Patent Application
20230303976
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 97,590 Byte XML file named “766736.XML,” dated Feb. 27, 2023.
Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) or cells that have been genetically engineered to express an anti-cancer antigen T cell receptor (TCR) can produce positive clinical responses in some cancer patients. Nevertheless, obstacles to the successful use of ACT for the widespread treatment of cancer and other diseases remain. For example, T cells and TCRs that specifically recognize cancer antigens may be difficult to identify and/or isolate from a patient. Accordingly, there is a need for improved methods of obtaining cancer-reactive T cells and TCRs.
An embodiment of the invention provides a method of isolating T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising obtaining a bulk population of peripheral blood mononuclear cells (PBMCs) from a sample of peripheral blood from a patient; selecting T cells that express programmed cell death 1 (PD-1) from the bulk population; separating the T cells that express PD-1 from cells that do not express PD-1 to obtain a T cell population enriched for T cells that express PD-1; identifying one or more genes in the nucleic acid of a cancer cell of the patient, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence; inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequence; co-culturing T cells from the population enriched for T cells that express PD-1 with the autologous APCs that present the mutated amino acid sequence; and selecting the T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a major histocompatibility complex (MHC) molecule expressed by the patient.
Another embodiment of the invention provides a method of isolating T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising obtaining a first population of PBMCs from a sample of peripheral blood from a patient; selecting T cells that express PD-1 from the bulk population; separating the T cells that express PD-1 from cells that do not express PD-1 to obtain a T cell population enriched for T cells that express PD-1; isolating nucleotide sequence(s) that encode(s) one or more TCR(s), or antigen-binding portion(s) thereof, from the T cells of the population enriched for T cells that express PD-1; introducing the nucleotide sequence(s) encoding the TCR(s), or antigen binding portion(s) thereof, into further population(s) of PBMCs to obtain T cells that express the TCR(s), or antigen binding portion(s) thereof; identifying one or more genes in the nucleic acid of a cancer cell of the patient, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence; inducing autologous APCs of the patient to present the mutated amino acid sequence; co-culturing the T cells that express the TCR(s), or antigen binding portion(s) thereof, with the autologous APCs that present the mutated amino acid sequence; and selecting the T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a MHC molecule expressed by the patient.
Another embodiment of the invention provides an isolated or purified TCR comprising the amino acid sequences of (a) SEQ ID NOs: 5-10; (b) SEQ ID NOs: 13-18; (c) SEQ ID NOs: 21-26; (d) SEQ ID NOs: 29-34; or (e) SEQ ID NOs: 37-42.
Another embodiment of the invention provides an isolated or purified polypeptide comprising the amino acid sequences of (a) SEQ ID NOs: 5-10; (b) SEQ ID NOs: 13-18; (c) SEQ ID NOs: 21-26; (d) SEQ ID NOs: 29-34; or (e) SEQ ID NOs: 37-42.
An isolated or purified protein comprising (a) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 5-7 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 8-10; (b) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 13-15 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 16-18; (c) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 21-23 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 24-26; (d) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 29-31 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 32-34; or (e) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 37-39 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 40-42.
Additional embodiments of the invention provide related nucleic acids, recombinant expression vectors, host cells, populations of cells, pharmaceutical compositions, and methods of treating or preventing cancer.
An embodiment of the invention provides a method of isolating T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. The invention provides many advantages. For example, the inventive methods may, advantageously, obtain cancer antigen-reactive T cells from a patient's peripheral blood, which is a more accessible and abundant source of T cells as compared to other tissues such as, for example, tumor. By obtaining cancer antigen-reactive T cells from the peripheral blood, the inventive methods may, advantageously, obtain cancer antigen-reactive T cells without using invasive techniques such as, for example, surgery or biopsy, which may be required when obtaining T cells from other tissues such as, for example, a tumor. Cancer antigen-reactive T cells are not frequently found in the peripheral blood. Nevertheless, the inventive methods overcome this obstacle, and effectively and efficiently identify and enrich for these infrequent, cancer antigen-reactive T cells from the peripheral blood. In addition, the inventive methods make it possible to administer ACT to patients that have no tumors available for TIL harvest. The inventive methods may also reduce the cost of ACT, making ACT available for a larger number of patients.
Moreover, the inventive methods may rapidly assess a large number of mutations restricted by all of the patient's MHC molecules at one time, which may identify the full repertoire of the patient's mutation-reactive T cells. Additionally, by distinguishing immunogenic cancer mutations from (a) silent cancer-specific mutations (which do not encode a mutated amino acid sequence) and (b) cancer-specific mutations that encode a non-immunogenic amino acid sequence, the inventive methods may identify one or more cancer-specific, mutated amino acid sequences that may be targeted by a T cell, a TCR, or an antigen-binding portion thereof. The mutated amino acid sequences could be used to synthesize peptides and immunize patients to treat or prevent cancer recurrence. In addition, the invention may provide T cells, TCRs, and antigen-binding portions thereof, having antigenic specificity for mutated amino acid sequences encoded by cancer-specific mutations that are unique to the patient, thereby providing “personalized” T cells, TCRs, and antigen-binding portions thereof, that may be useful for treating or preventing the patient's cancer. The inventive methods may also avoid the technical biases inherent in traditional methods of identifying cancer antigens such as, for example, those using cDNA libraries, and may also be less time-consuming and laborious than those methods. For example, the inventive methods may select mutation-reactive T cells without co-culturing the T cells with tumor cell lines, which may be difficult to generate, particularly for e.g., epithelial cancers. Without being bound to a particular theory or mechanism, it is believed that the inventive methods may identify and isolate T cells and TCRs, or antigen-binding portions thereof, that target the destruction of cancer cells while minimizing or eliminating the destruction of normal, noncancerous cells, thereby reducing or eliminating toxicity. Accordingly, the invention may also provide T cells, TCRs, or antigen-binding portions thereof, that successfully treat or prevent cancer such as, for example, cancers that do not respond to other types of treatment such as, for example, chemotherapy alone, surgery, or radiation.
The method may comprise obtaining a bulk population of PBMCs from a sample of peripheral blood of a patient by any suitable method known in the art. Suitable methods of obtaining a bulk population of PBMCs may include, but are not limited to, a blood draw and/or a leukapheresis. The bulk population of PBMCs obtained from a peripheral blood sample may comprise T cells, including tumor-reactive T cells.
The method may comprise selecting T cells that express PD-1 from the bulk population. In an embodiment of the invention, the T cells that express PD-1 may be PD-1hi cells. In a preferred embodiment, selecting T cells that express PD-1 from the bulk population comprises selecting T cells that co-express (a) PD-1 and (b) any one or more of CD3, CD4, CD8, T cell immunoglobulin and mucin domain 3 (TIM-3), and CD27. In an embodiment of the invention, the cells that express CD3, CD4, CD8, TIM-3, or CD27 may be CD3hi, CD4hi, CD8hi, TIM-3hi, or CD27hi cells, respectively. The method may comprise specifically selecting the cells in any suitable manner. Preferably, the selecting is carried out using flow cytometry. The flow cytometry may be carried out using any suitable method known in the art. The flow cytometry may employ any suitable antibodies and stains. For example, the specific selection of PD-1, CD3, CD4, CD8, TIM-3, or CD27 may be carried out using anti-PD-1, anti-CD3, anti-CD4, anti-CD8, anti-TIM-3, or anti-CD27 antibodies, respectively. Preferably, the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected. The antibody or antibodies may be conjugated to a bead (e.g., a magnetic bead) or to a fluorochrome. Preferably, the flow cytometry is fluorescence-activated cell sorting (FACS).
In an embodiment of the invention, selecting may comprise specifically selecting PD-1+ T cells that are also positive for expression of (i) any one of CD4, CD8, TIM-3, and CD27; (ii) both of CD8 and TIM-3; (iii) both of CD8 and CD27; (iv) both of TIM-3 and CD27; (v) all three of CD8, TIM-3, and CD27; (vi) both of CD4 and TIM-3; (vii) both of CD4 and CD27; or (viii) all three of CD4, TIM-3, and CD27. In another embodiment of the invention, any one or more of the populations of (i)-(viii) may also co-express CD3.
In an embodiment of the invention, selecting T cells that express PD-1 from the bulk population comprises selecting any one or more of (a) CD8+PD-1+; (b) PD-1+TIM-3+; (c) PD-1+CD27+; (d) CD8+PD-1hi; (e) CD8+PD-1+TIM-3+; (0 CD8+PD-1+CD27hi; (g) CD8+PD-1+CD27+; (h) CD8+PD-1+TIM-3−; (i) CD8+PD-1+CD27−; (j) CD4+PD-1+; (k) CD4+PD-1hi; (l) CD4+PD-1+TIM-3+; (m) CD4+PD-1+CD27hi; (n) CD4+PD-1+CD27+; (o) CD4+PD-1+TIM-3−; and (p) CD4+PD-1+CD27− T cells. In another embodiment of the invention, any one or more of the populations of (a)-(p) may also co-express CD3.
As used herein, the term “positive” (which may be abbreviated as “+”), with reference to expression of the indicated cell marker, means that the cell expresses the indicated cell marker at any detectable level, which may include, for example, expression at a low (but detectable) level as well as expression at a high (hi) level. The term “negative” (which may be abbreviated as “−”), as used herein with reference to expression of the indicated cell marker, means that the cell does not express the indicated cell marker at a detectable level. The term “high” (which may be abbreviated as “hi”), as used herein with reference to expression of the indicated cell marker, refers to a subset of cells that are positive for expression of the indicated cell marker which stain more brightly for the indicated cell marker using one of the following methods (e.g., FACS, flow cytometry, immunofluorescence assays or microscopy) than other cells that are positive for expression of the indicated cell marker. For example, cells with a “high” level of expression of the indicated cell marker may stain more brightly than about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%, or a range of any two of the foregoing values, of the other cells that are positive for expression of the indicated cell marker.
