposon backbone were identified as DNA fragments of 1.3 kb and 5.2 kb, respectively, by agarose gel electrophoresis in a 0.8% agarose gel run at 150 volts for 45 minutes and stained with ethidium bromide for visualization under ultraviolet light exposure. Bands were excised and purified (Qiaquick Gel Extraction kit, Qiagen, Valencia, CA), then ligated using T4 DNA ligase (Promega, Madison, WI) at a molar ratio of insert to backbone of 3:1. Heat shock transformation of TOP10 chemically competent bacteria (Invitrogen, Grand Island, NY) and selection on kanamycin-containing agar plates cultured at 37° C. for 12-16 hours identified bacteria clones positive for transposon backbone. Six clones were selected for mini-culture in TB media with kanamycin selection at 37° C. for 8 hours. Preparation of DNA from mini-cultures was done via MiniPrep kit (Qiagen) and subsequent analytical digestion with restriction enzymes and analysis of fragment size by agarose gel electrophoresis identified clones positive for CetuxCD28mZ (CoOp)/pSBSO (
Nimotuzumab-derived CAR transposon. Nimotuzumab-derived CAR is composed of the following: a signal peptide from human GMCSFR2 signal peptide (amino acids 1-19, NP_001155003.1), variable light chain of nimotuzumab (PDB:3GKW_L) whitlow linker (GenBank: AAE37780.1), variable heavy chain of nimotuzumab (PDB:3GKW_H), human IgG4 (amino acids 161-389, AAG00912.1), human CD28 transmembrane and signaling domains (amino acids 153-220, NP_006130), and human CD3-ζ intracellular domain (amino acids 52 through 164, NP_932170.1). Sequence of GMCSFR2, variable light chain, whitlow linker, variable heavy chain and partial IgG4 were human codon optimized and generated by GeneART as 0841503/pMK. 08541503/pMK and previously described CD19CD28mZ/pSBSO (Singh et al., 2013; Singh et al., 2008) underwent double digestion with NheI and XmnI restriction enzymes, ligation, transformation, large scale amplification and purification of plasmid NimoCD28mZ(CoOp)/pSBSO (
SB11 transposase. The hyperactive SB11 transposase under control of CMV promoter (Kan-CMV-SB11) was used as previously described (Singh et al., 2008; Davies et at., 2010).
pGEM/GFP/A64. GFP under control of of a T7 promoter followed by 64 A-T base pairs and a SpeI site was use to in vitro transcribe GFP RNA. The cloning of pGEM/GFP/A64 has been previously described (Boczkowski et al., 2000).
Cetuximab-derived CAR/pGEM-A64. Cetuximab-derived CAR was cloned into an intermediate vector, pSBSO-MCS, by NheI and XmnI double digestion of CetuxCD28mZ(CoOp)/pSBSO and CD19CD28mZ(CoOp)/pSBSO-MCS. Cetux-CAR insert and pSBSO-MCS backbone were isolated by extraction from agarose gel after electrophoresis and ligated, transformed, and amplified on large-scale as described in generation of CetuxCD28mZ(CoOp)/pSBSO. CetuxCD28mZ(CoOp) was cloned into pGEM/GFP/A64 plasmid to place Cetux-CAR under control of a T7 promoter for in vitro transcription of RNA with artificial poly-A tail 64 nucleotides in length. CetuxCD28mZ(CoOp)/pSBSO-MCS was digested with NheI and EcoRV at 37° C. while pGEM/GFP/A64 was sequentially digested with XbaI at 37° C. then SmaI at 25° C. Digested Cetux-CAR insert and pGEM/A64 backbone were separated by electrophoresis in 0.8% agarose gel run at 150 volts for 45 minutes and visualized by ethidium bromide staining and UV light exposure. Fragments were excised from gel and purified by Qiaquick Gel Extraction (Qiagen) and ligated using T4 DNA ligase (Promega) at 3:1 insert to vector molar ratio and incubated at 16° C. overnight. Dam -/- C2925 chemcially competent bacteria (Invitrogen) were transformed by heat shock and cultured overnight at 37° C. on ampicillin-containing agar for selection of clones containing pGEM/A64 backbone. Eight clones were selected for small-scale DNA amplification by inoculation in TB media with ampicillin antibiotic selection and cultured on a shaker at 37° C. for 8 hours. Purification of DNA was performed using MiniPrep kit (Qiagen) and analytical restriction enzyme digest and subsequent electrophoresis determined which clones expressed correct ligation product, CetuxCD28mZ/pGEM-A64 (
Nimotuzumab-derived CAR/pGEM-A64. NimoCD28mZ(CoOp)/pSBSO was digested sequentially with NheI at 37° C. and SfiI at 50° C. while pGEM/GFP/A64 was digested sequentially with XbaI at 37° C. and SfiI at 50° C. NimoCD28mZ(CoOp) was cloned into pGEM/GFP/A64 plasmid to place Nimo-CAR under control of a T7 promoter for in vitro transcription of RNA with artificial poly A tail 64 nucleotides in length. Digested Nimo-CAR insert and pGEM/A64 backbone were separated by electrophoresis in 0.8% agarose gel run at 150 volts for 45 minutes and visualized by ethidium bromide staining and UV light exposure. Fragments were excised from gel and purified by Qiaquick Gel Extractions (Qiagen) and ligated using T4 DNA ligase (Promega) at 3:1 insert to vector molar ratio and incubated at 16° C. overnight. Dam-/- C2925 chemically competent bacteria (Invitrogen) were transformed by heat shock and cultured overnight at 37° C. on ampicillin-containing agar for selection of clones containing pGEM/A64 backbone. Eight clones were selected for small-scale DNA amplification by inoculation in TB media with ampicillin antibiotic selection and cultured on a shaker at 37° C. for 8 hours. Purification of DNA was performed using MiniPrep kit (Qiagen) and analytical restriction enzyme digest and subsequent electrophoresis determined which clones expressed correct ligation product, NimoCD28mZ/pGEM-A64 (
Truncated EGFR transposon. Truncated EGFR was cloned into a SB transposon linked via self-cleavable peptide sequence F2A to a gene for neomycin resistance. A codon-optimized truncated form of human EGFR (accession NP_005219.2) containing only extracellular and transmembrane domains, 0909312 ErbB1/pMK-RQ, was synthesized by GeneArt (Regensburg, Germany). ErbB1/pMK-RQ was digested with NheI and SmaI at 37° C. while tCD19-F2A-Neo/pSBSO was sequentially digested with NheI at 37° C., then NruI at 37° C. with a purification step between (Qiaquick Gel Extraction kit, Qiagen). tEGFR insert and F2A-Neo/pSBSO backbone were separated by gel electrophoresis on 0.8% agarose gel run at 150 volts for 45 minutes. Bands of predicted sizes were isolated (Qiaquick Gel Extraction kit, Qiagen) and ligated with T4 DNA Ligase (Promega) overnight at 16° C. TOP10 chemically competent cells (Invitrogen) were heat-shock transformed with ligation production and cultured overnight on agar containing kanamycin. Five clones were inoculated for small scale DNA amplification by culture in TB containing kanamycin for 8 hours. DNA purification by Mini Prep kit (Qiagen) and subsequent analytical restriction enzyme digest identified clones positive for tErbB1-F2A-Neo/pSBSO (
CAR-L transposon. A previously described 2D3 hybridoma (94) was used to derive the scFv sequence of CAR-L. Briefly, RNA was extracted from hybridoma by RNeasy Mini Kit (Qiagen), according to manufacturer’s instructions. Reverse transcription via Superscript III First Strand kit (Invitrogen) generated a cDNA library. PCR using degenerate primers for the FR1 region amplified mouse variable heavy and light chains, which were subsequently ligated into TOPO TA vector. CAR-L was constructed as a codon optimized sequence, as follows: Following a human GMCSFR signal peptide (amino acid 1-22; NP_758452.1), 2D3-derived scFv was fused to human CD8α extracellular domain (amino acid 136-182; NP_001759.3) and transmembrane and intracellular domains of human CD28 (amino acid 56-123; NP_001230006.1) and terminates in human intracellular domain of CD3ζ (amino acid. 48-163; NP_ 000725.1). The CAR-L protein was synthesized at GeneArt, then excised and ligated into a SB transposon with a self-cleavable 2A peptide fused to a Zeomycin resistance gene, designated CAR-L-2A-Zeo (
All cell lines were maintained in complete media Dulbecco’s modified eagle media (DMEM) (Life Technologies, Grand Island, NY), supplemented with 10% heat inactivated fetal bovine serum (FBS) (HyClone, ThermoScientific) and 2 mM Glutamax-100 (Gibco, Life Technologies) at 5% CO2, 95% humidity and 37° C., unless otherwise noted. Adherent cell lines were routinely cultured to 70-80% confluency, then passaged 1:10 following dissociation with 0.05% Trypsin-EDTA (Gibco). Identity of cell lines was validated by STR DNA fingerprinting using the AmpF_STR Identifier kit according to manufacturer’s instructions (Applied Biosystems, cat# 4322288). The STR profiles were compared to known ATCC fingerprints (ATCC.org), and to the Cell Line Integrated Molecular Authentication database (CLIMA) version 0.1.200808 (on the world wide web at bioinformatics.istge.it/clima/) (Nucleic Acids Research 37:D925-D932 PMCID: PMC2686526). The STR profiles matched known DNA fingerprints.
