Patent Application
20200181238
The Sequence Listing is concurrently submitted herewith with the specification as an ASCII formatted text file via EFS-Web with a file name of Sequence Listing.txt with a creation date of Aug. 9, 2017, and a size of 10.9 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.
The present invention relates to transduced T cells expressing human somatostatin receptor 2 (SSTR2), and its application for monitoring T cell distribution in a patient by PET/CT imaging. The present invention also relates to a method for treating cancer and monitoring CAR T cell distribution in a patient using transduced CAR T cells.
Adoptive cell transfer (ACT) of cytotoxic T lymphocytes is being studied as a potent treatment strategy for cancers that are refractory to standard chemotherapy and radiation therapy. Clinical advances have been made in patients with metastatic melanoma using autologous tumor-infiltrating lymphocytes (TILs) and in several B-cell malignancies using autologous chimeric antigen receptor (CAR)-modified T cells1. Methods used to predict or monitor the activity of infused T cells in patients provide useful but limited data related to treatment efficacy. Current practices involve serum profiling of cytokines associated with T cell activation, direct enumeration of tumor-specific T cell numbers in peripheral circulation, and tumor biopsies2,3. Changes in serum cytokine levels, while useful, likely reflect a broader, systemic immune response, illustrating not only the activation of adoptively transferred T cells, but also their effects on neighboring immune cells and dying tumor cells4. Similarly, while the quantification of adoptively transferred cells in circulation provides useful information regarding their proliferation, researchers and clinicians are blind as to whether the dynamism in T cell numbers relates to expansion at the primary tumor site, metastatic foci, or at off-tumor sites5.
The imaging modalities with the highest potential for whole-body visualization of cell trafficking in humans are magnetic resonance imaging (MM), single-photon emission computed tomography (SPECT), PET/CT, or PET/MRI techniques for detection of labeled cells and coregistration of anatomical information of the body8-10. PET (positron emission tomography) is particularly amenable to clinical use as it enables non-invasive, highly sensitive, repetitive, and quantitative imaging of positron-emitting, target-specific probes. The introduction of microPET for small animal imaging has similarly made PET amenable to pre-clinical studies11. On-going activity of ACT against both on- and off-tumor sites can therefore be monitored in vivo by quantitative, radiotracer-based imaging of T cell distribution and expansion upon interaction with target antigen-expressing cells2,10,12. However, previous attempts to systemically monitor ACT in patients have yet to be adopted13. Passive labeling of T cells with positron emitting probes ex vivo has been used to monitor the early-stage migration of infused T cells but suffers from potential inaccuracies due to signals from dead or dying cells, probe dilution upon cell division, and a limited ability to track cells over extended periods of time due to short probe half-life10.
As used herein, “about” refers to ±10% of the recited value.
As used herein, “adoptive T cell therapy” involves the isolation and ex vivo expansion of tumor specific T cells to achieve greater number of T cells than what could be obtained by vaccination alone. The tumor specific T cells are then infused into patients with cancer in an attempt to give their immune system the ability to overwhelm remaining tumor via T cells which can attack and kill cancer.
As used herein, “affinity” is the strength of binding of a single molecule (e.g., I domain) to its ligand (e.g., ICAM-1). Affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions.
A “binding molecular,” refers to a molecule that is capable to bind another molecule of interest.
A “chimeric antigen receptor (CAR)” means a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular domain. The “extracellular domain capable of binding to an antigen” means any oligopeptide or polypeptide that can bind to a certain antigen. The “intracellular domain” means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.
A “domain” means one region in a polypeptide which is folded into a particular structure independently of other regions.
An “elongation factor 1 (EF-1) α promoter” is derived from the human EEF1A1 gene that expresses the α subunit of eukaryotic elongation factor 1. EF-1 α promoter offers a broad host range.
An “integrin” or “integrin receptor” (used interchangeably) refers to any of the many cell surface receptor proteins, also referred to as adhesion receptors which bind to extracellular matrix ligands or other cell adhesion protein ligands thereby mediating cell-cell and cell-matrix adhesion processes. Binding affinity of the integrins to their ligands is regulated by conformational changes in the integrin. Integrins are involved in physiological processes such as, for example, embryogenesis, hemostasis, wound healing, immune response and formation/maintenance of tissue architecture. Integrin subfamilies contain a beta-subunit combined with different alpha-subunits to form adhesion protein receptors with different specificities.
As used herein, “I domain” refers to the inserted or I domain of the αL subunit of LFA-1, and is an allosteric mediator of ligand binding to LFA-1. I domain is a native ligand of ICAM-1. The ligand binding site of the I domain, known as a metal ion-dependent adhesion site (MIDAS), exists as two distinct conformations allosterically regulated by the C-terminal α7 helix. A wild-type (WT) I domain encompasses amino acid residues 130-310 of the 1145 amino acid long mature αL integrin subunit protein (SEQ ID NO: 1, which is the amino acid residues 26-1170 of GenBank Accession No. NP 002200). All numbering of amino acid residues as used herein refers to the amino acid sequence of the mature αL integrin (SEQ ID NO: 1), wherein residue 1 of SEQ ID NO: 1 corresponds to residue 26 of the sequence of GenBank Accession No. NP_002200.
“Lymphocyte function-associated antigen-1”, “LFA-1”, “αLβ2 integrin” or “CD18/CD11a” refers to a member of the leukocyte integrin subfamily. LFA-1 is found on all T-cells and also on B-cells, macrophages, neutrophils and NK cells and is involved in recruitment to the site of infection. It binds to ICAM-1 on antigen-presenting cells and functions as an adhesion molecule.
As used herein, a “tumor antigen” means a biological molecule having antigenecity, expression of which causes cancer.
A “single chain variable fragment (scFv)” means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the scFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for preparing an scFv are known to a person skilled in the art.