In an embodiment of the invention, selecting T cells that express PD-1 may comprise selecting combinations of PD-1+ cells, each PD-1+ cell expressing any one, two, or more different markers as described herein. In this regard, the method may produce a cell population that is enriched for tumor-reactive cells that comprises a mixture of PD-1+ cells, each PD-1+ cell expressing any one, two, or more different markers described herein. In an embodiment of the invention, selecting T cells that express PD-1 comprises selecting a combination of (i) both PD-1+CD8+ cells and PD-1+TIM-3+ cells; (ii) both PD-1+CD8+ cells and PD-1+CD27+ cells; (iii) both PD-1+TIM-3+ cells and PD-1+CD27+ cells; (iv) all of PD-1+CD8+ cells, PD-1+TIM-3+ cells, and PD-1+CD27+ cells; (v) both PD-1+CD4+ cells and PD-1+TIM-3+ cells; (vi) both PD-1+CD4+ cells and PD-1+CD27+ cells; (vii) all of PD-1+CD4+ cells, PD-1+TIM-3+ cells, and PD-1+CD27+ cells, or (viii) a combination of any of the populations of (i)-(vii). In another embodiment of the invention, any one or more of the populations of (i)-(vii) may also co-express CD3. In another embodiment of the invention, selecting T cells that express PD-1 comprises selecting a combination of any two or more of (a) CD8+PD-1+; (b) PD-1+TIM-3+; (c) PD-1+CD27+; (d) CD8+PD-1hi; (e) CD8+PD-1+TIM-3+; (f) CD8+PD-1+CD27hi; (g) CD8+PD-1+CD27+; (h) CD8+PD-1+TIM-3−; (i) CD8+PD-1+CD27−; (j) CD4+PD-1+; (k) CD4+PD-1hi; (l) CD4+PD-1+TIM-3+; (m) CD4+PD-1+CD27hi; (n) CD4+PD-1+CD27+; (o) CD4+PD-1+TIM-3−; and (p) CD4+PD-1+CD27− cells. In another embodiment of the invention, any one or more of the populations of (a)-(p) may also co-express CD3.
The method may comprise separating the T cells that express PD-1 from cells that do not express PD-1 to obtain a T cell population enriched for T cells that express PD-1. In this regard, the selected cells may be physically separated from unselected cells, i.e., the cells that do not express PD-1. The selected cells may be separated from unselected cells by any suitable method such as, for example, sorting.
The method may comprise identifying one or more genes in the nucleic acid of a cancer cell of a patient, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence. The cancer cell may be obtained from any bodily sample derived from a patient which contains or is expected to contain tumor or cancer cells. The bodily sample may be any tissue sample such as blood, a tissue sample obtained from the primary tumor or from tumor metastases, or any other sample containing tumor or cancer cells. The nucleic acid of the cancer cell may be DNA or RNA.
In order to identify cancer-specific mutations, the method may further comprise sequencing nucleic acid such as DNA or RNA of normal, noncancerous cells and comparing the nucleic acid sequence of the cancer cell with the sequence of the normal, noncancerous cell. The normal, noncancerous cell may be obtained from the patient or a different individual.
The cancer-specific mutation may be any mutation in any gene which encodes a mutated amino acid sequence (also referred to as a “non-silent mutation”) and which is expressed in a cancer cell but not in a normal, noncancerous cell. Non-limiting examples of cancer-specific mutations that may be identified in the inventive methods include missense, nonsense, insertion, deletion, duplication, frameshift, and repeat expansion mutations. In an embodiment of the invention, the method comprises identifying at least one gene containing a cancer-specific mutation which encodes a mutated amino acid sequence. However, the number of genes containing such a cancer-specific mutation that may be identified using the inventive methods is not limited and may include more than one gene (for example, about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values). Likewise, in an embodiment of the invention, the method comprises identifying at least one cancer-specific mutation which encodes a mutated amino acid sequence. However, the number of such cancer-specific mutations that may be identified using the inventive methods is not limited and may include more than one cancer-specific mutation (for example, about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values). In an embodiment in which more than one cancer-specific mutation is identified, the cancer-specific mutations may be located in the same gene or in different genes.
In an embodiment, identifying one or more genes in the nucleic acid of a cancer cell comprises sequencing the whole exome, the whole genome, or the whole transcriptome of the cancer cell. Sequencing may be carried out in any suitable manner known in the art. Examples of sequencing techniques that may be useful in the inventive methods include Next Generation Sequencing (NGS) (also referred to as “massively parallel sequencing technology”) or Third Generation Sequencing. NGS refers to non-Sanger-based high-throughput DNA sequencing technologies. With NGS, millions or billions of DNA strands may be sequenced in parallel, yielding substantially more throughput and minimizing the need for the fragment-cloning methods that are often used in Sanger sequencing of genomes. In NGS, nucleic acid templates may be randomly read in parallel along the entire genome by breaking the entire genome into small pieces. NGS may, advantageously, provide nucleic acid sequence information of a whole genome, exome, or transcriptome in very short time periods, e.g., within about 1 to about 2 weeks, preferably within about 1 to about 7 days, or most preferably, within less than about 24 hours. Multiple NGS platforms which are commercially available or which are described in the literature can be used in the context of the inventive methods, e.g., those described in Zhang et al., J. Genet. Genomics, 38(3): 95-109 (2011) and Voelkerding et al., Clinical Chemistry, 55: 641-658 (2009).
Non-limiting examples of NGS technologies and platforms include sequencing-by-synthesis (also known as “pyrosequencing”) (as implemented, e.g., using the GS-FLX 454 Genome Sequencer, 454 Life Sciences (Branford, CT), ILLUMINA SOLEXA Genome Analyzer (Illumina Inc., San Diego, CA), or the ILLUMINA HISEQ 2000 Genome Analyzer (Illumina), or as described in, e.g., Ronaghi et al., Science, 281(5375): 363-365 (1998)), sequencing-by-ligation (as implemented, e.g., using the SOLID platform (Life Technologies Corporation, Carlsbad, CA) or the POLONATOR G.007 platform (Dover Systems, Salem, NH)), single-molecule sequencing (as implemented, e.g., using the PACBIO RS system (Pacific Biosciences (Menlo Park, CA) or the HELISCOPE platform (Helicos Biosciences (Cambridge, MA)), nano-technology for single-molecule sequencing (as implemented, e.g., using the GRIDON platform of Oxford Nanopore Technologies (Oxford, UK), the hybridization-assisted nano-pore sequencing (HANS) platforms developed by Nabsys (Providence, RI), and the ligase-based DNA sequencing platform with DNA nanoball (DNB) technology referred to as probe-anchor ligation (cPAL)), electron microscopy-based technology for single-molecule sequencing, and ion semiconductor sequencing.
The method may comprise inducing autologous APCs of the patient to present the mutated amino acid sequence. The APCs may include any cells which present peptide fragments of proteins in association with MHC molecules on their cell surface. The APCs may include, for example, any one or more of macrophages, dendritic cells (DCs), langerhans cells, B-lymphocytes, and T-cells. Preferably, the APCs are DCs. By using autologous APCs from the patient, the inventive methods may, advantageously, identify T cells, TCRs, and antigen-binding portions thereof, that have antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation that is presented in the context of an MHC molecule expressed by the patient. The MHC molecule can be any MHC molecule expressed by the patient including, but not limited to, MHC Class I, MHC Class II, HLA-A, HLA-B, HLA-C, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR molecules. The inventive methods may, advantageously, identify mutated amino acid sequences presented in the context of any MHC molecule expressed by the patient without using, for example, epitope prediction algorithms to identify MHC molecules or mutated amino acid sequences, which may be useful only for a select few MHC class I alleles and may be constrained by the limited availability of reagents to select mutation-reactive T cells (e.g., an incomplete set of MHC tetramers). Accordingly, in an embodiment of the invention, the inventive methods advantageously identify mutated amino acid sequences presented in the context of any MHC molecule expressed by the patient and are not limited to any particular MHC molecule. Preferably, the autologous APCs are antigen-negative autologous APCs.
Inducing autologous APCs of the patient to present the mutated amino acid sequence may be carried out using any suitable method known in the art. In an embodiment of the invention, inducing autologous APCs of the patient to present the mutated amino acid sequence comprises pulsing the autologous APCs with peptides comprising the mutated amino acid sequence or a pool of peptides, each peptide in the pool comprising a different mutated amino acid sequence. Each of the mutated amino acid sequences in the pool may be encoded by a gene containing a cancer specific mutation. In this regard, the autologous APCs may be cultured with a peptide or a pool of peptides comprising the mutated amino acid sequence in a manner such that the APCs internalize the peptide(s) and display the mutated amino acid sequence(s), bound to an MHC molecule, on the cell membrane. In an embodiment in which more than one gene is identified, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence, the method may comprise pulsing the autologous APCs with a pool of peptides, each peptide in the pool comprising a different mutated amino acid sequence. Methods of pulsing APCs are known in the art and are described in, e.g., Solheim (Ed.), Antigen Processing and Presentation Protocols (Methods in Molecular Biology), Human Press, (2010). The peptide(s) used to pulse the APCs may include the mutated amino acid(s) encoded by the cancer-specific mutation. The peptide(s) may further comprise any suitable number of contiguous amino acids from the endogenous protein encoded by the identified gene on each of the carboxyl side and the amino side of the mutated amino acid(s). The number of contiguous amino acids from the endogenous protein flanking each side of the mutation is not limited and may be, for example, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or a range defined by any two of the foregoing values. Preferably, the peptide(s) comprise(s) about 12 contiguous amino acids from the endogenous protein on each side of the mutated amino acid(s).
In an embodiment of the invention, inducing autologous APCs of the patient to present the mutated amino acid sequence comprises introducing a nucleotide sequence encoding the mutated amino acid sequence into the APCs. The nucleotide sequence is introduced into the APCs so that the APCs express and display the mutated amino acid sequence, bound to an MHC molecule, on the cell membrane. The nucleotide sequence encoding the mutated amino acid may be RNA or DNA. Introducing a nucleotide sequence into APCs may be carried out in any of a variety of different ways known in the art as described in, e.g., Solheim et al. supra. Non-limiting examples of techniques that are useful for introducing a nucleotide sequence into APCs include transformation, transduction, transfection, and electroporation. In an embodiment in which more than one gene is identified, the method may comprise preparing more than one nucleotide sequence, each encoding a mutated amino acid sequence encoded by a different gene, and introducing each nucleotide sequence into a different population of autologous APCs. In this regard, multiple populations of autologous APCs, each population expressing and displaying a different mutated amino acid sequence, may be obtained.