OKT3-loaded K562 clone 4. K562 clone 4 was received as a gift from Carl June, M.D. at the University of Pennsylvania and has been previously described (Suhoski et al., 2007; Paulos et al., 2008). Clone 4 are modified to express tCD19, CD86, CD137L, CD64 and a membrane IL15-GFP fusion protein and have been manufactured as a working cell bank for pre-clinical and clinical studies under PACT. K562 clone 4 can be made to express anti-CD3 antibody, OKT3, through binding to the CD64 high affinity Fc receptor. To load OKT3 onto K562 clone 4, cells are cultured overnight in X-VIVO serum free media (Lonza, Cologne, Germany) with 1× 20% N-Acetylcysteine at a density of 1×106 cells/mL. This step clears the Fc receptors for optimal binding of OKT3. The following day, cells are washed and resuspended at 1×106 cells/mL in X-VIVO media with 1× 20% N-Acetylcysteine and irradiated at achieve 100 Gy. Cells are washed and resuspended at 1×106 cells/mL in PBS and OKT3 (eBioscience, San Diego, CA) is added at a concentration of 1 mg/mL and incubated on roller at 4° C. for 30 minutes. Cells are washed again, stained to verify expression of costimulatory molecules and OKT3 by flow cytometry, and cryopreserved.
tEGFR+ K562 clone 27. K562 clone 27 was derived from K562 clone 9, gift from Carl June, M.D. at the University of Pennsylvania. K562 clone 9 was lentivirally transduced, as previously described (Suhoski et al., 2007; Paulos et al., 2008), to express tCD19, CD86, CD137L, and CD64. Clone 27 were modified from clone 9 to stably express a membrane tethered IL15-IL15Rα fusion protein (Hurton, L. V., 2014) via SB transfection, cloned by limiting dilution, and verified to have high expression of all transgenes by flow cytometry. K562 clone 27 was modified to express truncated EGFR by SB transfection of tErbB1-F2A-Neo/pSBSO. K562 clone 27 expressing EGFR were incubated with PE-labeled EGFR-specific antibody (BD Biosciences, Carlsbad, CA, cat# 555997) and anti-PE beads (Miltenyi Biotec, Auburn, CA), then separated from non-labeled cells by flow through a magnetic column (Miltenyi Biotec). Following magnetic selection, tEGFR+ K562 clone 27 were cultured in the presence of 1 mg/mL G418 (Invivogen, San Diego, CA) to maintain high EGFR expression.
EL4, CD19+ EL4, tEGFR+ EL4, and CAR-L+ EL4. EL4 were obtained from ATCC and modified to express tCD19-F2A-Neo, tEGFR-F2A-Neo or CAR-L-F2A-Neo by SB non-viral gene modification. EL4 were electroporated in using Amaxa Nucelofector (Lonza) and primary mouse T cell kit (Lonza) according to manufacturer’s instructions. Briefly, 2×106 EL4 cells were centrifuged at 90xg for 10 minutes and resuspended in 100 uL primary mouse T cell buffer with 3 µg transposon (tCD19-F2A-Neo, tEGFR-F2A-Neo, or CAR-L-2A-Zeo) and 2 ug SB11 transposase and electroporated using Amaxa program X-001. Following electroporation, cells were immediately transferred to pre-warmed and supplemented primary mouse T cell media, supplied with kit (Lonza). The following day, 1 mg/mL G418 was added to select for EL4 cells modified to express transgenes. Expression was verified by flow cytometry 7 days post-modification.
U87, U87low, U87med, and U87high. U87, formally designated U87MG, were obtained from ATCC (Manassas, VA). U87low and U87med were generated to overexpress EGFR by electroporation with tErbB1-F2A-Neo/pSBSO and SB11 using Amaxa Nucleofector and cell line Nucleofector kit T (Lonza, cat#VACA-1002), according to manufacturer’s instructions. Briefly, U87 cells were cultured to 80% confluency, then harvested by dissociation in 0.05% Trypsin-EDTA (Gibco) and counted via trypan blue exclusion using and automated cell counter (Cellometer, Auto T4 Cell Counter, Nexcelcom, Lawrence, MA).1x106 U87 cells were suspended in 100 µL cell line kit T electroporation buffer in the presence of 3 µg of tErbB1-F2A-Neo/pSBSO transposon and 2 µg SB11 transposase, transferred to a cuvette and electroporated via program U-029. Immediately following electroporation, cells were transferred to 6-well plate and allowed to recover in complete DMEM media. The following day, 0.35 mg/mL G418 (Invivogen) was added to select for transgene expression. After propagation to at least 1x106 cells, flow cytometry was performed to assess EGFR expression. Electroporated U87 cells demonstrated modest increase in EGFR expression relative to unmodified U87 and were designated U87low. To generate U87med cells, U87 cells were lipofectamine-transferred with tErbB1-F2A-Neo and SB11 using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. The following day, 0.35 mg/mL G418 was added to culture to select for neomycin resistance. After propagation of cells to significant number, flow cytometrey revealed a two-peak population, with mutually exclusive modest or high EGFR overexpression, relative to U87 cells. Cells were stained with anti-EGFR-PE and FACS sorted for the top 50% of highest peak. Careful subcloning when cells reached no greater than 70% confluence and flow cytometry analysis was routinely performed to ensure cells maintained EGFR expression. U87high are U87-172b cells overexpressing wtEGFR, and were a kind gift from Oliver Bölger, Ph.D.
U87-ffLuc-mKate and U87med-ffLuc-mKate. U87 and U87med cells were lentivirally transduced to express ffLuc-mKate transgene (
Human renal cortical epithelial cells (HRCE). HRCE were obtained from Lonza, described to be taken from proximal and distal renal tubules of healthy individuals, and were cultured in complete Renal Growth Media (Lonza, cat# CC-3190) supplemented with recombinant human epidermal growth factor (rhEGFR), epinephrine, insulin, triiodothyronine, hydrocortisone, transferrin, 10% heat-inactivated FBS (HyClone), and 2 mM Glutamax-100 (Gibco). HRCE have finite lifespan in vitro, therefore, all assays were performed with cells that underwent less than 10 population doublings. Cells were cultured to 70-80% confluency, then detached by 0.05% Trypsin-EDTA (Gibco) and passaged 1:5 in fresh, complete Renal Growth Media.
NALM-6, T98G, LN18 and A431. NALM-6, T98G, LN18, and A431 were all obtained from ATCC and cultured as described for cell lines.