“Somatostatin receptor type 2 (SSTR2)” is a receptor for somatostatin-14 and -28. Somatostatin acts at many sites to inhibit the release of many hormones and other secretory proteins. The biologic effects of somatostatin are probably mediated by a family of G protein-coupled receptors that are expressed in a tissue-specific manner. SSTR2 is a member of the superfamily of receptors having seven transmembrane segments and is expressed in highest levels in cerebrum and kidney. A full molecule of human SSTR2 has 369 amino acids and its sequence is shown as GenBank Accession No. NP_001041. A “truncated SSTR2”, as used herein, refers to a C-terminus shortened human SSTR2, which contains 1-314 amino acid residues of human SSTR2 with a deletion of the C-terminus beyond amino acid 31466.
A “2A peptide” is used by several families of viruses, the best known foot-and-mouth disease virus of the Picornaviridae family, for producing multiple polypeptides. The mechanism by the 2A sequence for generating two proteins from one transcript is by ribosome skipping—a normal peptide bond is impaired at 2A, resulting in two discontinuous protein fragments from one translation event. 2A peptides include porcine teschovirus-1 (P2A, SEQ ID NO: 2), equine rhinitis A virus (E2A, SEQ ID NO: 3), thosea asigna virus (T2A, SEQ ID NO: 4), or foot-and-mouth disease virus (F2A, SEQ ID NO: 5). Table 1 shows the sequences of T2A, P2A, E2A, and F2A.
“A vector” is a nucleic acid molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. A vector is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the backbone of the vector.
The inventor has discovered a method to map the physical distribution and expansion of adoptively transferred T cells throughout the body in a longitudinal manner with transduced T cells having sufficient surface reporter molecules to provide high sensitivity of PET/CT imaging. The method significantly improves real-time monitoring of T cell activity against tumor, reduces potential toxicity from off-tumor site targeting, and contributes to exploring adjuvant therapies to enhance adoptive T cell efficacy against solid cancers.
The present invention is directed to transduced T cells that efficiently express surface reporters, e.g., sodium iodide symporter (NIS)17, prostate-specific membrane antigen (PSMA)18, or human somatostatin receptor 2 (SSTR2)19. In one embodiment, the present invention is directed to transduced T cell expressing at least 100,000 molecules of the surface reporter per T cell, or at least 300,000 molecules per T cells, or at least 600,000 molecules per T cells; preferably 1 million molecules per T cell. The high level of expression of surface reporter molecules improves PET/CT imaging sensitivity, i.e., improves a minimum number of T cells that can be detected by labelled NIS, PSMA, SSTR2 on the surface of the T cells. The site density of the expression of the surface reporter molecules on T cells, for example, can be determined by incubating non-transduced and SSTR-transduced T cells with DOTATOC/DOTATATE and then measuring the DOTATOC/DOTATATE uptake. The values obtained are used for Scatchard analysis to determine the affinity (KD) and site density (surface reporter molecules per T cell). For imaging purpose in the present invention, a full molecule of human SSTR2 (access number NP_001041) or a truncated SSTR2, containing 1-314 amino acid residues of the full molecule of human SSTR2 can be used.
In one embodiment, the T cell has been transduced with a lentivirus vector that expresses a human surface reporter such as human SSTR2, PSMA, or NIS. The present invention provides a lentivirus vector comprising an elongation factor 1a promoter and a nucleic acid molecule encoding SSTR2, PSMA, or NIS; the lentivirus vector comprises the human SSTR2 gene, PSMA gene, or NIS gene operably linked to (e.g., downstream of) the elongation factor-1α promoter, as illustrated in
The present invention is directed to a method for monitoring T cell distribution in a patient. The method can be used in adoptive T cell therapy, or in hematopoietic stem cell transplant. In a first embodiment, the surface reporter (e.g. SSTR2) is pre-labelled in vitro. The first method comprises the steps of: incubating transduced T cells that efficiently express a surface reporter with a radioactive label that binds to the surface reporter, intravenously infusing the labelled T cells into a patient, and detecting the labelled T cell distribution by PET/CT imaging. In one embodiment, the labelled T cells administered to the patient are in an amount of 104-108, or 106-108, or 106-107 cells/kg of the patient.
In a second embodiment, the surface reporter (e.g. SSTR2) is labelled post-infusion in vivo. The second method comprises the steps of: intravenously infusing the transduced T cells into a patient, injecting to the patient a radioactive label that binds to the surface reporter at least one hour prior to PCT/CT imaging, and detecting the labelled T cell distribution by PET/CT imaging. In one embodiment, the transduced T cells administered to the patient are in an amount of 104-108, or 106-108, or 106-107 cells/kg of the patient.
The present invention provides stable transduction of T cells with a specific reporter gene, which allows for extended longitudinal studies using serial infusions of reporter-specific probes. Additionally, as only live cells are capable of continually expressing the reporter gene, observed signals are limited to these cells only.
SSTR2 is a preferred surface reporter for the present invention for several reasons. One is to take advantage of the FDA-approved PET radiotracer DOTATOC or DOTATATE, which is currently in use in clinics to probe for overexpressed SSTR2 in neuroendocrine tumors43. SPECT-based imaging is also available using 111In-DTPAOC (Octreoscan)44. SSTR2 displays restricted basal expression in tissues and all major organs except in the kidneys and cerebrum making it ideal for detection of adoptively transferred T cells targeting a multitude of solid tumors. SSTR2 can potentially function as a dual reporter-suicide gene by conjugation of the therapeutic high-energy radioisotopes 177Lutetium, 90Yttrium, or 213Bismuth to DOTATOC instead of 68Gallium25,45, thus enabling elimination of SSTR2 expressing T cells in the case of CAR toxicity. SSTR2 is surface expressed and therefore does not require prior radioligand internalization into the cell. It has previously been shown that SSTR2 facilitates rapid radiotracer uptake and this combined with swift renal clearance of unbound DOTATOC means that high quality, clinical-grade images can be obtained at one hour post DOTATOC injection46. DOTATOC also has a short half-life of 68 min which, combined with its rapid clearance, delivers a low radiation dose to the patient. The fact that SSTR2 is of human origin should also limit its immunogenicity which has plagued experiments using non-human genetic reporters48,49.