In an embodiment in which more than one gene is identified, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence, the method may comprise introducing a nucleotide sequence encoding the more than one gene. In this regard, in an embodiment of the invention, the nucleotide sequence introduced into the autologous APCs is a tandem minigene (TMG) construct, each minigene comprising a different gene, each gene including a cancer-specific mutation that encodes a mutated amino acid sequence. Each minigene may encode one mutation identified by the inventive methods flanked on each side of the mutation by any suitable number of contiguous amino acids from the endogenous protein encoded by the identified gene, as described herein with respect to other aspects of the invention. The number of minigenes in the construct is not limited and may include for example, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, or more, or a range defined by any two of the foregoing values. The APCs express the mutated amino acid sequences encoded by the TMG construct and display the mutated amino acid sequences, bound to an MHC molecule, on the cell membranes. In an embodiment, the method may comprise preparing more than one TMG construct, each construct encoding a different set of mutated amino acid sequences encoded by different genes, and introducing each TMG construct into a different population of autologous APCs. In this regard, multiple populations of autologous APCs, each population expressing and displaying mutated amino acid sequences encoded by different TMG constructs, may be obtained.
The method may comprise co-culturing T cells from the population enriched for T cells that express PD-1 with the autologous APCs that present the mutated amino acid sequence. The T cells from the population enriched for T cells that express PD-1 are obtained from peripheral blood as described herein with respect to other aspects of the invention. The T cells can express PD-1 and any of the other cell markers described herein with respect to other aspects of the invention. The method may comprise co-culturing the T cells that express PD-1 and autologous APCs so that the T cells encounter the mutated amino acid sequence presented by the APCs in such a manner that the T cells specifically bind to and immunologically recognize a mutated amino acid sequence presented by the APCs. In an embodiment of the invention, the T cells are co-cultured in direct contact with the autologous APCs.
The method may comprise selecting the T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a MHC molecule expressed by the patient. The phrase “antigenic specificity,” as used herein, means that a T cell, TCR, or the antigen-binding portion thereof, expressed by the T cell, can specifically bind to and immunologically recognize the mutated amino acid sequence encoded by the cancer-specific mutation. The selecting may comprise identifying the T cells that have antigenic specificity for the mutated amino acid sequence and separating them from T cells that do not have antigenic specificity for the mutated amino acid sequence. Selecting the T cells having antigenic specificity for the mutated amino acid sequence may be carried out in any suitable manner. In an embodiment of the invention, the method comprises expanding the numbers of T cells that express PD-1, e.g., by co-culturing with a T cell growth factor, such as interleukin (IL)-2 or IL-15, or as described herein with respect to other aspects of the invention, prior to selecting the T cells that have antigenic specificity for the mutated amino acid sequence. In an embodiment of the invention, the method does not comprise expanding the numbers of T cells that express PD-1 with a T cell growth factor, such as IL-2 or IL-15 prior to selecting the T cells that have antigenic specificity for the mutated amino acid sequence.
For example, upon co-culture of the T cells that express PD-1 with the APCs that present the mutated amino acid sequence, T cells having antigenic specificity for the mutated amino acid sequence may express any one or more of a variety of T cell activation markers which may be used to identify those T cells having antigenic specificity for the mutated amino acid sequence. Such T cell activation markers may include, but are not limited to, PD-1, lymphocyte-activation gene 3 (LAG-3), TIM-3, 4-1BB, OX40, and CD107a. Accordingly, in an embodiment of the invention, selecting the T cells that have antigenic specificity for the mutated amino acid sequence comprises selecting the T cells that express any one or more of PD-1, LAG-3, TIM-3, 4-1BB, OX40, and CD107a. Cells expressing one or more T cell activation markers may be sorted on the basis of expression of the marker using any of a variety of techniques known in the art such as, for example, FACS or magnetic-activated cell sorting (MACS) as described in, e.g., Turcotte et al., Clin. Cancer Res., 20(2): 331-43 (2013) and Gros et al., J. Clin. Invest., 124(5): 2246-59 (2014).
In another embodiment of the invention, selecting the T cells that have antigenic specificity for the mutated amino acid sequence comprises selecting the T cells (i) that secrete a greater amount of one or more cytokines upon co-culture with APCs that present the mutated amino acid sequence as compared to the amount of the one or more cytokines secreted by a negative control or (ii) in which at least twice as many of the numbers of T cells secrete one or more cytokines upon co-culture with APCs that present the mutated amino acid sequence as compared to the numbers of negative control T cells that secrete the one or more cytokines. The one or more cytokines may comprise any cytokine the secretion of which by a T cell is characteristic of T cell activation (e.g., a TCR expressed by the T cells specifically binding to and immunologically recognizing the mutated amino acid sequence). Non-limiting examples of cytokines, the secretion of which is characteristic of T cell activation, include IFN-γ, IL-2, and tumor necrosis factor alpha (TNF-α), granulocyte/monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.
For example, the T cells may be considered to have “antigenic specificity” for the mutated amino acid sequence if the T cells secrete at least twice as much IFN-γ upon co-culture with (a) antigen-negative APCs pulsed with a concentration of a peptide comprising the mutated amino acid sequence (e.g., about 0.001 ng/mL to about 10 μg/mL, e.g., 0.001 ng/ml, 0.005 ng/mL, 0.01 ng/ml, 0.05 ng/ml, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 100 ng/mL, 1 μg/mL, 5 μg/mL, or 10 μg/mL) or (b) APCs into which a nucleotide sequence encoding the mutated amino acid sequence has been introduced as compared to the amount of IFN-γ secreted by a negative control. The negative control may be, for example, autologous T cells (e.g., derived from PBMCs) co-cultured with (a) antigen-negative APCs pulsed with the same concentration of an irrelevant peptide (e.g., the wild-type amino acid sequence, or some other peptide with a different sequence from the mutated amino acid sequence) or (b) APCs into which a nucleotide sequence encoding an irrelevant peptide sequence has been introduced. The T cells may also have “antigenic specificity” for the mutated amino acid sequence if the T cells secrete a greater amount of IFN-γ upon co-culture with antigen-negative APCs pulsed with higher concentrations of a peptide comprising the mutated amino acid sequence as compared to a negative control, for example, the negative control described above. IFN-γ secretion may be measured by methods known in the art such as, for example, enzyme-linked immunosorbent assay (ELISA).
Alternatively or additionally, the T cells may be considered to have “antigenic specificity” for the mutated amino acid sequence if at least twice as many of the numbers of T cells secrete IFN-γ upon co-culture with (a) antigen-negative APCs pulsed with a concentration of a peptide comprising the mutated amino acid sequence or (b) APCs into which a nucleotide sequence encoding the mutated amino acid sequence has been introduced as compared to the numbers of negative control T cells that secrete IFN-γ. The concentration of peptide and the negative control may be as described herein with respect to other aspects of the invention. The numbers of cells secreting IFN-γ may be measured by methods known in the art such as, for example, ELISPOT.
While T cells having antigenic specificity for the mutated amino acid sequence may both (1) express any one or more T cells activation markers described herein and (2) secrete a greater amount of one or more cytokines as described herein, in an embodiment of the invention, T cells having antigenic specificity for the mutated amino acid sequence may express any one or more T cell activation markers without secreting a greater amount of one or more cytokines or may secrete a greater amount of one or more cytokines without expressing any one or more T cell activation markers.
In another embodiment of the invention, selecting the T cells that have antigenic specificity for the mutated amino acid sequence comprises selectively growing the T cells that have antigenic specificity for the mutated amino acid sequence. In this regard, the method may comprise co-culturing the T cells with autologous APCs in such a manner as to favor the growth of the T cells that have antigenic specificity for the mutated amino acid sequence over the T cells that do not have antigenic specificity for the mutated amino acid sequence. Accordingly, a population of T cells is provided that has a higher proportion of T cells that have antigenic specificity for the mutated amino acid sequence as compared to T cells that do not have antigenic specificity for the mutated amino acid sequence.
In an embodiment of the invention in which T cells are co-cultured with autologous APCs expressing multiple mutated amino acid sequences (e.g., multiple mutated amino acid sequences encoded by a TMG construct or multiple mutated amino acid sequences in a pool of peptides pulsed onto autologous APCs), selecting the T cells may further comprise separately assessing T cells for antigenic specificity for each of the multiple mutated amino acid sequences. For example, the inventive method may further comprise separately inducing autologous APCs of the patient to present each mutated amino acid sequence encoded by the construct (or included in the pool), as described herein with respect to other aspects of the invention (for example, by providing separate APC populations, each presenting a different mutated amino acid sequence encoded by the construct (or included in the pool)). The method may further comprise separately co-culturing T cells with the different populations of autologous APCs that present each mutated amino acid sequence, as described herein with respect to other aspects of the invention. The method may further comprise separately selecting the T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a MHC molecule expressed by the patient, as described herein with respect to other aspects of the invention. In this regard, the method may comprise determining which mutated amino acid sequence encoded by a TMG construct that encodes multiple mutated amino acid sequences (or included in the pool) are immunologically recognized by the T cells (e.g., by process of elimination).
The method may further comprise isolating a nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, from the selected T cells, wherein the TCR, or the antigen-binding portion thereof, has antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation. In an embodiment of the invention, prior to isolating the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, the numbers selected T cells that have antigenic specificity for the mutated amino acid sequence may be expanded. Expansion of the numbers of T cells can be accomplished by any of a number of methods as are known in the art as described in, for example, U.S. Pat. Nos. 8,034,334; 8,383,099; U.S. Patent Application Publication No. 2012/0244133; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In an embodiment, expansion of the numbers of T cells is carried out by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMC (e.g., irradiated allogeneic PBMC). In another embodiment of the invention, the numbers of selected T cells that have antigenic specificity for the mutated amino acid sequence are not expanded prior to isolating the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof. For example, the TCR, or antigen binding portion thereof, may be isolated from a single cell.
The “the antigen-binding portion” of the TCR, as used herein, refers to any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the antigen-binding portion specifically binds to the mutated amino acid sequence encoded by the gene identified as described herein with respect to other aspects of the invention. The term “antigen-binding portion” refers to any part or fragment of the TCR of the invention, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). Antigen-binding portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to the mutated amino acid sequence, or detect, treat, or prevent cancer, to a similar extent, the same extent, or to a higher extent, as compared to the parent TCR. In reference to the parent TCR, the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent TCR.