T cell modification and culture. Peripheral blood mononuclear cells were obtained from healthy donors from Gulf Coast Regional Blood Bank and isolated by Ficoll-Paque (GE Healthcare, Milwaukee, WI) and cryopreserved. All T cell cultures were maintained in complete RPMI-1640 (HyClone), supplemented with 10% FBS (HyClone) and 2 mM Glutamax (Gibco).
Electroporation with SB Transposon/Transposase. SB electroporation was performed as previously described (Singh et al., 2008). PBMC were thawed on the day of electroporation and rested in cytokine-free media complete RPMI-1640 at a density of 1x106 cells/mL for 2 hours. Following resting period, cells were centrifuged at 200xg for 8 minutes, then resuspended in media and counted by trypan blue exclusion using an automated cell counter (Cellometer, Auto T4 Cell Counter, Nexcelcom). PBMC were centrifuged again and resuspended at 2×108 /mL in human T cell electroporation buffer (Lonza, cat# VPA-1002), then 100 µL of cell suspension was mixed with 15 µg transposon (either Cetux- or Nimo-CAR) and 5 µg SB11 transposase, transferred to electroporation cuvette, and electroporated via Amaxa Nucleofector (Lonza) using program U-014 for unstimulated human T cells. Following electroporation, cells were immediately transferred to phenol-free RPMI supplemented with 20% heat-inactivated FBS (HyClone), and 2 mM Glutamax-100 (Gibco) to recover overnight. The next day, cells were analyzed by flow cytometry for CD3 and Fc (to determine CAR expression) to determine transient expression of transposon.
Stimulation and Culture of CAR+ T cells. Twenty-four hours after electroporation, cells were stimulated with 100 Gy-irradiated EGFR+ K562 clone 27 artificial antigen presenting cells (aAPC) at a ratio of 2 CAR+ T cells:1 aAPC. T cells were restimulated every 7-9 days following evaluation of CAR expression by flow cytometry. Throughout culture period, T cells received 30 ng/mL IL-21 (Peprotech, Rocky Hill, NJ) added to culture every 2-3 days. IL-2 (Aldeleukin, Novartis, Switzerland) was added to culture after second stimulation cycle at 50 U/mL, every 2-3 days. At day 14, cultures were evaluated for the presence of NK cells, designated as CD3negCD56+ cells present in culture. If NK cells represented >10% of cell population, NK cell depletion was performed by labeling NK cells with CD56-specific magnetic beads (Miltenyi Biotec) and sorting on LS column (Miltenyi Biotec). Flow cytometry of negative flow through containing CAR+ T cells verified successful depletion of NK cell subset from culture. Cultures were evaluated for function when CAR was expressed on >85% of CD3+ T cells, usually following 5 stimulation cycles.
In vitro transcription of RNA. CetuxCD28mZ/pGEM-A64, NimoCD28mZ/pGEM-A64, or GFP/pGEM-A64 was digested with SpeI at 37° C. for 4 hours to provide linear template for in vitro RNA transcription. Complete linearization of template confirmed by agarose gel electrophoresis in 0.8% agarose gel and presence of single band and remaining digest purified by QiaQuick PCR Purification (Qiagen) and eluted in low volume to achieve concentration of 0.5 µg/µL. In vitro transcription reaction was performed using T7 mMACHINE mMESSAGE Ultra (Ambion, Life Technologies, cat# AM1345) according to manufacturer’s protocol and incubated at 37° C. for 2 hours. After transcription of mRNA, DNA template was degraded by addition of supplied Turbo DNAse at 1 unit/µg DNA template and incubated an additional 30 minutes at 37° C. Transcribed RNA was purified using RNeasy Mini kit (Qiagen). Concentration and purity (OD 260/280 value = 2.0-2.2) were determined by spectrophotometry and frozen in single-thaw aliquots at -80° C. Quality of RNA product evaluated by gel electrophoresis on formaldehyde-containing agarose gel (1% agarose, 10% 10x MOPS Running Buffer, 6.7% formaldehyde) at 75 volts for 80 minutes in 1xMOPS Running Buffer and visualization of single, delineated band.
Polyclonal T-cell expansion. Numeric expansion of T cells independent of antigen was achieved by culture with 100 Gy-irradiated K562 clone 4 loaded with OKT3 delivering proliferative stimulus through cross-linking CD3. aAPC were added at a density of 10:1 or 1:2 T cells: aAPC every 7-10 days, 50 U/mL IL-2 was added every 2-3 days. Media changes were performed throughout culture to keep T cells at a density between 0.5-2×106 cells/mL.
RNA electro-transfer to T cells. T cells underwent stimulation 3-5 days prior to RNA transfer by co-culture with 100 Gy-irradiated OKT3-loaded K562 clone 4 as described above. Prior to electro-transfer, T cells were harvested and counted by trypan blue exclusion using an automated cell counter (Cellometer, Auto T4 Cell Counter, Nexcelcom). During preparation of cells, RNA was removed from -80° C. freezer and thawed on ice. T cells were centrifuged at 90xg for 10 minutes, and supernatant was carefully aspirated to ensure complete removal without disruption of cell pellet. T cells were suspended in P3 Primary Cell 4D-Nucleofector buffer (Lonza, cat # V4XP-3032) to a concentration of 1×108/mL and 20 µL of each T cell suspension was mixed with 3 µg of in vitro transcribed RNA, then transferred to Nucleofector cuvette strip (Lonza, cat # V4XP-3032). Cells were electroporated in Amaxa 4D Nucleofector (Lonza) using program DQ-115, then allowed to rest in cuvette up to 15 minutes. Following rest period, warm recovery media, phenol-free RPMI 1640 (HyClone) supplemented with 2 mM Glutamax-100 (Gibco) and 20% heat-inactivated FBS (HyClone), was added to cuvette and cells were gently transferred to 6 well plate containing recovery media and transferred to a tissue culture incubator. After 4 hours, 50 U/mL IL-2 and 30 ng/mL IL-21 were added to the T cells. Four to twenty-four hours after RNA transfer, T cells were analyzed for expression of CAR by flow cytometry for Fc. All functional assays were carried out at 24 hours post-RNA transfer.
Acquisition and analysis. Flow cytometry data were collected on FACS Calibur (BD Biosciences, San Jose, CA) and acquired using CellQuest software (version 3.3, BD Biosciences). Analysis of flow cytometry data was performed using FlowJo software (version x.0.6, TreeStar, Ashland, OR).
Surface Immunostaining and Antibodies. Immunostaining of up to 1x106 cells was performed with monoclonal antibodies conjugated to the following dyes at the following dilutions (unless otherwise stated): fluorescein (FITC, 1:25), phycoerythrin (PE, 1:40), peridinin chlorophyll protein conjugated to cyanine dye (PerCPCy5.5, 1:25), allophycocyanin (APC, 1:40), AlexaFluor488 (1:20), AlexaFluor647 (1:20). All antibodies were purchased from BD Biosciences, unless otherwise stated. Antibodies specific for the following were used: CD3 (clone SK7), CD4 (clone RPA-T4), CD8 (clone SK1), CD19 (HIB19), CD27 (clone L128), CD28 (clone L293), CD45RA (clone HI100), CD45RO (clone HI100), CD56 (clone B159), CD62L (clone DREG-56), CCR7 (clone GD43H7, Biolegend, San Diego, CAR PerCPCy5.5 diluted 1:45), EGFR (clone EGFR.1, PE diluted 1:13.3), Fc (to detect CAR, clone HI10104, Invitrogen), IL15 (clone 34559, R&D Systems, Minneapolis, MN, PE diluted 1:20), murine F(ab′)2 (to detect OKT3 loaded on K562, Jackson Immunoresearch, West Grove, PA, cat# 115-116-072, PE diluted 1:100), TNF-α (clone mAb11, PE diluted 1:40) and IFN-γ (clone 27, APC diluted 1:66.7), pErk1/2 (clone 20A, AlexaFluor 647), pp38 (clone 36/p38, PE) and Ki-67 (clone B56, FITC, 1:20, BD Biosciences). Surface molecules were stained in FACS buffer (PBS, 2% FBS, 0.5% sodium azide) for 30 minutes in the dark at 4° C.