The inventor has discovered that co-expression of CAR with separate population-specific reporter genes, for example SSTR2 and PSMA, followed by sequential, time-delayed injection of cognate PET radiotracers, reveals dynamics of, and interactions between, these populations in both clinical and pre-clinical studies. The present invention is directed to transduced T cells that efficiently express surface receptors, and chimeric antigen receptor (CAR). In one embodiment, the transduced T cells express at least 100,000 molecules, or at least 300,000 molecules, or at least 600,000 molecules, or at least 1 million molecules, of reporters (e.g., SSTR2 or PSMA) per T cell. In one embodiment, the CAR is specific to an intracellular adhesion molecule-1 (ICAM-1), which is overexpressed in a range of malignant cancers27-32 such as thyroid cancer, gastric cancer, pancreatic cancer, or breast cancer. In another embodiment, the CAR is specific to a tumor antigen such as CD19.
In another aspect of the invention, the inventor has designed a single lentivirus vector to engineer human primary T cells to express both a human cell surface reporter (e.g. SSTR2, PSMA or NIS) and a CAR, which is specific to ICAM-1 or CD19.
The present invention provides a lentivirus vector encoding human SSTR2 and chimeric antigen receptor (CAR) specific to human ICAM-1 or human CD19. In one embodiment, the lentivirus vector further encodes an amino acid cleavage sequence C-terminal to the human SSTR2 or the CAR, wherein the amino acid cleavage sequence comprises a 2A peptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), thosea asigna virus (T2A), or foot-and-mouth disease virus (F2A).
In the lentivirus vector of the present invention, the CAR comprises (i) a binding domain to human ICAM-1 or human CD19, (ii) a transmembrane domain, (iii) at least one co-stimulating domain, and (iv) an activating domain. The binding domain to human ICAM-1 in CAR is scFv of anti-human ICAM-1, or an I domain of the αL subunit of human lymphocyte function-associated antigen-1. The binding domain to human CD19 in CAR is scFv of anti-human CD19.
In one embodiment, the CAR of the present invention comprises (i) a human I domain that binds specifically to ICAM-1. The I domain may be a wild type I domain, or a mutant thereof having 1 to 3 mutations. I domain specific to ICAM-1 can be built using the I domain derived from LFA-1 (
Table 2 shows measured affinities of LFA-1 I domains of wild type and mutants to ICAM-1. A majority of mutations are changing hydrophobic bulky side chains (F, L, I) into more hydrophilic (A, S, T), thereby disrupting the structure of more compact, low affinity I domain conformation. For example, substitution of Phe-292 located in the C-terminal α7-helix with Ala (F292A) and Gly (F292G) provides to affinities (KD) of ˜20 μM and 0.1 respectively (Table 2). The combination of F292G with another comparably activating mutation in Phe-265 (F265S/F292G) provides an affinity of 6 nM, approximately 200,000-fold higher than the wild-type (WT) I domain (KD=1.5 mM) (
In one embodiment, the CAR of the present invention comprises I domain that is a wild-type human I domain, a mutant of wild-type human I domain having 1 to 3 amino acid mutations, or a sequence having at least 95%, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to the sequence of the wild-type I domain or the mutant, having an affinity of binding human ICAM-1 of 1 mM or stronger. In one embodiment, the mutant may have one or more mutations at the amino acid residue 265, 288, 289, 292, 295, 309, or 311 of the wild-type I domain. For example, the mutant may have one or more mutations of I288N, I309T, L295A, F292A, F292S, L289G, F292G, F265S, F265S/F292G, or F265S/F292G/G311C, of the wild-type I domain. In general, combining two I domain mutations produces a mutant with a higher affinity than that of each parent mutant. For example, combining two mutants each having about 100 μM Kd typically produces a mutant having about 1 to about 10 μM Kd range. F292G is a very potent point mutation; combining F292G with other mutations increases I domain affinity to ICAM-1 to stronger than 100 nM Kd. The above numbering of the amino acid residues is in reference to the mature amino acid sequence of SEQ ID NO: 1, and residue number 1 corresponds to the amino acid residue 26 of GenBank Accession No. NP_002200.
In one embodiment, the CAR of the present invention comprises I domain that binds ICAM-1 at an affinity between 1 mM to 1 nM Kd, preferably 1-200 μM Kd or 1-20 μM Kd.
In one embodiment, the CAR of the present invention comprises I domain that binds to ICAM-1 at an affinity between about 120 nM to about 1 nM Kd, e.g., F292G, F265S, F265S/F292G, and F265S/F292G/G311C.
In one embodiment, the CAR of the present invention comprises I domain that binds to ICAM-1 at an affinity between about 20 μM to about 120 nM Kd, e.g., F292A, F292S, and 1289G.
In one embodiment, the CAR of the present invention comprises I domain that binds to ICAM-1 at an affinity between about 200 μM to about 20 μM Kd, e.g., I288N, I309T, L295A, and F292A.
In one embodiment, the CAR of the present invention comprises I domain that binds to ICAM-1 at an affinity between about 1 μM to about 100 μM Kd, e.g., L296A, F292A and F292S.
In one embodiment, the CAR of the present invention comprises I domain that binds to ICAM-1 at an affinity between about 1 mM to about 200 μM Kd, e.g., wild-type and I288N.
In one embodiment, the CAR of the present invention comprises I domain that binds to ICAM-1 at an affinity between about 1 mM to about 100 μM Kd, e.g., wild-type, I288N, and I309T. The affinities in the above embodiments refer to the interaction between I domain and ICAM-1 in solution.