The antigen-binding portion can comprise an antigen-binding portion of either or both of the α and β chains of the TCR of the invention, such as a portion comprising one or more of the complementarity determining region (CDR)1, CDR2, and CDR3 of the variable region(s) of the α chain and/or 13 chain of the TCR of the invention. In an embodiment of the invention, the antigen-binding portion can comprise the amino acid sequence of the CDR1 of the α chain (CDR1a), the CDR2 of the α chain (CDR2a), the CDR3 of the α chain (CDR3a), the CDR1 of the β chain (CDR1(3), the CDR2 of the β chain (CDR2(3), the CDR3 of the β chain (CDR3β), or any combination thereof. Preferably, the antigen-binding portion comprises the amino acid sequences of CDR1α, CDR2α, and CDR3α; the amino acid sequences of CDR1β, CDR2β, and CDR3β; or the amino acid sequences of all of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the inventive TCR.
In an embodiment of the invention, the antigen-binding portion can comprise, for instance, the variable region of the inventive TCR comprising a combination of the CDR regions set forth above. In this regard, the antigen-binding portion can comprise the amino acid sequence of the variable region of the α chain (Vα), the amino acid sequence of the variable region of the β chain (Vβ), or the amino acid sequences of both of the Vα and Vβ of the inventive TCR.
In an embodiment of the invention, the antigen-binding portion may comprise a combination of a variable region and a constant region. In this regard, the antigen-binding portion can comprise the entire length of the α or β chain, or both of the α and β chains, of the inventive TCR.
Isolating the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, from the selected T cells may be carried out in any suitable manner known in the art. For example, the method may comprise isolating RNA from the selected T cells and sequencing the TCR, or the antigen-binding portion thereof, using established molecular cloning techniques and reagents such as, for example, 5′ Rapid Amplification of cDNA Ends (RACE) polymerase chain reaction (PCR) using TCR-α and -β chain constant primers.
In an embodiment of the invention, the method may comprise cloning the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, into a recombinant expression vector using established molecular cloning techniques as described in, e.g., Green et al. (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed. (2012). For purposes herein, the term “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA (e.g., complementary DCA (cDNA)) and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring, non-naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages does not hinder the transcription or replication of the vector.
The recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of transposon/transposase, the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, e.g., a retroviral vector.
The TCR, or the antigen-binding portion thereof, isolated by the inventive methods may be useful for preparing cells for adoptive cell therapies. In this regard, an embodiment of the invention provides a method of preparing a population of cells that express a TCR, or an antigen-binding portion thereof, having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising isolating a TCR, or an antigen-binding portion thereof, as described herein with respect to other aspects of the invention, and introducing the nucleotide sequence encoding the isolated TCR, or the antigen-binding portion thereof, into host cells to obtain cells that express the TCR, or the antigen-binding portion thereof.
Introducing the nucleotide sequence (e.g., a recombinant expression vector) encoding the isolated TCR, or the antigen-binding portion thereof, into host cells may be carried out in any of a variety of different ways known in the art as described in, e.g., Green et al. supra. Non-limiting examples of techniques that are useful for introducing a nucleotide sequence into host cells include transformation, transduction, transfection, and electroporation.
The host cell into which the nucleotide sequence encoding the TCR, or antigen binding portion thereof, is introduced may be any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5a cell. For purposes of producing the TCR, or antigen binding portion thereof, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a PBL or a PBMC. More preferably, the host cell is a T cell.
In an embodiment of the invention, the PBMC include T cells. The T cells may be any type of T cell. Without being bound to a particular theory or mechanism, it is believed that less differentiated, “younger” T cells may be associated with any one or more of greater in vivo persistence, proliferation, and antitumor activity as compared to more differentiated, “older” T cells. Accordingly, the inventive methods may, advantageously, identify and isolate a TCR, or an antigen-binding portion thereof, that has antigenic specificity for the mutated amino acid sequence and introduce the TCR, or an antigen-binding portion thereof, into “younger” T cells that may provide any one or more of greater in vivo persistence, proliferation, and antitumor activity as compared to “older” T cells (e.g., effector cells in a patient's tumor).
In an embodiment of the invention, the host cells are autologous to the patient. In this regard, the TCRs, or the antigen-binding portions thereof, identified and isolated by the inventive methods may be personalized to each patient. However, in another embodiment, the inventive methods may identify and isolate TCRs, or the antigen-binding portions thereof, that have antigenic specificity against a mutated amino acid sequence that is encoded by a recurrent (also referred to as “hot-spot”) cancer-specific mutation. In this regard, the method may comprise introducing the nucleotide sequence encoding the isolated TCR, or the antigen-binding portion thereof, into host cells that are allogeneic to the patient. For example, the method may comprise introducing the nucleotide sequence encoding the isolated TCR, or the antigen-binding portion thereof, into the host cells from another patient whose tumors express the same mutation in the context of the same MHC molecule.
In an embodiment of the invention, the method further comprises expanding the numbers of host cells that express the TCR, or the antigen-binding portion thereof. The numbers of host cells may be expanded, for example, as described herein with respect to other aspects of the invention. In this regard, the inventive methods may, advantageously, generate a large number of T cells having antigenic specificity for the mutated amino acid sequence.
Another embodiment of the invention provides a TCR, or an antigen-binding portion thereof, isolated by any of the methods described herein with respect to other aspects of the invention. An embodiment of the invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (α) chain of a TCR, a beta (β) chain of a TCR, a gamma (γ) chain of a TCR, a delta (δ) chain of a TCR, or a combination thereof. Another embodiment of the invention provides an antigen-binding portion of the TCR comprising one or more CDR regions, one or more variable regions, or one or both of the α and β chains of the TCR, as described herein with respect to other aspects of the invention. The polypeptides of the inventive TCR, or the antigen-binding portion thereof, can comprise any amino acid sequence, provided that the TCR, or the antigen-binding portion thereof, has antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.
In an embodiment of the invention, the TCR, or antigen binding portion thereof, has antigenic specificity for MAGE-A6E168K. The phrase “antigenic specificity,” as used herein, means that the TCR can specifically bind to and immunologically recognize the particular antigen under discussion. Wild-type, non-mutated MAGE-A6 comprises the amino acid sequence of SEQ ID NO: 74. MAGE-A6E168K comprises the amino acid sequence of SEQ ID NO: 74 except that the glutamic acid at position 168 of SEQ ID NO: 74 is substituted with lysine. In an embodiment of the invention, the TCR has antigenic specificity for the MAGE-A6E168K amino acid sequence of SEQ ID NO: 77.
The anti-MAGE-A6E168K TCR, or antigen binding portion thereof, comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 5 or 13 (CDR1 of a chain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 6 or 14 (CDR2 of a chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7 or 15 (CDR3 of a chain), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 8 or 16 (CDR1 of β chain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 9 or 17 (CDR2 of β chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10 or 18 (CDR3 of β chain). In this regard, the inventive TCR, or antigen binding portion thereof, can comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 5-10 and SEQ ID NOs: 13-18. In an especially preferred embodiment, the TCR, or antigen binding portion thereof, comprises the amino acid sequences of (i) all of SEQ ID NOs: 5-10 or (ii) all of SEQ ID NOs: 13-18.
In an embodiment of the invention, the TCR, or antigen binding portion thereof, has antigenic specificity for PDS5AY1000F;H1007Y. Wild-type, non-mutated PDS5A comprises the amino acid sequence of SEQ ID NO: 75. PDS5AY1000F;H1007Y comprises the amino acid sequence of SEQ ID NO: 75 except that the tyrosine at position 1000 of SEQ ID NO: 75 is substituted with phenylalanine and the histidine at position 1007 of SEQ ID NO: 75 is substituted with tyrosine. In an embodiment of the invention, the TCR, or antigen binding portion thereof, has antigenic specificity for the PDS5AY1000F;H1007Y amino acid sequence of SEQ ID NO: 78.
In an embodiment of the invention, the anti-PDS5AY1000F;H1007Y TCR, or antigen binding portion thereof comprises the amino acid sequence of SEQ ID NO: 21 (CDR1 of a chain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 22 (CDR2 of a chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23 (CDR3 of a chain), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 24 (CDR1 of β chain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 25 (CDR2 of β chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26 (CDR3 of β chain). In this regard, the inventive TCR, or antigen binding portion thereof, can comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 21-26. In an especially preferred embodiment, the TCR, or antigen binding portion thereof, comprises the amino acid sequences of all of SEQ ID NOs: 21-26.
In an embodiment of the invention, the TCR, or antigen binding portion thereof, has antigenic specificity for MED13P1691S. Wild-type, non-mutated MED13 comprises the amino acid sequence of SEQ ID NO: 76. MED13P1691S comprises the amino acid sequence of SEQ ID NO: 76 except that the proline at position 1691 of SEQ ID NO: 76 is substituted with serine. In an embodiment of the invention, the TCR, or antigen binding portion thereof, has antigenic specificity for the MED13P1691S amino acid sequence of SEQ ID NO: 79.
In an embodiment of the invention, the anti-MED13P169S TCR, or antigen binding portion thereof, comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 29 or 37 (CDR1 of a chain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 30 or 38 (CDR2 of a chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31 or 39 (CDR3 of a chain), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 32 or 40 (CDR1 of β chain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 33 or 41 (CDR2 of β chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 34 or 42 (CDR3 of β chain). In this regard, the inventive TCR, or antigen binding portion thereof, can comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 29-34 and SEQ ID NOs: 37-42. In an especially preferred embodiment, the TCR, or antigen binding portion thereof, comprises the amino acid sequences of (i) all of SEQ ID NOs: 29-34 or (ii) all of SEQ ID NOs: 37-42.