Quantitative Flow Cytometry. Quantitative flow cytometry was performed using Quantum Simply Cellular polystyrene beads (Bangs Laboratories, Fishers, IN). Five bead populations are provided, four populations with increasing amounts of anti-murine IgG, and therefore a known antibody binding capacity (ABC) and one blank population. EGFR-PE (BD Biosciences, cat#555997) was incubated with beads at a saturated concentration (1:3 dilution, per manufacturer’s recommendation) synchronously with immunostaining of target cells. MFI of EGFR-PE binding to microspheres was used to create a standard curve, to which a linear regression was fit using QuickCal Data Analysis Program (version 2.3, Bangs Laboratories) (
Intracellular cytokine staining and flow cytometry. T cells were co-cultured with target cells at a ratio of 1:1 for 4-6 hours in the presence of GolgiStop diluted 4000x (BD Biosciences). Unstimulated T cells served as negative controls, while T cells treated with Leukocyte Activation Cocktail, containing PMA/Ionomycin and brefeldin A (BD Biosciences) diluted 1000x served as positive controls. An EGFR-specific monoclonal antibody (clone LA1, Millipore) was used to block interaction of CAR and EGFR interaction. Intracellular cytokine staining was performed after surface immunostaining by fixation/permeabilization in Cytofix/Cytoperm buffer (BD Biosciences) for 20 minutes in the dark at 4° C., followed by staining of intracellular cytokine in 1x Perm/Wash Buffer (BD Biosciences) for 30 minutes, in the dark at 4° C. Antibodies used were TNF-α (BD Biosciences, clone mAb11, PE diluted 1:40) and IFN-γ (BD Biosciences, clone 27, APC diluted 1:66.7). Following intracellular cytokine staining, cells were fixed with 0.5% paraformaldehyde (CytoFix, BD Biosciences) until samples were acquired on FACS Calibur.
Measuring phosphorylation by flow cytometry. T cells were co-cultured with target cells at a ratio of 1:1 for 45 minutes, unless otherwise indicated. Following activation, T cells centrifuged 300xg for 5 min and supernatant decanted. T cells were lysed and fixed by addition of 20 volumes of 1x PhosFlow Lyse/Fix buffer (BD Biosciences), pre-warmed to 37° C. and incubated at 37° C. for 10 minutes. Following centrifugation, T cells are permeabilized by addition of ice-cold PhosFlow Perm III Buffer (BD Biosciences) while vortexing and incubated on ice in the dark for 20 minutes. After incubation, cells were washed with FACS Buffer and resuspended in 100 µL staining solution. Staining solution was composed of antibodies against CD4 (clone SK3, FITC), CD8 (clone SK1, PerCPCy5.5), pErk1/2 (clone 20A, AlexaFluor 647), pp38 (clone 36/p38, PE) and FACS buffer, all present at the same ratio and incubated for 20 minutes in the dark at room temperature. Cells were fixed with 0.5% paraformaldehyde and analyzed by flow cytometry within 24 hours.
Viability Staining. Staining for Annexin V (BD Biosciences) and 7-AAD (BD Biosciences) was used to determine cell viability and was performed in 1x Annexin Binding buffer, with staining for CD4 or CD8, for 20 minutes, in the dark, at room temperature. Percentage of viable cells was determined as %AnnexinVneg7-AADneg in CD4 or CD8 gated T cell population.
Staining for cellular proliferation marker Ki-67. Proliferation marker Ki-67 was measured by intracellular flow cytometry. T cells were co-cultured with adherent target cells at a ratio of 1:5 for 36 hours, then T cells were harvested from culture by removing supernatant and centrifugation at 300xg. T cells were then fixed and permeabilized by drop-wise addition of ice-cold 70% ethanol while vortexing at high speed. T cells were then stored at -20° C. for 2-24 hours before staining. Cells were stained with Ki-67 (clone B56, FITC, 1:20, BD Biosciences), CD4 (clone RPA-T4), and CD8 (clone SK1) in 100 µL FACs Buffer for 30 min in the dark at room temperature, then immediately analyzed by flow cytometry.
CAR downregulation. CAR+ T cells and targets were harvested and counted by trypan blue exclusion using an automated cell counter (Cellometer, Auto T4 Cell Counter, Nexcelcom), then mixed at a 1:1 ratio in a 12-well plate, and individual wells were harvested at each time point to measure CAR surface expression on T cells. Negative controls for downregulation were T cells plated without stimuli. Staining for T cells by CD3, CD4 and CD8 expression and co-staining for CAR by Fc was analyzed on flow cytometer. Percent downregulation of CAR was calculated as [CAR expression following stimuli]/[CAR expression without stimuli] x 100.
Secondary activation and cytokine production. CAR+ T cells and adherent targets were harvested and counted by trypan blue exclusion using an automated cell counter (Cellometer, Auto T4 Cell Counter, Nexcelcom), then mixed at a ratio of 1:1 in a 12-well plate. After 24 hours of co-culture, T cells were harvested from culture by removing supernatant and washing adherent cells with PBS. T cells were spun at 300xg for 5 minutes, then resuspended in media and counted by trypan blue exclusion using an automated cell counter (Cellometer, Auto T4 Cell Counter, Nexcelcom). T cells were stimulated with targets at 1:1 ratio and intracellular cytokine production analysis as described above.
Long-term cytotoxicity assay. The day prior to initiation of assay, adherent U87 and U87high cells were harvested, counted, and 40,000 target cells were plated in each well of a 6-well plate in complete DMEM and incubated in tissue culture incubator overnight. On the day of assay, CAR+ T cells were harvested, counted by trypan blue exclusion, and added at a 1:5 E:T ratio to plated target cells. Negative control wells had no T cells added. At each assay time point, T cells were removed by discarding supernatant and washing the well with PBS. Adherent cells were dissociated from wells by 0.05% Trypsin-EDTA (Gibco). Microscopy was performed to visually ensure complete detachment of cells from well. Harvested cells were spun down and resuspended in 100 µL of media, then counted by trypan blue exclusion using a hemacytometer. Percent surviving cells was calculated as [cell number after T cell co-culture]/[cell number with no T cell co-culture] x 100.
Chromium release assay. Specific cytotoxicity was assessed via standard 4 hour chromium release assay, as previously described (Singh et al., 2008). Target cells were harvested and counted by trypan blue exclusion using an automated cell counter (Cellometer, Auto T4 Cell Counter). No less than 250,000 cells were aliquoted, then centrifuged at 300xg for 5 minutes and supernatant was discarded. Next, 0.1 µCi of 51Cr was added to each target and incubated for 1-1.5 hours in a tissue culture incubator at 37° C. 100,000 T cells per well were plated in triplicate and serially diluted at 1:2 ratio to give a final effector to target (E:T) ratio of 20:1, 10:1, 5:1, 2.5:1 and 1.25:1 in a 96-well V-bottom plate (Corning, Corning, NY) and placed in a tissue culture incubator. Media only was placed in wells for minimum chromium release control. Following labeling with chromium, targets were washed three times with 10 mL PBS, then resuspended at a final concentration of 125,000 cells/mL, thoroughly mixed, and 100 µL was added to each row, included all T-cell containing rows, a minimum release row, and a maximum release row. Plates were centrifuged at 300xg for 3 minutes. Following centrifugation, 100 µL of 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) was added to maximum release row, and plates were placed in tissue culture incubator for 4 hours. Following incubation, plates were then harvested by careful removal of 50 µL supernatant, without disrupting cell pellet, and transferred to LumaPlate-96 (Perkin-Elmer, Waltham, MA) and allowed to dry overnight. The following day, plates were sealed with Top-Seal (Perkin-Elmer) and scintillation measured on TopCount NXT (Perkin-Elmer). Percent specific lysis was calculated as [(51Cr released - minimum) / (maximum- minimum)] x 100 where maximum and minimum values were averaged for each triplicate.