The CAR of the present invention comprises (ii) a transmembrane domain which spans the membrane. The transmembrane domain may be derived from a natural polypeptide, or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. For example, a transmembrane domain of a T cell receptor α or β chain, a CD3 zeta chain, CD28, CD3-epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or a GITR can be used. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. In preferred embodiments, the transmembrane domain is derived from CD28 or CD8, which give good receptor stability.
The CAR of the present invention comprises (iii) one or more co-stimulatory domains selected from the group consisting of human CD28, 4-1BB (CD137), ICOS-1, CD27, OX 40 (CD137), DAP10, and GITR (AITR). In embodiment, the CAR is a third generation and comprises two co-stimulating domains such as CD28 and 4-1BB.
The endodomain (the activating domain) is the signal-transmission portion of the CAR. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta (CD3 Z or CD3ζ), which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling may be needed. For example, one or more co-stimulating domains can be used with CD3-Zeta to transmit a proliferative/survival signal.
The CAR of the present invention may comprise a signal peptide N-terminal to the I domain so that when the CAR is expressed inside a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed. The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. As an example, the signal peptide may derive from human CD8 or GM-CSF, or a variant thereof having 1 or 2 amino acid mutations provided that the signal peptide still functions to cause cell surface expression of the CAR.
The CAR of the present invention may comprise a spacer sequence as a hinge to connect I domain with the transmembrane domain and spatially separate antigen binding domain from the endodomain. A flexible spacer allows to the binding domain to orient in different directions to enable its binding to a tumor antigen. The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge or a CD8 stalk, or a combination thereof. A human CD28 or CD8 stalk is preferred.
In one embodiment, the lentivirus vector includes a third generation of CAR, and the vector encodes a fusion protein comprising human SSTR2, porcine teschovirus-1 2A, a binding domain (scFv of anti-human ICAM-1, scFv of anti-human CD19, or human I domain), transmembrane domain of CD28, cytoplasmic domain of CD28, CD137, and CD 3ζ, from N-terminus to C-terminus (see
The present invention provides a nucleic acid encoding the CAR described above. The nucleic acid encoding the CAR can be prepared from an amino acid sequence of the specified CAR by a conventional method. A base sequence encoding an amino acid sequence can be obtained from the aforementioned NCBI RefSeq IDs or accession numbers of GenBenk for an amino acid sequence of each domain, and the nucleic acid of the present invention can be prepared using a standard molecular biological and/or chemical procedure. For example, based on the base sequence, a nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR).
The nucleic acid encoding the CAR of the present invention can be inserted into a vector, and the vector can be introduced into a cell. For example, a virus vector such as a retrovirus vector (including an oncoretrovirus vector, a lentivirus vector, and a pseudo type vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a Sendai virus vector, an Epstein-Barr virus (EBV) vector, and a HSV vector can be used. As the virus vector, a virus vector lacking the replicating ability so as not to self-replicate in an infected cell is preferably used.
For example, when a retrovirus vector is used, the process of the present invention can be carried out by selecting a suitable packaging cell based on a LTR sequence and a packaging signal sequence possessed by the vector and preparing a retrovirus particle using the packaging cell. Examples of the packaging cell include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. A retrovirus particle can also be prepared using a 293 cell or a 293T cell having high transfection efficiency. Many kinds of retrovirus vectors produced based on retroviruses and packaging cells that can be used for packaging of the retrovirus vectors are widely commercially available from many companies.
The present invention provides T cells modified to express a reporter molecule (e.g., SSTR2, PMSA, or NIS) and the CAR as described above. CAR-T cells of the present invention bind to ICAM-1 or CD19 via anti-ICAM-1 antibody, or I domain, or anti-CD19 antibody of CAR, thereby a signal is transmitted into the cell, and as a result, the cell is activated. The activation of the cell expressing the CAR is varied depending on the kind of a host cell and an intracellular domain of the CAR, and can be confirmed based on, for example, release of a cytokine, improvement of a cell proliferation rate, change in a cell surface molecule, killing target cells, or the like as an index.
T cells modified to express SSTR2 and CAR can be used as a therapeutic agent for a disease. The therapeutic agent comprises the T cells expressing the I domain-CAR as an active ingredient, and may further comprise a suitable excipient. Examples of the excipient include pharmaceutically acceptable excipients known to a person skilled in the art.
The invention is further directed to a method for treating cancer and monitoring CAR T cell distribution in a patient. The method comprises the steps of: incubating the transduced CAR T cells with a radioactive label that binds to SSTR2, intravenously infusing the labelled CAR T cells into a patient, detecting the labelled CAR T cell distribution by PET/CT imaging, and infiltrating the labelled CAR T cells into cancer cells to kill cancer cells. In this method, SSTR2 is pre-labelled in vitro. In one embodiment, the labelled CAR T cells is administered in an amount of 104-108, or 106-108, or 106-107 cells/kg of the patient.
In an alternative method, the method comprises the steps of: intravenously infusing the transduced CAR T cells into a patient, injecting to the patient a radioactive label that binds to SSTR2 at least one hour prior to PCT/CT imaging, detecting the labelled CAR T cell distribution by PET/CT imaging, and infiltrating the labelled CAR T cells into cancer cells to kill cancer cells. In this method, SSTR2 is labelled post-infusion in vivo. In one embodiment, the transduced CAR T cells is administered in an amount of 104-108, or 106-108, or 106-107 cells/kg of the patient.
Adoptively transferred T cells have been shown to penetrate and distribute throughout tumor tissue12. The utility of any imaging modality capable of monitoring ACT therefore depends upon a limit-of-detection threshold sensitive enough to enable monitoring of meaningful T cell activity at low tissue densities. Moreover, evaluation of this detection limit in a physiologically relevant model must also account for the dynamic sensitivity and specificity of the infused imaging agent for the reporter in question, whose density at the tumor site will vary exponentially as its cognate T cells expand and contract in response to target interaction. Therefore, to approximate the DOTATOC-based detection limit of tumor infiltrating SSTR2-transduced T cells, the inventor utilized mosaic tumor xenografts of Jurkat T cells with increasing ratios of SSTR2 expression to create a standard by which quantitative PET signals can be related to T cells of known density within tumors. Using the lentivirus developed in this study, the surface expression of SSTR2 in transduced Jurkat T cells was in the range of several million per cell, a level significantly higher than previously published19 (36,000 copies per Jurkat T cell) and therefore extending the lower-limit of SSTR2+ T cell detection.