In an embodiment of the invention, the TCR can comprise an amino acid sequence of a variable region of a TCR comprising the CDRs set forth above. In this regard, the TCR can comprise the amino acid sequence of SEQ ID NO: 11 or 19 (the variable region of an α chain of an anti-MAGE-A6E168K TCR); SEQ ID NO: 12, wherein X at position 2 of SEQ ID NO: 12 is Gly or Ala (the variable region of a β chain of an anti-MAGE-A6E168K TCR); SEQ ID NO: 20, wherein X at position 2 of SEQ ID NO: 20 is Gly or Ala (the variable region of a β chain of an anti-MAGE-A6E168K TCR); both SEQ ID NOs: 11 and 12; both SEQ ID NOs: 19 and 20; SEQ ID NO: 27 (the variable region of an α chain of the anti-PDS5AY1000F;H1007Y TCR); SEQ ID NO: 28, wherein X at position 2 of SEQ ID NO: 28 is Gly or Ala (the variable region of a β chain of the anti-PDS5AY1000FF;H1007Y TCR); both SEQ ID NOs: 27 and 28; SEQ ID NO: 35 or 43 (the variable region of an α chain of an anti-MED13P169S TCR); SEQ ID NO: 36, wherein X at position 2 of SEQ ID NO: 36 is Gly or Ala (the variable region of a β chain of an anti-MED13P169S TCR); SEQ ID NO: 44, wherein X at position 2 of SEQ ID NO: 44 is Gly or Ala (the variable region of a β chain of an anti-MED13P169S TCR); both SEQ ID NOs: 35 and 36; or both SEQ ID NOs: 43 and 44. Preferably, the inventive TCR comprises the amino acid sequences of (a) SEQ ID NOs: 11-12; (b) SEQ ID NOs: 19-20; (c) SEQ ID NOs: 27-28; (d) SEQ ID NOs: 35-36; or (e) SEQ ID NOs: 43-44.
The inventive TCRs may further comprise a constant region derived from any suitable species such as, e.g., human or mouse. As used herein, the term “murine” or “human,” when referring to a TCR or any component of a TCR described herein (e.g., complementarity determining region (CDR), variable region, constant region, alpha chain, and/or beta chain), means a TCR (or component thereof) which is derived from a mouse or a human, respectively, i.e., a TCR (or component thereof) that originated from or was, at one time, expressed by a mouse T cell or a human T cell, respectively.
In an embodiment of the invention, the constant region is a human constant region. In this regard, the TCR can comprise SEQ ID NO: 61 (constant region of a human α chain); SEQ ID NO: 62 (constant region of a human β chain); SEQ ID NO: 63 (constant region of a human β chain); both SEQ ID NO: 61 and SEQ ID NO: 62; or both SEQ ID NOs: 61 and 63. The TCR may comprise any of the CDR regions as described herein with respect to other aspects of the invention. In another embodiment of the invention, the TCR may comprise any of the variable regions described herein with respect to other aspects of the invention.
In an embodiment of the invention, the TCR further comprises a murine constant region. For example, the TCR may be a chimeric TCR comprising a human variable region and a murine constant region. In this regard, the TCR can comprise SEQ ID NO: 47 (constant region of a murine α chain); SEQ ID NO: 48 (constant region of a murine β chain); or both SEQ ID NO: 47 and SEQ ID NO: 48. The chimeric TCR may comprise any of the CDR regions as described herein with respect to other aspects of the invention. In another embodiment of the invention, the chimeric TCR may comprise any of the variable regions described herein with respect to other aspects of the invention. In an embodiment of the invention, the TCR comprises a murine constant region, optionally with one, two, three, or four amino acid substitution(s) in the constant region of one or both of the alpha and beta chains, as described herein with respect to other aspects of the invention. In an embodiment of the invention, the TCR comprises a murine constant region, optionally with one, two, three, or four amino acid substitution(s) in the murine constant region of the alpha chain and one amino acid substitution in the murine constant region of the beta chain, as described herein with respect to other aspects of the invention.
In some embodiments, the TCRs comprising the substituted amino acid sequence(s) advantageously provide one or more of increased recognition of mutated amino acid sequence-positive targets, increased expression by a host cell, and increased anti-tumor activity as compared to the parent TCR comprising an unsubstituted amino acid sequence. In general, the substituted amino acid sequences of the murine constant regions of the TCR a and β chains, SEQ ID NOs: 45 and 46, respectively, correspond with all or portions of the unsubstituted murine constant region amino acid sequences SEQ ID NOs: 47 and 48, respectively, with SEQ ID NO: 45 having one, two, three, or four amino acid substitution(s) when compared to SEQ ID NO: 47 and SEQ ID NO: 46 having one amino acid substitution when compared to SEQ ID NO: 48. In this regard, an embodiment of the invention provides a TCR comprising the amino acid sequences of one or both of (a) SEQ ID NO: 45 (constant region of alpha chain), wherein (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (b) SEQ ID NO: 46 (constant region of beta chain), wherein X at position 57 is Ser or Cys. In an embodiment of the invention, the TCR comprising SEQ ID NO: 45 does not comprise SEQ ID NO: 47 (unsubstituted murine constant region of alpha chain). In an embodiment of the invention, the TCR comprising SEQ ID NO: 46 does not comprise SEQ ID NO: 48 (unsubstituted murine constant region of beta chain).
In an embodiment of the invention, the substituted amino acid sequence includes cysteine substitutions in the constant region of one or both of the α and β chains to provide a cysteine-substituted TCR. Opposing cysteines in the α and the 13 chains provide a disulfide bond that links the constant regions of the α and the β chains of the substituted TCR to one another and which is not present in a TCR comprising the unsubstituted human constant region or the unsubstituted murine constant region. In this regard, the TCR is a cysteine-substituted TCR in which one or both of the native Thr48 of SEQ ID NO: 47 and the native Ser57 of SEQ ID NO: 48 may be substituted with Cys. Preferably, both of the native Thr48 of SEQ ID NO: 47 and the native Ser57 of SEQ ID NO: 48 are substituted with Cys. In an embodiment, the cysteine-substituted TCR comprises an alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein X at position 48 is Cys, X at position 112 is the native Ser, X at position 114 is the native Met, and X at position 115 is the native Gly, and a beta chain constant region comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 57 is Cys. The cysteine-substituted TCRs of the invention may include the substituted constant region in addition to any of the CDRs or variable regions described herein.
In an embodiment of the invention, the substituted amino acid sequence includes substitutions of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the α and β chains with a hydrophobic amino acid to provide a hydrophobic amino acid-substituted TCR. The hydrophobic amino acid substitution(s) in the TM domain of the TCR may increase the hydrophobicity of the TM domain of the TCR as compared to a TCR that lacks the hydrophobic amino acid substitution(s) in the TM domain. In this regard, the TCR may be a hydrophobic amino acid-substituted TCR in which one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 47 may, independently, be substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably with Leu, Ile, or Val. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 47 are, independently, substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably with Leu, Ile, or Val. In an embodiment, the hydrophobic amino acid-substituted TCR comprises an alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein X at position 48 is the native Thr, X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, X at position 114 is Met, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and a beta chain constant region comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 57 is the native Ser, wherein the hydrophobic amino acid-substituted TCR comprising SEQ ID NO: 45 does not comprise SEQ ID NO: 47 (unsubstituted murine constant region of alpha chain). Preferably, the hydrophobic amino acid-substituted TCR comprises an alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein X at position 48 is the native Thr, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val, and a beta chain constant region comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 57 is the native Ser. The hydrophobic amino acid-substituted TCRs of the invention may include the substituted constant region in addition to any of the CDRs or variable regions described herein.
In an embodiment of the invention, the substituted amino acid sequence includes the cysteine substitutions in the constant region of one or both of the α and β chains in combination with the substitution(s) of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the α and β chains with a hydrophobic amino acid (also referred to herein as “cysteine-substituted, hydrophobic amino acid-substituted TCR”). In this regard, the TCR is a cysteine-substituted, hydrophobic amino acid-substituted TCR in which the native Thr48 of SEQ ID NO: 47 is substituted with Cys; one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 47 are, independently, substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably with Leu, Ile, or Val; and the native Ser57 of SEQ ID NO: 48 is substituted with Cys. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 47 are, independently, substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably with Leu, Ile, or Val. In an embodiment, the cysteine-substituted, hydrophobic amino acid-substituted TCR comprises an alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 45, wherein X at position 48 is Cys, X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, X at position 114 is Met, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and a beta chain constant region comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 56 is Cys, wherein the cysteine-substituted, hydrophobic amino acid-substituted TCR comprising SEQ ID NO: 45 does not comprise SEQ ID NO: 47 (unsubstituted murine constant region of alpha chain). Preferably, the cysteine-substituted, hydrophobic amino acid-substituted TCR comprises an alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 49 and a beta chain constant region comprising the amino acid sequence of SEQ ID NO: 50. The cysteine-substituted, hydrophobic amino acid-substituted, TCRs of the invention may include the substituted constant region in addition to any of the CDRs or variable regions described herein. In an especially preferred embodiment, the cysteine-substituted, hydrophobic amino acid-substituted TCR comprises a full-length alpha chain comprising the amino acid sequence of SEQ ID NO: 51, 53, 55, 57, or 59 and a full-length beta chain comprising the amino acid sequence of SEQ ID NO: 52, 54, 56, 58, or 60. In this regard, the Cys-substituted, hydrophobic amino acid-substituted TCR can comprise the amino acid sequences of (a) both of SEQ ID NOs: 51-52; (b) both of SEQ ID NOs: 53-54; (c) both of SEQ ID NOs: 55-56; (d) both of SEQ ID NOs: 57-58; or (e) both of SEQ ID NOs: 59-60.
Also provided by the invention is a polypeptide comprising an antigen-binding portion of any of the TCRs described herein. The term “polypeptide” as used herein includes oligopeptides and refers to a single chain of amino acids connected by one or more peptide bonds. The antigen-binding portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acids do not interfere with the biological function of the antigen-binding portion, e.g., specifically binding to a mutated amino acid sequence; and/or having the ability to detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent TCR.
The polypeptide can comprise an antigen-binding portion of either or both of the α and β chains of the TCRs of the invention, such as an antigen-binding portion comprising one of more of CDR1, CDR2, and CDR3 of the variable region(s) of the α chain and/or β chain of a TCR of the invention. In an embodiment of the invention, the polypeptide can comprise an antigen-binding portion comprising the amino acid sequence of SEQ ID NO: 5, 13, 21, 29, or 37 (CDR1 of a chain), SEQ ID NO: 6, 14, 22, 30, or 38 (CDR2 of a chain), SEQ ID NO: 7, 15, 23, 31, or 39 (CDR3 of a chain), SEQ ID NO: 8, 16, 24, 32, or 40 (CDR1 of β chain), SEQ ID NO: 9, 17, 25, 33, or 41 (CDR2 of β chain), SEQ ID NO: 10, 18, 26, 34, or 42 (CDR3 of β chain), or a combination thereof. Preferably, the inventive polypeptide comprises the amino acid sequences of (a) all of SEQ ID NOs: 5-10; (b) all of SEQ ID NOs: 13-18; (c) all of SEQ ID NOs: 21-26; (d) all of SEQ ID NOs: 29-34; or (e) all of SEQ ID NOs: 37-42.