High-throughput gene expression and CDR3 sequencing
Analysis of gene expression by direct imaging of mRNA transcripts. Direct imaging and quantification of mRNA molecules was performed as previously described (319-322). Cells prior to or following expansion were positively sorted for CD4 and CD8 expression by incubating with CD4 and CD8 magnetics beads (Miltenyi Biotec), respectively, and sorting on LS column. Flow cytometry was used to verify purity of CD4 and CD8 separated populations. 1x106 T cells were lysed in 165 pL of RLT Buffer (Qiagen) and frozen at -80° C. in single-thaw aliquots. RNA lysates were thawed and hybridized with multiplexed target-specific, color-coded reporter and biotinylated capture probes at 65° C. for 12 hours. Lymphocyte specific mRNA transcripts of interest were identified and two CodeSets generated from RefSeq accessions were used to generate reporter and capture probe pairs, a Lymphocyte CodeSet, and TCR Va and Vβ CodeSet. The Lymphocyte CodeSet contained probes for the following genes: ABCB1; ABCG2; ACTB; ADAM19; AGER; AHNAK; AIF1; AIM2; AIMP2; AKIP1; AKT1; ALDH1A1; ANXA1; ANXA2P2; APAF1; ARG1; ARRB2; ATF3; ATM; ATP2B4; AXIN2; B2M; B3GAT1; BACH2; BAD; BAG1; BATF; BAX; BCL10; BCL11B; BCL2; BCL2L1; BCL2L1; BCL2L11; BCL2L11; BCL6; BCL6B; BHLHE41; BID; BIRC2; BLK; BMI1; BNIP3; BTLA; C21orf33; CA2; CA9; CARD9; CASP1; CAT; CBLB; CCBP2; CCL3; CCL4; CCL5; CCNB1; CCND1; CCR1; CCR2; CCR4; CCR5; CCR6; CCR7; CD160; CD19; CD19R-scfv; CD19RCD28; CD2; CD20-scfv rutuximab); CD226; CD244; CD247; CD27; CD274; CD276; CD28; CD300A; CD38; CD3D; CD3E; CD4; CD40LG; CD44; CD45R-scfv; CD47; CD56R-scfv; CD58; CD63; CD69; CD7; CD80; CD86; CD8A; CDH1; CDK2; CDK4; CDKN1A; CDKN1B; CDKN2A; CDKN2C; CEBPA; CFLAR; CFLAR; CHPT1; CIITA; CITED2; CLIC1; CLNK; c-MET-scfv; CREB1; CREM; CRIP1; CRLF2; CSAD; CSF2; CSNK2A1; CTGF; CTLA4; CTNNA1; CTNNB1; CTNNBL1; CTSC; CTSD; CX3CL1; CX3CR1; CXCL10; CXCL12; CXCL9; CXCR1; CXCR3; CXCR4; DAPL1; DEC1; DECTIN-1R; DGKA; DOCKS; DOK2; DPP4; DUSP16; EGFR-scfv (NIMO CAR); EGLN1; EGLN3; EIF1; ELF4; ELOF1; ENTPD1; EOMES; EPHA2; EPHA4; EPHB2; ETV6; FADD; FAM129A; FANCC; FAS; FASLG; FCGR3B; FGL2; FLT1; FLT3LG; FOS; FOXO1; FOXO3; FOXP1; FOXP3; FYN; FZD1; G6PD; GABPA; GADD45A; GADD45B; GAL3ST4; GAS2; GATA2; GATA3; gBAD-1R-scfv; GEMIN2; GFI1; GLIPR1; GLO1; GNLY; GSK3B; GZMA; GZMB; GZMH; HCST; HDAC1; HDAC2; HER2-scfv; HERV-K 6H5-scfv; HLA-A; HMGB2; HOPX; HOXA10; HOXA9; HOXB3; HOXB4; HPRT1; HRH1; HRH2; Human CD19R-scfv; ICOS; ICOSLG; ID2; ID3; IDOl; IFNA1; IFNG; IFNGR1; IGF1R; IKZF1; IKZF2; IL10; IL10RA; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL15; IL15RA; IL17A; IL17F; IL17RA; IL18; IL18R1; IL18RAP; IL1A; IL1B; IL2; IL21R; IL22; IL23A; IL23R; IL27; IL2RA; IL2RB; IL2RG; IL4; IL4R; IL5; IL6; IL6R; IL7R; IL9; IRF1; IRF2; IRF4; ITCH; ITGA1; ITGA4; ITGA5; ITGAL; ITGAM; ITGAX; ITGB1; ITGB7; ITK; JAK1; JAK2; JAK3; JUN; JUNB; KIR2DL1; KIR2DL2; KIR2DL3; KIR2DL4; KIR2DL5A; KIR2DS1; KIR2DS2; KIR2DS3; KIR2DS4; KIR2DS5; KIR3DL1; KIR3DL2; KIR3DL3; KIR3DS1; KIT; KLF10; KLF2; KLF4; KLF6; KLF7; KLRAP1; KLRB1; KLRC1; KLRC2; KLRC3; KLRC4; KLRD1; KLRF1; KLRG1; KLRK1; LAG3; LAIR1; LAT; LAT2; LCK; LDHA; LEF1; LGALS1; LGALS3; LIFR; LILRB1; LOC282997; LRP5; LRP6; LRRC32; LTA; LTBR; LYN; MAD1Ll; MAP2K1; MAPK14; MAPK3; MAPK8; MBD2; MCL1; MIF; MMP14; MPL; MTOR; MXD1; MYB; MYC; MYO6; NANOG; NBEA; NCAM1; NCL; NCR1; NCR2; NCR3; NCRNA00185; NEILl; NEIL2; NFAT5; NFATC1; NFATC2; NFATC3; NFKB1; NOS2; NOTCH1; NR3C1; NR4A1; NREP; NRIP1; NRP1; NT5E; OAZ1; OPTN; P2RX7; PAX5; PDCD1; PDCD1LG2; PDE3A; PDE4A; PDE7A; PDK1; PDXK; PECAM1; PHACTR2; PHC1; POLR1B; POLR2A; POP5; POUSF1; PPARA; PPP2R1A; PRDM1; PRF1; PRKAA2; PRKCQ; PROM1; PTGER2; PTK2; PTPN11; PTPN4; PTPN6; PTPRK; RAB31; RAC1; RAC2; RAF1; RAP1GAP2; RARA; RBPMS; RHOA; RNF125; RORA; RORC; RPL27; RPS13; RUNX1; RUNX2; RUNX3; S100A4; S100A6; SATB1; SCML1; SCML2; SEL1L; SELL; SELPLG; SERPINE2; SH2B3; SH2D2A; SIT1; SKAP1; SKAP2; SLA2; SLAMF1; SLAMF7; SLC2A1; SMAD3; SMAD4; SNAIl; SOCS1; SOCS3; SOD1; SOX13; SOX2; SOX4; SOX5; SPIl; SPN; SPRY2; STAT1; STAT3; STAT4; STAT5A; STAT5B; STAT6; STMN1; SYK; TAL1; TBP; TBX21; TBXA2R; TCF12; TCF3; TCF7; TDGF1; TDO2; TEK; TERF1; TERT; TF; TFRC; TGFA; TGFB1; TGFB2; TGFBR1; Thymidine Kinase; TIE1; TLR2; TLR8; TNF; TNFRSF14; TNFRSF18; TNFRSF1B; TNFRSF4; TNFRSF9; TNFSF10; TNFSF11; TNFSF14; TOX; TP53; TRAF1; TRAF2; TRAF3; TSC22D3; TSLP; TXK; TYK2; TYROBP; UBASH3A; VAX2; VEGFA; WEE1; XBP1; XBP1; YY1AP1; ZAP70; ZBTB16; ZC2HC1A; ZEB2; ZNF516. The TCR Va and Vβ CodeSet contained probes for the following genes: TRAV1-1; TRAV1-2; TRAV2; TRAV3; TRAV4; TRAV5; TRAV6; TRAV7; TRAV8-1; TRAV8-2; TRAV8-3; TRAV8-6; TRAV9-1; TRAV9-2; TRAV10; TRAV11; TRAV12-1; TRAV12-2; TRAV12-3; TRAV13-1; TRAV13-2; TRAV14; TRAV16; TRAV17; TRAV18; TRAV19; TRAV20; TRAV21; TRAV22; TRAV23; TRAV24; TRAV25; TRAV26-1; TRAV26-2; TRAV27; TRAV29; TRAV30; TRAV34; TRAV35; TRAV36; TRAV38-1; TRAV38-2; TRAV39; TRAV40; TRAV41; TRBV2; TRBV3-1; TRBV4-1; TRBV4-2; TRBV4-3; TRBV5-1; TRBV5-4; TRBV5-5; TRBV5-6; TRBV5-8; TRBV6-1; TRBV6-2; TRBV6-4; TRBV6-5; TRBV6-6; TRBV6-8; TRBV6-9; TRBV7-2; TRBV7-3; TRBV7-4; TRBV7-6; TRBV7-7; TRBV7-8; TRBV7-9; TRBV9; TRBV10-1; TRBV10-2; TRBV10-3; TRBV11-1; TRBV11-2; TRBV11-3; TRBV12-3; TRBV12-5; TRBV13; TRBV14; TRBV15; TRBV16; TRBV18; TRBV19; TRBV20-1; TRBV24-1; TRBV25-1; TRBV27; TRBV28; TRBV29-1; TRBV30. Following hybridization, samples were processed in nCounter Prep (NanoString Technologies, Seattle, WA), and analyzed in nCounter Digital Analyzer (NanoString Technologies). Reference genes were identified that span wide range of RNA expression levels: ACTB, G6PD, OA21, POLR1B, RPL27, RPS13, and TBP and were used to normalize data. Normalization to positive-, negative-, and house-keeping genes was using nCounter RCC Collector (version 1.6.0, NanoString Technologies). A statistical test developed for digital gene expression profiling was used to determine differential expression of genes between sample pairs (O′Connor et al., 2012; Audic et al., 1997). After normalization, significant differential gene expression in the Lymphocyte CodeSet was identified by a combination of p<0.01 and a fold change greater than 1.5 in at least ⅔ pairs, as previously described (O′Connor et al., 2012). Heat-mapping of normalized values for differentially RNA transcripts was performed by hierarchical clustering and TreeView software, version 1.1 (Eisen et al., 1998). After normalization, percentage of TCR Vα and Vβ were derived from count data as previously described (Zhang et al., 2012).