The inventor observed that with a known threshold of radiotracer uptake, one can detect tumor-infiltrating T cells down to a minimum density of 0.8% or ˜4×106 cells/cm3, with 95% specificity and 87% sensitivity. This compares favorably to a previous report using PET to detect tumor-infiltrating T cells that used flow cytometry-based detection in a separate, equally treated cohort of mice as a reference to purport a tumor-infiltrating T cell detection limit of 0.5% within tumors53.
To demonstrate the feasibility of SSTR2 reporter-based imaging to predict and monitor tumor-directed T cell activity, the inventor chose ICAM-1 positive anaplastic thyroid cancer cells as a target model and engineered T cells to express both ICAM-1-specific CAR and SSTR2 using a single lentiviral vector. A potential drawback to the use of genetic reporters for imaging receptor-modified ACT is that the T cells in question require the stable expression of two separate genes, which can substantially reduce the percentage of cells co-expressing both genes. However, with the use of a self-cleaving ‘P2A’ sequence39, the inventor demonstrated that both CAR and reporter genes can be successfully expressed on the same individual cell without compromising the level of expression otherwise achievable using independent vectors. T cells expressing the R6.5 CAR efficiently and specifically lysed ICAM-1 expressing 8505C and HeLa tumor cell lines, as monitored by bioluminescence, within 24 hr whilst leaving ICAM-1 negative HEK293 cells largely untouched. Furthermore, efficient killing was also observed at lower E:T ratios, thus demonstrating both specificity and high activity of the R6.5 CAR for ICAM-1 expressing target cells. This targeting efficacy was replicated in vivo as treatment of 8505C tumor-bearing mice with SSTR2-R6.5-CAR T cells resulted in a significant reduction in tumor burden across all treated mice compared to those that received T cells expressing SSTR2 only. Tumor lysis occurred 1-2 weeks post treatment with the time required for CAR mediated tumor reduction correlating with burden at time of treatment. Expanding SSTR2-R6.5-CAR T cells at the tumor site were visualized by increasing DOTATOC signals. PET imaging of SSTR2-R6.5-CAR T cells was sensitive enough to visualize their perfusion throughout the lungs such that the lung footprint and outline became distinguishable by the presence of T cells alone. This was confirmed by histological analysis of tissue sections, which demonstrated the ubiquitous presence of human CD3+ T cells throughout the lungs of treated mice. Despite the small size (several mm) of metastatic nodules in the liver, localized DOTATOC accumulation was also recorded which coincided with the emergence, followed by attenuation, of distinct liver tumor nodules as detected by bioluminescence imaging. Extrapolation of the image quality and sensitivity obtained in the current study to similar scenarios in humans would likely enable equal if not better monitoring of CAR tracking to primary, metastatic and critically, to on-target, off-tumor-sites. Visualization of CAR expansion may even draw attention to previously undetected metastatic sites. Therefore, sufficiently sensitive reporter imaging of ACT may provide additional prognostic capabilities.
Due to reported dose-limiting toxicity, the number of modified T cells infused to patients is typically in the range of 1-10×106 T cells/kg2,3,41,54 and target-mediated expansion and persistence of T cells is therefore a prerequisite for substantive tumor destruction to occur. Indeed, it has been reported that higher peak expansion of infused T cells correlates with increased rates of disease remission2,54. Tumor-bearing mice in the current study were treated with approximately 1.5×106 SSTR2-CAR+ T cells at days 7-15 post xenograft. Subsequent longitudinal monitoring of both tumor growth and CAR expansion at the tumor site enabled several observations to be made. Timely infusion of CAR T cells resulted in survival of all subjects without any weight loss, while a later treatment led to uniform death despite the evidence of tumor killing by CAR T cells.
Survivors exhibited a biphasic pattern of DOTATOC uptake within the lungs, with a similar luminescence pattern observed regarding primary lung tumors. A notable exception to this correlative pattern was that peak DOTATOC signal, and therefore peak T cell expansion, lagged behind peak tumor burden by several days. This expansion of T cells past the onset of target elimination may result from enduring cognate antigen mediated signals causing continued CAR T cell expansion before eventual exhaustion and contraction occurred55. Swift contraction of CAR T cells following peak expansion indicates that target antigen density has fallen to levels no longer capable of sustaining CAR expansion and that tumor elimination was achieved without immediate relapse. The biphasic pattern of DOTATOC uptake in survivors stood in stark contrast to the unrelenting increases in both T cell and tumor burdens in non-survivors where tumor growth was evidently surpassing the rate of killing by T cells. It would be interesting to investigate whether a similar pattern is observed in additional tumor models and in clinical studies. Finally, DOTATOC uptake values obtained in the longitudinal study enabled comparisons with uptake values derived from the SSTR2-titrated Jurkat model. This indicated that peak CAR T cell density in 8505C tumors ranged from below 1% at infusion to ˜10% in survivors and to well over 10% in mice with high tumor burden.
In summary, the inventor utilized a genetic reporter, somatostatin receptor 2 (SSTR2) and positron emission tomography (PET) to quantitatively and longitudinally visualize whole body T cell distribution and anti-tumor dynamics. SSTR2-based PET was applied to an ACT model using chimeric antigen receptor (CAR) T cells specific to intercellular adhesion molecule-1 that is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in the survival of all subjects bearing rapidly growing tumors, while later T cell infusions led to uniform death. Quantitative, longitudinal PET imaging of T cells revealed a biphasic expansion and contraction response among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, non-survivors displayed unrelenting increases of both tumor burden and T cell number, indicating that the rate of tumor growth was outpacing that of T cell killing. The inventor demonstrates that the prognostic utility of SSTR2-based longitudinal imaging, directly relating biphasic CAR T cell actions to tumor elimination, may apply to close monitoring of ACT efficacy and overall response in patients.