In an embodiment of the invention, the inventive polypeptide can comprise, for instance, the variable region of the inventive TCR comprising a combination of the CDR regions set forth above. In this regard, the polypeptide can comprise the amino acid sequence of SEQ ID NO: 11 or 19 (the variable region of an α chain of an anti-MAGE-A6E168K TCR); SEQ ID NO: 12, wherein X at position 2 of SEQ ID NO: 12 is Gly or Ala (the variable region of a β chain of an anti-MAGE-A6E168K TCR); SEQ ID NO: 20, wherein X at position 2 of SEQ ID NO: 20 is Gly or Ala (the variable region of a β chain of an anti-MAGE-A6E168K TCR); both SEQ ID NOs: 11 and 12; both SEQ ID NOs: 19 and 20; SEQ ID NO: 27 (the variable region of an α chain of the anti-PDS5AY1000F;H1007YTCR); SEQ ID NO: 28, wherein X at position 2 of SEQ ID NO: 28 is Gly or Ala (the variable region of a β chain of the anti-PDS5AY1000F;H1007Y TCR); both SEQ ID NOs: 27 and 28; SEQ ID NO: 35 or 43 (the variable region of an α chain of an anti-MED13P169S TCR); SEQ ID NO: 36, wherein X at position 2 of SEQ ID NO: 36 is Gly or Ala (the variable region of a β chain of an anti-MED13P169S TCR); SEQ ID NO: 44, wherein X at position 2 of SEQ ID NO: 44 is Gly or Ala (the variable region of a β chain of an anti-MED13P169S TCR); both SEQ ID NOs: 35 and 36; or both SEQ ID NOs: 43 and 44. Preferably, the inventive polypeptide comprises the amino acid sequences of (a) SEQ ID NOs: 11-12; (b) SEQ ID NOs: 19-20; (c) SEQ ID NOs: 27-28; (d) SEQ ID NOs: 35-36; or (e) SEQ ID NOs: 43-44.
The inventive polypeptide may further comprise a constant region derived from any suitable species such as, e.g., human or mouse, described herein or any of the substituted constant regions described herein. In this regard, the polypeptide can comprise the amino acid sequence of SEQ ID NO: 45 (constant region of a chain, substituted as described herein with respect to other aspects of the invention), SEQ ID NO: 47 (the unsubstituted constant region of a murine α chain), SEQ ID NO: 46 (constant region of β chain, substituted as described herein with respect to other aspects of the invention), SEQ ID NO: 48 (the unsubstituted constant region of a murine β chain), SEQ ID NO: 49 (constant region of a cysteine-substituted, hydrophobic amino acid-substituted α chain), SEQ ID NO: 50 (constant region of a cysteine-substituted β chain), both SEQ ID NOs: 45 and 46, both SEQ ID NOs: 47 and 48, both SEQ ID NOs: 49 and 50, SEQ ID NO: 61 (constant region of a human α chain); SEQ ID NO: 62 (constant region of a human β chain); SEQ ID NO: 63 (constant region of a human β chain); both SEQ ID NO: 61 and SEQ ID NO: 62; or both SEQ ID NOs: 61 and 63.
In an embodiment of the invention, the inventive polypeptide can comprise the entire length of an α or β chain of one of the TCRs described herein. In this regard, the inventive polypeptide can comprise an amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. Preferably, the polypeptide comprises the amino acid sequences of (a) both of SEQ ID NOs: 51-52; (b) both of SEQ ID NOs: 53-54; (c) both of SEQ ID NOs: 55-56; (d) both of SEQ ID NOs: 57-58; or (e) both of SEQ ID NOs: 59-60.
The invention further provides a protein comprising at least one of the polypeptides described herein. By “protein” is meant a molecule comprising one or more polypeptide chains.
In an embodiment of the invention, the protein may comprise the CDR sequences of the inventive TCR. In this regard, the protein of the invention can comprise: (a) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 5-7 and a second polypeptide chain comprising the amino acid sequences of: SEQ ID NOs: 8-10; (b) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 13-15 and a second polypeptide chain comprising the amino acid sequences of: SEQ ID NOs: 16-18; (c) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 21-23 and a second polypeptide chain comprising the amino acid sequences of: SEQ ID NOs: 24-26; (d) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 29-31 and a second polypeptide chain comprising the amino acid sequences of: SEQ ID NOs: 32-34; or (e) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 37-39 and a second polypeptide chain comprising the amino acid sequences of: SEQ ID NOs: 40-42.
In an embodiment of the invention, the protein may comprise the variable region sequences of the inventive TCR. In this regard, the protein of the invention can comprise: (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 11 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 12, wherein X at position 2 of SEQ ID NO: 12 is Gly or Ala; (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 19 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 20, wherein X at position 2 of SEQ ID NO: 20 is Gly or Ala; (c) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 28, wherein X at position 2 of SEQ ID NO: 28 is Gly or Ala; (d) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 35 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 36, wherein X at position 2 of SEQ ID NO: 36 is Gly or Ala; or (e) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 43 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 44, wherein X at position 2 of SEQ ID NO: 44 is Gly or Ala.
In an embodiment of the invention, the inventive protein may further comprise TCR constant region sequences. In this regard, the first polypeptide chain of the inventive protein may further comprise the amino acid sequence of SEQ ID NO: 45 (constant region of the alpha chain, substituted as described herein with respect to other aspects of the invention), SEQ ID NO: 47 (the unsubstituted constant region of a murine α chain), or SEQ ID NO: 49 (constant region of a cysteine-substituted, hydrophobic amino acid-substituted a chain); and the second polypeptide chain of the inventive protein may further comprise the amino acid sequence of SEQ ID NO: 46 (constant region of β chain, substituted as described herein with respect to other aspects of the invention), SEQ ID NO: 48 (the unsubstituted constant region of a murine β chain), SEQ ID NO: 50 (constant region of a cysteine-substituted β chain) SEQ ID NO: 61 (constant region of a human α chain); SEQ ID NO: 62 (constant region of a human β chain); SEQ ID NO: 63 (constant region of a human β chain); both SEQ ID NO: 61 and SEQ ID NO: 62; or both SEQ ID NOs: 61 and 63. In a preferred embodiment of the invention, the protein comprises: (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 46; (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 48; or (c) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 49 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 50.
In an embodiment of the invention, the protein may comprise the full length alpha and beta chains of the inventive TCR. In this regard, the protein may comprise (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 51 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 52; (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 53 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 54; (c) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 55 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 56; (d) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 57 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 58; or (e) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 59 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 60. In this instance, the protein of the invention can be a TCR. Alternatively, if, for example, the protein comprises a single polypeptide chain comprising SEQ ID NOs: 5-10; or if the first and/or second polypeptide chain(s) of the protein further comprise(s) other amino acid sequences, e.g., an amino acid sequence encoding an immunoglobulin or a portion thereof, then the inventive protein can be a fusion protein. In this regard, the invention also provides a fusion protein comprising at least one of the inventive polypeptides described herein along with at least one other polypeptide. The other polypeptide can exist as a separate polypeptide of the fusion protein, or can exist as a polypeptide, which is expressed in frame (in tandem) with one of the inventive polypeptides described herein. The other polypeptide can encode any peptidic or proteinaceous molecule, or a portion thereof, including, but not limited to an immunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1α, CD1b, CD1c, CD1d, etc.
The fusion protein can comprise one or more copies of the inventive polypeptide and/or one or more copies of the other polypeptide. For instance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copies of the inventive polypeptide and/or of the other polypeptide. Suitable methods of making fusion proteins are known in the art, and include, for example, recombinant methods.
The protein of the invention can be a recombinant antibody comprising at least one of the inventive polypeptides described herein. As used herein, “recombinant antibody” refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the invention and a polypeptide chain of an antibody, or a portion thereof. The polypeptide of an antibody, or portion thereof, can be a heavy chain, a light chain, a variable or constant region of a heavy or light chain, a single chain variable fragment (scFv), or an Fc, Fab, or F(ab)2′ fragment of an antibody, etc. The polypeptide chain of an antibody, or portion thereof, can exist as a separate polypeptide of the recombinant antibody. Alternatively, the polypeptide chain of an antibody, or portion thereof, can exist as a polypeptide, which is expressed in frame (in tandem) with the polypeptide of the invention. The polypeptide of an antibody, or portion thereof, can be a polypeptide of any antibody or any antibody fragment.
The TCRs, polypeptides, and proteins of the invention can be of any length, i.e., can comprise any number of amino acids, provided that the TCRs, polypeptides, or proteins retain their biological activity, e.g., the ability to specifically bind to a mutated amino acid sequence; detect cancer; or treat or prevent cancer in a mammal, etc. For example, the polypeptide can be in the range of from about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length. In this regard, the polypeptides of the invention also include oligopeptides.
The TCRs, polypeptides, and proteins of the invention of the invention can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.
Included in the scope of the invention are functional variants of the inventive TCRs, polypeptides, and proteins described herein. The term “functional variant,” as used herein, refers to a TCR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent TCR, polypeptide, or protein, which functional variant retains the biological activity of the TCR, polypeptide, or protein of which it is a variant. Functional variants encompass, for example, those variants of the TCR, polypeptide, or protein described herein (the parent TCR, polypeptide, or protein) that retain the ability to specifically bind to a mutated amino acid sequence for which the parent TCR has antigenic specificity or to which the parent polypeptide or protein specifically binds, to a similar extent, the same extent, or to a higher extent, as the parent TCR, polypeptide, or protein. In reference to the parent TCR, polypeptide, or protein, the functional variant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in amino acid sequence to the parent TCR, polypeptide, or protein.
The functional variant can, for example, comprise the amino acid sequence of the parent TCR, polypeptide, or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.
Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent TCR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent TCR, polypeptide, or protein.
The TCR, polypeptide, or protein can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the TCR, polypeptide, or protein, e.g., other amino acids, do not materially change the biological activity of the TCR, polypeptide, or protein.