High-throughput CDR3 deep-sequencing. TCRβ CDR3 regions were amplified and sequenced from DNA extracted from 1x106 T cells (Qiagen DNeasy Blood and Tissue Kit, Qiagen) and carried out on ImmunoSEQ platform (Adaptive Technologies, Seattle, WA), as previously described (Robins et al., 2009).
In vivo evaluation of T cells in intracranial glioma xenograft murine model
All animal experiments were carried out under guidance and regulation from the Institutional Animal Care and Use Committee (IACUC) at MD Anderson Cancer Center under the approved animal protocol ACUF 11-11-13131. All mice used were 7-8 week old female NOD.Cg-PrkdescidIL2Rγtm1Wjl/Sz strain (NSG) (Jackson Laboratory, Bar Harbor, ME).
Implantation of guide-screw. Mice aged 7-8 weeks were anesthetized using ketamine/xylazine cocktail (10 mg/mL ketamine, 0.5 mg/mL xylazine) dosed at 0.1 mL/10 g. Implantation of guide-screw was performed as previously described (Lal et al., 2000) Once unresponsive to stimuli, surgical area on head was prepared by shaving fur and treated with povidone-iodine (polyvinylpyrrolidone complexed with elemental iodine) antiseptic solution. Using surgically ascpetic technique, a 1 cm incision was made down the middle of the cranium. An opening was made using a 1 mm drill bit (DH#60, Plastics One, Roanoke, VA) extending 1 mm from drill (DH-0, Plastics One) using firm circular pressure. A guide-screw (Plastics One, cat # C212SG) with a 0.50 mm opening in the center and a 1.57 mm shaft diameter was inserted into the drill site using a screwdriver (SD-80, Plastics One). Incision sites were sutured and mice were given 0.01 mg/mL buprenorphine dosed at 0.1 mL/10 grams as post-surgical analgesic. Mice recovered from surgery on low-power heat source until full mobility was regained.
Implantation of U87-ffLucm-Kate or U87med-ffLuc-mKate tumor cells. Mice recovered from guide-screw implantation for 2-3 weeks before intracranial tumors were established, as previously described (Lal et al., 2000). U87-ffLuc-mKate or U87med-ffLuc-mKate were dissociated from tissue culture vessel following 10 minute incubation with Cell Dissociation Buffer, enzyme-free, PBS (Gibco) at room temperature. Cells were counted by trypan blue exclusion using hemacytometer and centrifuged at 200xg for 8 minutes. Following centrifugation, cells were resuspended in sterile PBS to a final concentration of 50,000 cells/µL. Mice were anesthetized with isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane), and prepared for incision as described above. While mice were undergoing surgical preparation, 26 gauge, 10 µL Hamilton syringes with blunt needle (Hamilton Company, Reno, NV cat# 80300) were prepared by placing plastic guard 2.5 mm from the end of syringe and loading 5 µL of cell suspension containing 250,000 cells. After incision site was opened, syringes were inserted into guide screw opening and cells were injected with constant slow pressure. After completion of injection, syringes were held in place an additional 30 seconds to allow intracranial pressure to dissipate, then slowly removed. Incisions were sutured and mice were removed from isoflurane exposure. Day of implantation is designated as day 0 of study. On day 1 and 4 tumors were imaged via non-invasive bioluminescent imaging, as described above to ensure successful tumor engraftment. Mice were then divided into three groups to evenly distribute relative tumor flux, and then randomly assigned to receive Cetux-CAR+ T-cell treatment, Nimo-CAR+ T-cell treatment and no treatment.
Non-invasive bioluminescent imaging of U87-ffLuc-mKate or U87med-ffLuc-mKate. Intracranial glioma was non-invasively and serially imaged and used as a measure of relative tumor burden. Ten minutes after sub-cutaneous injection of 215 µg D-luciferin potassium salt (Caliper Life Sciences, Perkin-Elmer), tumor flux (photons/s/cm2/steradian) was measured using Xenogen Spectrum (Caliper Life Sciences, Perkin-Elmer) and Living Image software (version 2.50, Caliper Life Sciences, Perkin-Elmer). Tumor flux was measured in a delineated region of interest encompassing entire cranial region of mice.
Delivery of CAR+ T cells to intracranially established U87-ffLuc-mKate or U87med-ffLuc-mKate glioma. Treatment of intracranial glioma xenografts began on day 5 of tumor establishment and continued weekly for a total of 3 T cell injections. CAR+ T cells having completed 3 stimulation cycles were confirmed to be >85% CAR-expressing by flow cytometry, then viable cells were counted by trypan blue exclusion using an automated cell counter (Cellometer, Auto T4 Cell Counter, Nexcelcom). CAR+ T cells were spun at 300xg for 5 minutes, and resuspended at a concentration of 0.6×106 /µL in sterile PBS. Mice were prepared for cranial incision as described above, and anesthetized by isoflurane exposure. While mice were being prepared, 26 gauge, 10 µL Hamilton syringes with blunt needle (Hamilton Company, cat# 80300) were prepared by placing plastic guard 2.5 mm from the end of syringe and loading 5 µL of cell suspension containing 3×106 T cells. Syringes were inserted into the guide-screw, extending 2.5 mm into intracranial space, and injected with slow, constant pressure. After syringe was emptied, it was held in place an addition 30 seconds to allow intracranial pressure to dissipate. Following injection, incisions were sutured closed and mice were removed from isoflurane exposure.
Assessing survival of mice. Mice were sacrificed when they displayed progressive weight loss (>25% of body mass), rapid weight loss (>10% loss of body mass within 48 hours) or hind limb paralysis, or any two of the following clinical symptoms of illness: ataxia, hunched posture, irregular respiration rate, ulceration of exposed tumor, or palpable tumor diameter exceeding 1.5 cm.