The inventor has demonstrated a clinically adaptable, quantitative imaging system capable of specifically detecting adoptively transferred CAR T cells and monitoring their target-specific expansion and contraction at the tumor site with unprecedented detail. A simple method for estimating the density of solid tumor-infiltrating T cells has also been established. The inventor anticipates that the SSTR2 system can be easily adapted to multiple ACT models and can facilitate efforts towards increasing our understanding of the parameters behind the success and failures of ACT with particular regard to monitoring systemic toxicities and the responses to solid tumors.
The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.
Parental HeLa, HEK 293 (ATCC), and 8505C (DSMZ, Germany) cells were transduced with lentivirus encoding Firefly Luciferase-F2A-GFP (Biosetta) followed by fluorescence activated cell sorting (FACS) to purify GFP expressing cells. HeLa-FLuc+GFP+ and HEK 293-FLuc+GFP+ cells were cultured in Advanced Dulbecco's Modified Eagle Medium containing 10% (v/v) fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine dipeptide (Gibco), and 100 U/ml Penicillin-Streptomycin (Pen/Strep) (Gibco). 8505C-FLuc+GFP+ cells were cultured in RPMI-1640 supplemented with 10% (v/v) FBS, 2 mM L-alanyl-L-glutamine dipeptide, and 100 U/ml Pen/Strep. Human peripheral blood was obtained from healthy volunteer donors by venipuncture. This protocol is approved by an Institutional Review Board of Weill Cornell Medicine (Permit Number: #1302013613). Peripheral blood mononuclear cells (PBMC) were isolated over Ficoll-Paque PLUS (GE Healthcare) and cultured in Optimizer CTS T-cell Expansion SFM (Thermo) supplemented with 5% human AB serum (Sigma), 2 mM L-alanyl-L-glutamine dipeptide, 100 U/ml Pen/Strep and 30 IU/ml human IL-2 (Cell Sciences). Non-adherent cells were removed after 24 hr and magnetically enriched for T cells with Dynabeads CD3/CD28 T cell expander (Thermo) at a 2:1 bead:T cell ratio. Dynabead-bound T cells were subsequently cultured in IL-2 containing media at a density of 1-2×106 cells/ml. All cells were incubated at 37° C. in a 5% CO2 humidified incubator.
The CAR gene specific to ICAM-1 was derived from the scFv sequence of a murine monoclonal anti-human R6.5 antibody56,57—itself derived from hybridoma (ATCC). The R6.5 specific scFv was then fused to the transmembrane and cytoplasmic domains of CD28, CD137, and CD3ζ of an independent third generation pLenti plasmid (a kind gift from Dr. Carl June at PENN35). A lentivirus vector (derived from CAR vector) encoding human SSTR2 (NM_001050) was constructed by synthesizing SSTR2 coding sequencing (IDT) and inserting it into the vector using Xba1 and Sal1 sites.
Genetic sequences encoding LFA-1 I domains of varying affinities to ICAM-1 were derived from a previous study62. I domain variants were fused at the C-terminus directly to the CD8 hinge, CD28 transmembrane domain, and the intracellular portions of the 3rd generation CAR architecture incorporating the cytoplasmic domains of CD28, CD137, and CD3ζ. The complete CAR inserts were then subcloned into a pLenti backbone35. A reporter gene for CAR T cell imaging, SSTR2, was linked to I domain at the N-terminus using a ‘ribosome skipping’ porcine teschovirus-1 2A (P2A) sequence to ensure comparable production of CAR and SSTR2 from the same mRNA.
Lentivirus particles were produced by transiently transfecting HEK 293 cells using calcium phosphate. Briefly, 10 μg transfer gene, 7.5 μg CMV-dR8.2 (Addgene) and 5 pCMV-VSVG (Addgene) were mixed and incubated with 2 M CaCl2 followed by 2×HBSS. Resulting solutions were added dropwise to 10 cm2 cell culture dishes seeded with 3.2×106 HEK 293 in 10 ml DMEM 24 hr previously. Transfection media was replaced after 6 hr. Media containing lentivirus was harvested at 48 and 72 hr post transfection, filtered through 0.45 μm filters and concentrated by ultracentrifugation at 75,000×g for 2 hr at 4° C. Lentivirus was then resuspended in serum containing media at an approximate titer of 108/ml and frozen at −80° C. Human T cells were transduced 24-72 hr post activation with CD3/CD28 Dynabeads either by spinfection at 1,000× g for 1 hr at 32° C. or by overnight incubation of lentivirus in the presence of Synperonic F108 (Sigma)58. T cells were also transduced a second time, 24 hr after initial transduction. The virus titer was adjusted to obtain a transduction level of approximately 50%. During and following transduction, media containing IL-2 was replaced with media containing human IL-7 (10 ng/ml) and IL-15 (5 ng/ml) (Peprotech) which was found to augment T cell persistence in vitro59,60 Jurkat T cells were transduced by a single incubation with lentivirus overnight in the presence of Synperonic F108.
SSTR2-transduced Jurkat T cells were incubated with or without octreotide, 1 μM (Sigma) for 30 min at 37° C. Subsequent internalization of SSTR2 was measured by flow cytometry analysis of SSTR2 expression. The site density of SSTR2 expression on Jurkats was determined by incubating non-transduced and SSTR2-transduced Jurkat T cells with DOTATOC (250 nM-8 nM) at either 37° C. or 4° C. for 30 min in PBS/0.1% BSA. After incubation, cells were washed three times and DOTATOC uptake was measured using a gamma counter (Packard, Cobra II Auto—Gamma). Values obtained were used for Scatchard analysis to estimate affinity and site density.
All animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This study's animal protocol was approved by the Institutional Laboratory Animal Use and Care Committee of Weill Cornell Medicine. SSTR2 expressing Jurkats were spiked with increasing numbers of non-transduced Jurkats to derive distinct cultures containing defined percentages of SSTR2 expression ranging from 100-0%. For each subcutaneous xenograft, 5×106 total cells were resuspended in 100 μl Matrigel Basement Membrane Matrix (Corning) and injected bilaterally into nonobese diabetic (NOD)/LtSz Prkdcscid Il2rgtm1Wjl/J (NSG) mice (Jackson Laboratory). Measurements of tumor size were made using an external digital caliper. Tumor volume was calculated by using the modified ellipsoid formula ½(Length×Width). Length was measured as the longer dimension. Each dimensional measurement was rounded to the nearest 0.5 cm.
DOTATOC (1,4,7,10-tetraazacyclododecane-NI,NII,NIII,NIIII-tetraacetic acid (D)-Phe1-Tyr3-octreotide, GMP grade) was obtained as a 1 mg lyophilized powder (ABX Pharmaceuticals). The DOTATOC vial content was diluted with 18 MW water to 2 ml (0.5 mg/ml solution) and stored at 4° C. as a stock solution. 68Ga was obtained by eluting an ITG 68Ge/68Ga generator (ITM) with 4 ml 0.05M HCl solution. To the eluted 68Ga3+, 50 μl of the DOTATOC stock solution (25 μg) was added, followed by 80 μl of a 3 N NaOAc solution for buffering. The mixture was immediately placed in a Thermomixer (Eppendorf) at 95° C. and incubated for 15 minutes. Following incubation the mixture was passed through a previously activated C-18 Sep-Pak Lite (Waters) to trap the labeled peptide. The labeling vial was washed with 5 ml 18 MW water and the resulting wash was also passed through the C-18 Sep-Pak. Finally, the Sep-Pak was washed with an extra 5 ml of 18 MW water to eliminate any remaining free 68Ga. The trapped 68Ga-DOTATOC was then slowly eluted from the C-18 Sep-Pak using 100 μl of ethanol followed by 900 μl of saline solution for injection, providing the final product in a 10% EtOH isotonic, injectable solution. The purity of the final product was checked by reverse phase HPLC.
2×105HeLa-FLuc+GFP+, 8505c-FLuc+GFP+ or HEK 293-FLuc+GFP+ cells were co-cultured with either non-transduced or CAR expressing T cells (SSTR2-R6.5 or R6.5) at varying E:T ratios as indicated. Co-cultures were carried out in ‘T cell media’ containing 150 μg/ml D-Luciferin (Gold Biotechnology) with no cytokine supplementation. Luminescence was measured using a plate reader (TECAN infinite M1000 PRO) with readings in each E:T condition normalized to the non-transduced T cell:target co-culture controls.
1×106 8505c-FLuc+GFP+ cells were injected into NSG mice via tail vein. 2-3×106 primary human T cells were injected via tail vein 7-15 days after tumor cell injection. Luminescence imaging of tumor xenografts in live mice was performed using a whole body optical imager (In-Vivo Extreme 4MP, Bruker). Mice were first anesthetized with 3% isoflurane at 2 L/min O2 and subsequent to this, maintained at 2% isoflurane at 2 L/min O2. Growth or reduction in tumor burden was estimated by integration of luminescence over the lungs or the entire mouse body. Ex-vivo fluorescence imaging of mouse liver, lungs, spleen and resected tumors were performed using a whole body optical imager (In-Vivo F Pro, Bruker).
Registered CT images were acquired using a micro-PET/CT scanner (Inveon, Siemens) at 1-2 hr post DOTATOC injection. Projection data was acquired in a cone-beam geometry with approximately 1s steps at 1 degree angular increments. At least 10 million coincidence events were acquired for PET per study using a 250 to 750 keV energy window and a 6 ns timing window. A reference was included using a tube containing 100 μl of 10% ID/cm3 for quantification of DOTATOC uptake in vivo. To compute DOTATOC uptake by Jurkat tumors, the ellipsoidal ROIs (Amide) were placed to enclose subcutaneous tumors that closely match overall tumor size and shape. For systemic 8505C tumor models, ellipsoids were drawn separately on the left and right sides of lungs to enclose much of five lobes of mouse lungs. The % ID/cm3 values, computed relative to the counts in a reference tube, can be approximated to a standard uptake value (SUV61) by dividing % ID/cm3 by four, assuming injection efficiency of 100% and 25 g of body weight. Visualization and analyses of PET/CT images were performed using Amide (amide.sourceforge.net).
Jurkat tumor xenografts or mouse organs (lungs and liver) were harvested from mice following completion of PET/CT imaging. Tissues were diced and flushed through an 80 μm cell strainer to yield single cell suspensions. Red blood cells were lysed by incubating cell suspension with 1× Ammonium-Chloride-Potassium lysing buffer (ThermoFisher), followed by washing and re-suspension in 1× HBSS containing 2% normal goat serum. Prior to staining, cells were blocked with mouse IgG at 2 μg/ml for 10 min. This was followed by live staining with 1 μg/ml Propidium Iodide (Invitrogen) in combination with 2 μg/ml murine anti-human CD3-Alexa Fluor 647 (Biolegend) or 2 μg/ml PE-conjugated murine anti-human SSTR2 (Clone #402038, R&D). Flow cytometry gates were determined first based on live cell gating (Propidium Iodide negative) and subsequently by staining of respective antibodies. ICAM-1 expression on tumor cell lines was determined using a murine anti-human R6.5 monoclonal antibody (10 μg/ml) derived from hybridoma (ATCC)57. R6.5-CAR expression on T cells was detected using FITC-conjugated goat anti-mouse F(ab′)2 secondary antibody (Thermo).