An embodiment of the invention provides a nucleic acid sequence comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. “Nucleic acid” as used herein includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. In an embodiment, the nucleic acid comprises complementary DNA (cDNA). It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
Preferably, the nucleic acids of the invention are recombinant. As used herein, the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.
The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook et al., supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).
The nucleic acid can comprise any nucleotide sequence which encodes any of the TCRs, polypeptides, or proteins described herein. In an embodiment of the invention, the nucleotide sequence may comprise, consist, or consist essentially of SEQ ID NO: 64 or 66 (the variable region of an α chain of an anti-MAGE-A6E168K TCR); SEQ ID NO: 65 or 67 (the variable region of a β chain of an anti-MAGE-A6E168K TCR); both SEQ ID NOs: 64 and 65; both SEQ ID NOs: 66 and 67; SEQ ID NO: 68 (the variable region of an α chain of the anti-PDS5AY100F;H1007Y TCR); SEQ ID NO: 69 (the variable region of a β chain of the anti-PDS5AY1000F;H1007Y TCR); both SEQ ID NOs: 68 and 69; SEQ ID NO: 70 or 72 (the variable region of an α chain of an anti-MED13P169S TCR); SEQ ID NO: 71 or 73 (the variable region of a β chain of an anti-MED13P169S TCR); both SEQ ID NOs: 70 and 71; or both SEQ ID NOs: 72 and 73. Preferably, the nucleotide sequence comprises (a) SEQ ID NOs: 64-65; (b) SEQ ID NOs: 66-67; (c) SEQ ID NOs: 68-69; (d) SEQ ID NOs: 70-71; or (e) SEQ ID NOs: 72-73.
In an embodiment of the invention, the nucleic acid comprises a codon-optimized nucleotide sequence. Without being bound to a particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.
The invention also provides a nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.
The nucleotide sequence which hybridizes under stringent conditions preferably hybridizes under high stringency conditions. By “high stringency conditions” is meant that the nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions which would distinguish a polynucleotide with an exact complementary sequence, or one containing only a few scattered mismatches from a random sequence that happened to have a few small regions (e.g., 3-10 bases) that matched the nucleotide sequence. Such small regions of complementarity are more easily melted than a full-length complement of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions would include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50-70° C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any of the inventive TCRs. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
The invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.
The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, the invention provides recombinant expression vectors comprising any of the nucleic acids of the invention. The recombinant expression vectors may be as described herein with respect to other aspects of the invention.
Another embodiment of the invention further provides a host cell comprising any of the recombinant expression vectors described herein and populations of host cells. The host cell, and populations thereof, may be as described herein with respect to other aspects of the invention.
The inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) can be isolated and/or purified. The term “isolated” as used herein means having been removed from its natural environment. The term “purified” as used herein means having been increased in purity, wherein “purity” is a relative term, and not to be necessarily construed as absolute purity. For example, the purity can be at least about 50%, can be greater than 60%, 70%, 80%, 90%, 95%, or can be 100%.
Another embodiment of the invention provides an isolated population of cells prepared according to any of the methods described herein with respect to other aspects of the invention. The population of cells can be a heterogeneous population comprising the host cells expressing the isolated TCR, or the antigen-binding portion thereof, in addition to at least one other cell, e.g., a host cell (e.g., a PBMC), which does not express the isolated TCR, or the antigen-binding portion thereof, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cells, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) expressing the isolated TCR, or the antigen-binding portion thereof. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell expressing the isolated TCR, or the antigen-binding portion thereof, such that all cells of the population express the isolated TCR, or the antigen-binding portion thereof. In one embodiment of the invention, the population of cells is a clonal population comprising host cells expressing the isolated TCR, or the antigen-binding portion thereof, as described herein. By introducing the nucleotide sequence encoding the isolated TCR, or the antigen binding portion thereof, into host cells, the inventive methods may, advantageously, provide a population of cells that comprises a high proportion of host cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence. In an embodiment of the invention, about 1% to about 100%, for example, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, or a range defined by any two of the foregoing values, of the population of cells comprises host cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence. Without being bound to a particular theory or mechanism, it is believed that populations of cells that comprise a high proportion of host cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence have a lower proportion of irrelevant cells that may hinder the function of the host cell, e.g., the ability of the host cell to target the destruction of cancer cells and/or treat or prevent cancer.
The inventive TCRs, or the antigen-binding portions thereof, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, and populations of cells (hereinafter, “inventive TCR material(s)”) can be formulated into a composition, such as a pharmaceutical composition. In this regard, the invention provides a pharmaceutical composition comprising any of the inventive TCRs, or the antigen-binding portions thereof, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells and a pharmaceutically acceptable carrier. The inventive pharmaceutical composition can comprise an inventive TCR, or an antigen-binding portion thereof, or population of cells in combination with another pharmaceutically active agent(s) or drug(s), such as a chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for the particular inventive TCR material under consideration. Such pharmaceutically acceptable carriers are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.
The choice of carrier will be determined in part by the particular inventive TCR material, as well as by the particular method used to administer the inventive TCR material. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. Suitable formulations may include any of those for oral, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or interperitoneal administration. More than one route can be used to administer the inventive TCR material, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
Preferably, the inventive TCR material is administered by injection, e.g., intravenously. When the inventive population of cells is to be administered, the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.
It is contemplated that the inventive TCR materials, and pharmaceutical compositions can be used in methods of treating or preventing cancer. Without being bound to a particular theory or mechanism, the inventive TCRs, or the antigen-binding portions thereof, are believed to bind specifically to a mutated amino acid sequence encoded by a cancer-specific mutation, such that the TCR, or the antigen-binding portion thereof, when expressed by a cell, is able to mediate an immune response against a target cell expressing the mutated amino acid sequence. In this regard, the invention provides a method of treating or preventing cancer in a patient, comprising administering to the patient any of the pharmaceutical compositions, TCRs, antigen-binding portions thereof, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells described herein, in an amount effective to treat or prevent cancer in the patient.
The terms “treat,” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a patient. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention can include promoting the regression of a tumor. Also, for purposes herein, “prevention” can encompass delaying the onset of the cancer, or a symptom or condition thereof.
For purposes of the invention, the amount or dose of the inventive TCR material or pharmaceutical composition administered (e.g., numbers of cells when the inventive population of cells is administered) should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the patient over a reasonable time frame. For example, the dose of the inventive TCR material or pharmaceutical composition should be sufficient to bind to a mutated amino acid sequence encoded by a cancer-specific mutation, or detect, treat or prevent cancer in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular inventive TCR material or pharmaceutical composition administered and the condition of the patient, as well as the body weight of the patient to be treated.
Many assays for determining an administered dose are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing the inventive TCR, or the antigen-binding portion thereof, or the inventive populations of cells, upon administration of a given dose of such T cells to a mammal among a set of mammals of which is each given a different dose of the cells, could be used to determine a starting dose to be administered to a patient. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a certain dose can be assayed by methods known in the art.
The dose of the inventive TCR material or pharmaceutical composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive TCR material or pharmaceutical composition. Typically, the attending physician will decide the dosage of the inventive TCR material or pharmaceutical composition with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive TCR material or pharmaceutical composition to be administered, route of administration, and the severity of the condition being treated.
In an embodiment in which the inventive population of cells is to be administered, the number of cells administered per infusion may vary, for example, in the range of one million to 200 billion cells; however, amounts below or above this exemplary range are within the scope of the invention. For example, the daily dose of inventive host cells can be about 1 million to about 200 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, about 60 billion cells, about 80 billion cells, about 100 billion cells, about 120 billion cells, about 130 billion cells, about 150 billion cells, about 160 billion cells, about 170 billion cells, about 180 billion cells, about 190 billion cells, about 200 billion cells, or a range defined by any two of the foregoing values), preferably about 10 million to about 200 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, about 100 billion cells, about 110 billion cells, about 120 billion cells, about 130 billion cells, about 140 billion cells, about 150 billion cells, about 160 billion cells, about 170 billion cells, about 180 billion cells, about 190 billion cells, about 200 billion cells, or a range defined by any two of the foregoing values), more preferably about 100 million cells to about 200 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, about 100 billion cells, about 110 billion cells, about 120 billion cells, about 130 billion cells, about 140 billion cells, about 150 billion cells, about 160 billion cells, about 170 billion cells, about 180 billion cells, about 190 billion cells, about 200 billion cells, or a range defined by any two of the foregoing values).
For purposes of the inventive methods, wherein populations of cells are administered, the cells can be cells that are allogeneic or autologous to the patient. Preferably, the cells are autologous to the patient.
Another embodiment of the invention provides any of the TCR materials or pharmaceutical compositions described herein for use in treating or preventing cancer in a patient.
The cancer may, advantageously, be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, cholangiocarcinoma, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, uterine cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, urinary bladder cancer, solid tumors, and liquid tumors. Preferably, the cancer is an epithelial cancer. In an embodiment, the cancer is cholangiocarcinoma, melanoma, colon cancer, or rectal cancer.
The mammal referred to in the inventive methods can be any mammal. As used herein, the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). Preferably, the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). Preferably, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). A more preferred mammal is the human. In an especially preferred embodiment, the mammal is the patient expressing the cancer-specific mutation.
In an embodiment of the invention, TCR(s), or antigen-binding portion(s) thereof, may be isolated from the T cells that express PD-1 immediately after separating the T cells that express PD-1 from cells that do not express PD-1. These TCR(s), or antigen-binding portion(s) thereof, may be cloned into a recombinant expression vector, and introduced into host cells to obtain expression of the TCR(s), or antigen binding portion(s) thereof, by the host cells. The host cells that express the TCR(s), or antigen binding portions thereof, could then be screened for antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
In this regard, an embodiment of the invention provides a method of isolating T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising obtaining a first population of PBMCs from a sample of peripheral blood from a patient; selecting T cells that express PD-1 from the bulk population; separating the T cells that express PD-1 from cells that do not express PD-1 to obtain a T cell population enriched for T cells that express PD-1; isolating nucleotide sequence(s) that encode(s) one or more TCR(s), or antigen-binding portion(s) thereof, from the T cells of the population enriched for T cells that express PD-1; introducing the nucleotide sequence(s) encoding the TCR(s), or antigen binding portion(s) thereof, into further population(s) of PBMCs to obtain T cells that express the TCR(s), or antigen binding portion(s) thereof; identifying one or more genes in the nucleic acid of a cancer cell of the patient, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence; inducing autologous APCs of the patient to present the mutated amino acid sequence; co-culturing the T cells that express the TCR(s), or antigen binding portion(s) thereof, with the autologous APCs that present the mutated amino acid sequence; and selecting the T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a MHC molecule expressed by the patient.