All statistical analyses were performed in GraphPad Prism, version 6.03. Statistical analyses of all in vitro cell culture experimentation, including flow cytometry analysis of cytokine production, viability, proliferation, and surface phenotype, kinetics of cell expansion, long term cytotoxicity, and chromium release assay by two-way ANOVA with donor-matching and Tukey’s post-test for multiple comparisons. Correlation of function with antigen density was performed by one-way ANOVA with post-test for linear trend. Analyses of in vivo bioluminescent imaging of tumor were performed using two-way ANOVA with repeated measures and Sidak’s post-test for multiple comparisons. Statistical analysis of animal survival data was performed by log-rank (Mantel-Cox) test. Significance of findings defined as follows: * p<0.05, ** p<0.01, *** p<0.001, **** p <0.0001.
Antigen-dependent stimulation through stable CAR expression achieved by DNA integration can be used to numerically expand CAR+ T cells to clinically feasible numbers. The transient nature of CAR expression via RNA transfer requires numeric expansion of T cells to clinically feasible numbers to be achieved prior to RNA transfer of CAR. To determine the ability of aAPC to numerically expand T cells independent of antigen, anti-CD3 (OKT3) was loaded onto K562 via stable expression of the high affinity Fc receptor CD64 (
T cells expanded with lower density of aAPC contained a higher proportion of CD8+ T cells than T cells expanded with more aAPC (10:1 = 53.9 ± 11.6% CD8, 1:2 = 28.1 ± 16.2% CD8, mean ± S.D., n=6) (p<0.001) (
To determine if expansion with low density or high density aAPC impacted T-cell phenotype, expression of a panel of mRNA transcripts (Lymphocyte-specific CodeSet) was analyzed by multiplex digital profiling using nCounter analysis (Nanostring Technologies, Seattle, WA). Significant differential gene expression was determined by a p<0.01 and fold change greater than 1.5 in sorted CD4+ or CD8+ T cells expanded with low density (10:1 T cell:aAPC) or high density (1:2 T cell:aAPC) aAPC. CD4+ and CD8+ T cells expanded with high density aAPC demonstrated increased expression of genes associated with T-cell activation, such as CD38 and granzyme A in CD4+ T cells and CD38 and NCAM-1 in CD8+ T cells (
To further evaluate differential phenotype of T cells expanded with low or high density aAPC, T cells were analyzed for phenotypic markers by flow cytometry and evaluated subsets by coexpression of CCR7 and CD45RA where CCR7+CD45RA+ indicates naïve phenotype, CCR7+CD45RAneg indicates central memory phenotype, CCR7negCD45RAneg indicates effector memory, and CCR7negCD45RA+ indicates a CD45RA+ effector memory phenotype (Geginat et al., 2003). CD4+ T cells expanded with low density aAPC contained significantly fewer T cells with effector memory phenotype (10:1=61.9 ± 9.1%, 1:2= 92.1 ± 3.9%, mean ± S.D., n=3) (p<0.05), but more central memory phenotype (10:1=36.5 ± 9.4%, 1:2=13.6 ± 2.4%, mean ± S.D., n=3) (p<0.05) T cells (
TCRα and TCRβ diversity was profiled prior to and following expansion with low and high density aAPC by multiplex digital profiling using nCounter analysis (Nanostring Technologies, Seattle, WA) and calculated the relative abundance of each TCRα and TCRβ chain as a percentage of total T-cell population. Following ex vivo expansion with low and high density aAPC, CD4+ and CD8+ T cells expressed diverse TCRα and TCRβ alleles, indicating that the resulting population maintained oligoclonal TCRα and TCRβ repertoire (
To determine the ability of T cells stimulated with low and high density aAPC to accept RNA by electro-transfer, in vitro transcribed RNA encoding green fluorescent protein (GFP) was electro-transferred using the Amaxa Nucleofector 4D transfection system (Lonza, Cologne, Germany) using a variety of electroporation programs, including program EO-115, the manufacturer’s recommended program for stimulated T cells 4 days following stimulation with aAPC. Plotting the mean fluorescent intensity (MFI) of GFP versus the viability of T cells determined by PI staining revealed an inverse correlation between GFP expression and T-cell viability following RNA transfer. Compared to T cells stimulated with low density aAPC, T cells stimulated with high density aAPC demonstrated both reduced expression of GFP by RNA transfer and reduced viability in response to every electroporation program tested (
To compare expression of CAR and function of CAR+ T cells manufactured by RNA and DNA modification, an EGFR-specific CAR was developed from the scFv of cetuximab, a clinically available anti-EGFR monoclonal antibody. The scFv of cetuximab was fused to an IgG4 hinge region, CD28 transmembrane and cytoplasmic domains, and CD3-ζ cytoplasmic domain to form a second generation CAR, termed Cetux-CAR, and expressed in a Sleeping Beauty transposon for permanent DNA integration as well as under a T7 promoter in the pGEM/A64 vector for in vitro transcription of RNA transcripts. RNA-modification of T cells was achieved by electro-transferring in vitro transcribed Cetux-CAR into T cells stimulated twice with OKT3-loaded K562 aAPC, four days following the second stimulation (
To compare the phenotype of T-cell populations expressing Cetux-CAR by RNA-modification or DNA-modification, phenotypic markers were analyzed by flow cytometry. CD4+ RNA-modified CAR+ T cells had significantly more T cells with central memory phenotype than CD4+ DNA-modified CAR+ T cells (CCR7+CD45RAneg) (DNA-modified=6.6 ± 1.9%, RNA-modified=49.6 ± 3.0%, mean ± S.D., n=3) (p<0.0001), but significantly fewer T cells with effector memory phenotype (CCR7negCD45RAneg) (DNA-modified=89.8 ± 2.6%, RNA- modified=48.1 ± 3.3%, mean ± S.D., n=3) (p<0.0001) (
Cytokine production of RNA-modified or DNA-modified CAR+ T cells was evaluated in response to a mouse T cell lymphoma cell line EL4 modified to express truncated EGFR, tEGFR+ EL4, or irrelevant antigen, CD19, and EGFR+ cell lines, including human glioblastoma cell lines U87, T98G, LN18 and human epidermoid carcinoma cell line A431. Fewer CD8+ CAR+ T cells modified by RNA transfer produced IFN-γ in response to all EGFR-expressing cell lines (
Because RNA-modified CAR+ T cells demonstrated reduced capacity to produce cytokine relative to DNA-modified CAR+ T cells, cytotoxicity of RNA-modified and DNA-modified T cells was compared to determine the cytotoxic potential of RNA-modified CAR+ T cells relative to DNA-modified CAR+ T cells. In response to CD19+ EL4 cells, RNA-modified and DNA-modified CAR+ T cells had low levels of background killing, although at high effector to target ratio (E:T = 20:1), RNA-modified CAR+ T cells demonstrated significantly more background lysis than DNA-modified CAR+ T cells (p<0.05) (
To determine the stability of CAR expression by RNA transfer, T cells were modified to express CAR by RNA transfer, and CAR expression was measured over time by flow cytometry. Following RNA transfer, expression of Cetux-CAR on T cells decreased over time, and 96 hours following electro-transfer, CAR was expressed at low levels (
Activity of T cells modified to express Cetux-CAR by RNA transfer was measured 24 and 120 hours after RNA transfer to determine the effect of loss of CAR expression on activity of T cells in response to EGFR-expressing cells. While RNA-modified T cells demonstrated equivalent production of IFN-γ by PMA/Ionomycin stimulation when assessed at 24 hours and 120 hours after RNA transfer, production of IFN-γ in response to tEGFR+ EL4 by T cells 24 hours after RNA transfer was abrogated 120 hours after RNA transfer (24 hrs=14.2 ± 2.5%, 120 hrs=1.1 ± 0.03%, mean ± S.D., n=3) (p=0.012) (
A second generation CAR derived from nimotuzumab, designated Nimo-CAR, was generated in a Sleeping Beauty transposon by fusing the scFv of nimotuzumab with an IgG4 hinge region, CD28 transmembrane domain and CD28 and CD3ζ intracellular domains, an identical configuration to Cetux-CAR. Cetux-CAR and Nimo-CAR were expressed in primary human T cells by electroporation of each transposon with SB11 transposase into peripheral blood mononuclear cells (PBMC). T cells with stable integration of Cetux-CAR or Nimo-CAR were selectively propagated by weekly recursive stimulation with γ-irradiated tEGFR+ K562 artificial antigen presenting cells (aAPC) (