Jurkat tumor xenografts were harvested, fixed in 4% paraformaldehyde in PBS, embedded in paraffin, and were cut to produce 5 μm sections (Microtome, Leica). Paraffin embedded sections were stained with hematoxylin and eosin (H&E) or hematoxylin only for CD3 immunostaining (performed by HistoWiz, Inc.). After euthanasia, mouse lungs were perfused via trachea with 4% paraformaldehyde, and each of five lobes was separated post fixation and embedded in paraffin. Liver tissue was harvested and processed identically for histology. Histological analysis was performed by an experienced pathologist.
We constructed a lentivirus vector for expression of SSTR2 by inserting the human SSTR2 gene downstream of the elongation factor-1α promoter35. With increasing lentivirus titer, 100% of Jurkat T cells were transduced to express SSTR2 as measured by antibody binding (
To examine the utility of SSTR2 for the detection of sparsely distributed T cells in tumors, we produced subcutaneous Jurkat T cell xenografts in mice with a mixture of SSTR2-transduced (referred to as SSTR2+) and non-transduced (wild-type) cells. These were titrated against each other immediately prior to xenografting to vary the levels of SSTR2 expressing cells within tumors from 0% to approximately 100%. Xenografted Jurkat T cell tumors began to show palpable growth at ˜30 days post xenograft and exhibited continuous growth for the next 25 days, reaching approximately 0.7 cm3 (
We next analyzed DOTATOC uptake values to determine the detection sensitivity and specificity of SSTR2-expressing T cell density within tumors. Regarding small, palpable tumors (below ˜65 mm3), DOTATOC uptake was slightly higher in those containing 1% SSTR2+ cells (0.64±0.4% ID/cm3; p=0.1 vs. 0% SSTR2+), but was significantly higher in tumors containing SSTR2+ T cell densities of 10% (0.83±0.5% ID/cm3) and 100% (3.4±1.7% ID/cm3), compared to uptake within 0% SSTR2+ tumors (0.44±0.2% ID/cm3) (
To express the observed SSTR2+ T cell tumor density as an absolute number per volume, we resected Jurkat tumors and determined the average total cell density to be 5.1±1.3×108/cm3 (n=4). Accordingly, 1% SSTR2+ tumors (comprising approximately 4×108/cm3 Jurkat T cells and 1×108/cm3 stroma) would contain ˜4×106 SSTR2 positive T cells per cm3. Based on the data obtained using the Jurkat model, we proceeded to utilize SSTR2-based PET imaging to estimate and longitudinally monitor the density of reporter-expressing CAR T cells infiltrating solid tumors.
One-way ANOVA and unpaired Student's t-test were performed using Prism 7 (GraphPad) on data indicated.
Radioiodine resistant, poorly differentiated thyroid cancers have been found to overexpress ICAM-1 at levels correlating with tumor malignancy and metastatic potential30. The anaplastic thyroid cancer cell line 8505C was also found to be ICAM-1 positive (
In order to test the SSTR2-based reporter system's ability to acquire in vivo visual mapping of CAR T cell localization and anti-tumor dynamics, NSG mice were first xenografted by systemic injection of 1×106 8505 C-FLuc+GFP+ cells. Bioluminescence imaging demonstrated that primary 8505C tumors localized to the lungs with metastases occurring within the liver and at more distant sites (
After confirming the relative absence of lung-specific DOTATOC uptake related to tumor burden itself or SSTR2-negative T cell expansion, we then treated tumor-bearing mice with SR T cells at day 7, 10, 13, and 15 post xenograft. Mice were subjected to longitudinal bioluminescence and concurrent PET/CT imaging to visualize the relationship between tumor burden and the dynamism of infused SR T cell numbers over the course of their localization, tumor engagement, and killing (
Assessment of CAR T cell-mediated tumor killing by ex vivo fluorescence imaging of the lungs and liver (performed on day 28 post-tumor xenograft) confirmed that tumor elimination had indeed occurred in survivors treated with SR T cells on 7-10 days post tumor xenograft (
In contrast to the more evenly distributed and synchronized growth of tumors in the lungs, metastatic lesions in the liver were distinctive, isolated and smaller, measuring only several mm in size (
To spatiotemporally monitor T cell distribution in real-time by PET/CT, we introduced an imaging reporter gene, SSTR2 into the I domain CAR vector using a ribosome skipping P2A sequence to ensure equal expression of CAR and the reporter on the surface of T cells (
Mice were xenografted with 8505C tumors as before, and were treated with NT or F292A CAR T cells. Whole-body luminescence imaging was performed to estimate tumor burden while PET/CT imaging was performed on the same day to track CAR T cell distribution (
Cytokine analysis of serum obtained from treated mice demonstrated a surge in IFN-γ, IL-6, and CXCL10 concentrations prior to peak T cell expansion, which also returned to background levels post tumor elimination and following contraction of T cell density in the lungs to background levels (
To demonstrate broad applicability of SSTR2-based imaging of CAR T cells, we used Burkitt lymphoma (Raji) xenograft, which is one of the tumor model being used by others for validation of CD19-specific CAR T cells63,64. Similar to findings of ICAM-1 overexpression in hematological cancer (including multiple myeloma65), we found overexpression of ICAM-1 (˜3×105/cell) in Raji cells. Our study confirmed the characteristics of Raji tumor growth, mainly appearing in the central nervous system (subarachnoid space, brain, and spine), liver, bones, and lymph nodes (
It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims.
This application is a divisional application of U.S. application Ser. No. 15/675,419, filed Aug. 11, 2017; which claims the benefit of U.S. Provisional Application Nos. 62/383,139, filed Sep. 2, 2016; and 62/419,817, filed Nov. 9, 2016; which are incorporated herein by reference in their entirety.
This invention was made with government support under grant CA178007 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62419817 | Nov 2016 | US | |
| 62383139 | Sep 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15675419 | Aug 2017 | US |
| Child | 16787539 | US |