In order to determine the impact of CAR scFv on T-cell function, electroporation and propagation of Cetux-CAR+ and Nimo-CAR+ T cells were established to result in phenotypically similar T-cell populations. Each donor yielded variable ratios of CD4+ and CD8+ T cells (Table 1), however, there was no statistical difference in the CD4/CD8 ratio between Cetux-CAR+ and Nimo-CAR+ T cells (p=0.44, student’s two-tailed t-test) (
To verify Cetux-CAR and Nimo-CAR were functional in response to stimulation with EGFR, CAR+ T cells were incubated with the A431 epidermoid carcinoma cell line, which is reported to express high levels of EGFR, about 1x106 molecules of EGFR/cell (Garrido et al., 2011). Cetux- and Nimo- CAR+ T cells produced IFN-γ during co-culture with A431, which was reduced in the presence of anti-EGFR monoclonal antibody that blocks binding to EGFR (
To investigate the impact of EGFR expression density on activation of Cetux-CAR+ and Nimo- CAR+ T cells, T-cell function was compared against cell lines with a range of EGFR expression density: NALM-6, U87, LN18, T98G, and A431. First, EGFR expression density was evaluated by quantitative flow cytometry (
To determine the impact of EGFR expression density on a syngeneic cellular background, a series of U87 cell lines expressing varying densities of EGFR was developed: unmodified, parental U87 (~30,000 molecules of EGFR/cell), U87low (130,000 molecules of EGFR/cell), U87med (340,000 molecules of EGFR/cell), and U87high (630,000 molecules of EGFR/cell) (
Because endogenous, low affinity T cell responses may require longer interaction with antigen to acquire effector function (Rossette et al., 2001), it was verified that the observed differences in T-cell activity between Cetux-CAR+ T cells and Nimo-CAR+ T cells was not due to a similar requirement for Nimo-CAR+ T cells. Extending interaction of CAR+ T cells with targets did not substantially increase cytokine production and did not alter the relationship of cytokine production between Cetux-CAR+ and Nimo-CAR+ CD8+ T cells (
Expression of CAR above a minimum density is required for CAR-dependent T cell activation, and increasing density of CAR expression has been shown to impact sensitivity of CAR to antigen (Weijtens et al., 2000; Turatti et al., 2007). Therefore, to determine if expressing Nimo-CAR with higher density improves recognition of low EGFR density, it was sought to overexpress Cetux-CAR and Nimo-CAR in human primary T cells. Load of DNA in electroporation transfection is limited due to toxicity of DNA to cells, however, transfer of RNA is relatively non-toxic and more amenable to overexpression by increasing amount of CAR RNA transcript delivered. Therefore, Cetux-CAR and Nimo-CAR were in vitro transcribed as RNA species and electro-transferred into human primary T cells. RNA transfer resulted in 2-5 fold increased expression of CAR when compared to donor-matched DNA-modified T cells (
To determine if Nimo-CAR+ T cells have reduced activation in response to low, basal EGFR levels on normal cells, the activity of Nimo-CAR+ T cells was evaluated in response to normal human renal cortical epithelial cells, HRCE. HRCE express ~15,000 molecules of EGFR per cell, lower than expression on tumor cell lines, including U87 (
Strength of endogenous TCR signal, impacted by affinity of binding and antigen density, can influence proliferation of T cells in response to antigenic stimulus (Gottschalk et al., 2012; Gottschalk et al., 2010). To evaluate proliferative response of Cetux-CAR+ T cells and Nimo-CAR+ T cells following stimulation with antigen, intracellular expression of Ki-67 was measured by flow cytometry after two days of co-culture with U87 or U87high in absence of exogenous cytokines. In response to low EGFR density on U87, Cetux-CAR+ and Nimo-CAR+ T cells demonstrated statistically similar proliferation (p>0.05) (
To determine if affinity of CAR or antigen density increases the propensity of CAR+ T cells to undergo AICD, Cetux-CAR+ and Nimo-CAR+ T cells were cocultured with U87 or U87high in the absence of exogenous cytokines and evaluated T-cell viability by annexin V and 7-AAD staining. In response to U87, Cetux-CAR+ T showed reduction in viability compared to unstimulated Cetux-CAR+ T cells, however, Nimo-CAR+ T cells did not show any appreciable change in viability (
Endogenous TCR can be downregulated following interaction with antigen, and the degree of downregulation is influenced by the strength of TCR binding (Cai et al., 1997). Similary, CAR can be downregulated following interaction with antigen, but the effect of affinity on CAR downregulation is unknown (James et al., 2008; James et al., 2010). Therefore, it was sought to determine if Cetux-CAR+ T cells have a higher propensity for antigen-induced downregulation. To accomplish this, Cetux-CAR+T cells and Nimo-CAR+ T cells were co-cultured with U87 or U87high and monitored CAR expression relative to unstimulated controls. In response to low EGFR density on U87, Cetux-CAR expression was significantly less than Nimo-CAR after 12 hours of interaction (Cetux-CAR=68.0±27.8%, Nimo-CAR=126.5±34.9%, mean±SD, n=3) (p<0.05) (
Strength of prior stimulus in endogenous CD8+ T cell responses can correlated with T-cell response upon re-challenge with antigen (Lim et al., 2002). Therefore, the ability of Cetux-CAR+ and Nimo-CAR+ T cells to respond to antigen re-challenge was evaluated. CAR+ T cells were co-cultured with U87 or U87high for 24 hours, then harvested and rechallenged with U87 or U87high to assess production of IFN-γ. Following initial challenge with U87 and U87high, Cetux-CAR+ T cells had reduced production of IFN-γ in response to rechallenge with both U87 and U87high (
To evaluate anti-tumor efficacy of Cetux-CAR+ T cells and Nimo-CAR+ T cells in vivo, an intracranial glioma xenograft of U87 cells modified to express firefly luciferase (ffLuc) reporter for serial, non-invasive imaging of relative tumor burden by bioluminescence (BLI) was established. The previously described guide-screw method was adopted for directed infusion of tumor and T cells into precise coordinates (Lal et al., 2000). The guide screw was implanted into the right frontal lobe of the cranium of NOD/Scid/IL2Rg-/- (NSG) mice and mice recovered for two weeks (
Four days after injection of U87med, mice were imaged by BLI to assess tumor burden (
Cetux-CAR+ T-cell treated mice showed significant toxicity resulting in death of 6/14 mice within 7 days of T-cell treatment from two independent experiments (p=0.0006) (
Mice were injected with U87, then four days later relative tumor burden was assessed by BLI (
Mice received T-cell treatment and tumor was assessed by BLI as previously described (
Cetux-CAR+ T cell treatment significantly extended survival in 3/6 mice compared to mice receiving no treatment (untreated median survival = 38.5 days, Cetux-CAR median survival = 53 days, p=0.0150) (
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The present application is a continuation of U.S. Application No. 16/600,806, filed Oct. 14, 2019, which is a divisional of U.S. Application No. 15/305,996, filed Oct. 21, 2016, as a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2015/027277, filed Apr. 23, 2015, which claims the priority benefit of U.S. Provisional Application No. 61/983,103, filed Apr. 23, 2014 and U.S. Provisional Application No. 61/983,298, filed Apr. 23, 2014, the entire contents of each of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61983298 | Apr 2014 | US | |
| 61983103 | Apr 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15305996 | Oct 2016 | US |
| Child | 16600806 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16600806 | Oct 2019 | US |
| Child | 18153025 | US |