Obtaining a first population of PBMCs from a sample of peripheral blood; selecting T cells that express PD-1; and separating the T cells that express PD-1 from cells that do not express PD-1 may be carried out as described herein with respect to other aspects of the invention.
The method may further comprise isolating nucleotide sequence(s) that encode(s) one or more TCR(s), or antigen binding portion(s) thereof, from the T cells of the population enriched for T cells that express PD-1. While the method may further comprise expanding the numbers of the T cells that express PD-1 prior to isolating the nucleotide sequence, in a preferred embodiment, the method comprises isolating the nucleotide sequence from the T cells without expanding the numbers of the T cells that express PD-1 prior to isolating the nucleotide sequence. For example, the TCR, or antigen binding portion thereof, may be isolated from a single cell. In an embodiment of the invention, the method comprises isolating nucleotide sequence(s) that encode(s) at least one TCR, or antigen binding portion thereof. However, the number of TCR(s), or antigen binding portion(s) thereof, that may be isolated using the inventive methods is not limited and may include more than one TCR(s), or antigen binding portion(s) thereof (for example, about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values). The nucleotide sequence(s) that encode(s) one or more TCR(s), or antigen binding portion(s) thereof, may otherwise be isolated as described herein with respect to other aspects of the invention.
The method may further comprise introducing the nucleotide sequence(s) encoding the TCR(s), or antigen binding portion(s) thereof, into further population(s) of PBMCs to obtain T cells that express the TCR(s), or antigen binding portion(s) thereof. Each TCR, or antigen binding portion thereof, isolated according to this embodiment of the invention may be introduced into a different population of PBMCs to provide multiple populations of cells, each population of cells expressing a different TCR or antigen binding portion thereof. Introducing the nucleotide sequence(s) encoding the TCR(s), or antigen binding portion(s) thereof, into further population(s) of PBMCs may otherwise be carried out as described herein with respect to other aspects of the invention.
Identifying one or more genes in the nucleic acid of a cancer cell of the patient; inducing APCs of the patient to present the mutated amino acid sequence; co-culturing the T cells with the autologous APCs that present the mutated amino acid sequence; and selecting the T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence may all be carried out as described herein with respect to other aspects of the invention. In an embodiment of the invention in which more than one TCR, or antigen binding portion thereof, is isolated and a nucleotide sequence encoding each TCR, or antigen binding portion thereof is introduced into a different population of cells, co-culturing may comprise separately co-culturing each population of cells (each expressing a different TCR, or antigen binding portion thereof) with the autologous APCs. Selecting may comprise determining which TCR, or antigen binding portion thereof, has antigenic specificity for the mutated amino acid sequence (e.g., by process of elimination). In an embodiment of the invention, the numbers of selected cells may be expanded as described herein with respect to other aspects of the invention. In an embodiment of the invention, the numbers of selected cells are not expanded.
In an embodiment of the invention, the method may further comprise isolating a nucleotide sequence that encodes a TCR, or an antigen-binding portion thereof, from the selected T cells that have antigenic specificity for the mutated amino acid sequence, wherein the TCR, or the antigen-binding portion thereof, has antigenic specificity for the mutated amino acid sequence. Isolating a nucleotide sequence that encodes a TCR, or an antigen-binding portion thereof, from the selected T cells may be carried out as described herein with respect to other aspects of the invention. Further embodiments of the invention may provide methods of preparing a population of cells that expresses the TCR, or antigen binding portion thereof; a TCR, or an antigen-binding portion thereof, isolated according to the inventive methods; isolated populations of cells prepared according to the inventive methods; pharmaceutical compositions comprising the inventive TCR, or antigen binding portion thereof, or the inventive population of cells; and methods of treating cancer using the inventive compositions, all of which may be as described herein with respect to other aspects of the invention.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
This example demonstrates the expression of PD-1 and TIM-3 in the CD8+ cell population in the peripheral blood of a melanoma patient and the purity of the cells separated according to PD-1 and TIM-3.
PBMC from melanoma patient 3713 were rested overnight in the absence of IL-2, stained with antibodies, and sorted according to expression of CD8 and CD3 by FACS. Then, the CD3+CD8+ cells were sorted according to expression of PD-1 and TIM-3 by FACS. The gates of the stained samples were set based on the isotype control. The frequency of the CD8+ PBMC populations expressing each of the markers is indicated in Table 1 below.
This example demonstrates that CD8+PD-1+, CD8+PD-1+TIM-3−, and CD8+PD-1+TIM-3+ cell populations, but not bulk CD8+, CD8+PD-1−, CD8+ TIM-3−, or CD8+ TIM-3+ cell populations, isolated from peripheral blood recognize target cells pulsed with unique, patient-specific mutated epitopes.
Pheresis from a melanoma patient (3713) was thawed and rested overnight in the absence of cytokines. CD8+ cells were sorted according to PD-1 and TIM-3 expression into the following populations: CD8+ bulk, CD8+PD-1−, CD8+PD-1+, CD8+ TIM-3−, CD8+ TIM-3+, CD8+PD-1+TIM-3−, and CD8+PD-1+TIM-3+. The numbers of the sorted cells were expanded in vitro for 15 days. On day 15, the cells were washed and co-cultured with target autologous B cells pulsed with wild type (wt) or mutated (mut) epitopes known to be recognized by the patient's tumor-infiltrating lymphocytes at a ratio of 2×104 effector cells: 1×105B cells. T cells were also co-cultured with the autologous tumor cell line (TC3713) in the absence or presence of HLA-I blocking antibody W6/32 or with an allogeneic tumor cell line (TC3903). T cells were also co-cultured with anti-CD3 antibody as a control. Reactivity was assessed by quantifying IFN-gamma spots 16 hours (h) after the co-culture by IFN-γ ELISpot. The results are shown in Tables 2A and 2B.
As shown in Tables 2A and 2B, CD8+PD-1+, CD8+PD-1+TIM-3−, and CD8+PD-1+TIM-3+ cell populations, but not bulk CD8+, CD8+PD-1−, CD8+ TIM-3−, or CD8+ TIM-3+ cell populations, isolated from peripheral blood recognized target cells pulsed with unique, patient-specific mutated epitopes.
This example demonstrates that CD8+PD-1+, CD8+PD-1+TIM-3−, CD8+PD-1+TIM-3+, and CD8+PD-1+CD27hi cell populations, but not bulk CD8+, CD8+ TIM-3−, CD8+ TIM-3+, CD8+PD-1-CD27hi, or CD8+PD-1− cell populations, isolated from peripheral blood recognize target cells electroporated with RNA encoding unique, patient-specific mutated epitopes.
Pheresis from melanoma patient 3903 was thawed and rested overnight in the absence of cytokines. CD8+ cells were enriched by bead separation and then sorted according to PD-1 and TIM-3 expression into the following populations: CD8+ bulk, CD8+PD-1−, CD8+PD-1+, CD8+ TIM-3−, CD8+ TIM-3+, CD8+PD-1+TIM-3−, CD8+PD-1+TIM-3+, CD8+PD-1-CD27hi, and CD8+PD-1+CD27hi. The numbers of sorted cells were expanded in vitro for 15 days. On day 15, the cells were washed and co-cultured with target autologous dendritic cells electroporated with RNA encoding mutated tandem minigenes (TMGs 1-26; each encoding multiple 25mers containing a mutation flanked by the endogenous sequence) identified by exome sequencing of a tumor from patient 3903. The effector cells were co-cultured with the target cells at a ratio of 2×104 effector cells: to about 1×105 DCs. The effector cells were also co-cultured with the autologous tumor cell line (TC3903) or with an allogeneic tumor cell line (TC3903). Reactivity was assessed by quantifying IFN-gamma spots 16 h after the co-culture by IFN-γ ELISpot. The results are shown in Tables 3A-3C.
As shown in Tables 3A-3C, CD8+PD-1+, CD8+PD-1+TIM-3−, CD8+PD-1+TIM-3+, and CD8+PD-1+CD27hi cell populations, but not bulk CD8+, CD8+ TIM-3−, CD8+ TIM-3+, CD8+PD-1-CD27hi, or CD8+PD-1− cell populations, isolated from peripheral blood recognized target cells electroporated with RNA encoding unique, patient-specific mutated epitopes. In melanoma patient 3903, CD8+ PBL subsets expressing PD-1 were enriched in cells recognizing TMG-9 (Tables 2A-2B). In this patient, further enrichment in mutation-specific cells from peripheral blood was observed when selecting CD8+ cells expressing PD-1 in combination with TIM-3 or CD27 (TMG-8, TMG-3, and weaker recognition of TMG-7 and TMG-11) (Tables 3A-3C).
CD8+ lymphocytes expressing PD-1 in the peripheral blood of patient 3903 were enriched in cells capable of recognizing the autologous tumor cell line (Tables 3A-3C).
The sorted cells were also co-cultured with autologous DCs electroporated with RNA encoding full-length MART-1, GP100, tyrosinase, NY-ESO-1, MAGE-A3, or SSX2. CD8+ lymphocytes expressing PD-1 in the peripheral blood of patient 3903 also recognized mutation-specific cells and cancer germline antigens SSX2 and MAGE-A3.
This example demonstrates that CD8+PD-1+, CD8+PD-1hi, CD8+PD-1+TIM-3+, CD8+PD-1+CD27hi, and CD8+PD-1+CD27− cell populations, but not bulk CD8+, CD8+ TIM-3−, CD8+ TIM-3+, or CD8+PD-1− cell populations, isolated from peripheral blood recognize target cells electroporated with RNA encoding unique, patient-specific mutated epitopes.
Pheresis from melanoma patient 3784 was thawed and rested overnight in the absence of cytokines. CD8+ cells were enriched by bead separation and then sorted according to PD-1 and TIM-3 expression into the following populations: CD8+ bulk, CD8+PD-1−, CD8+PD-1+, CD8+PD-1hi, CD8+ TIM-3−, CD8+ TIM-3+, CD8+PD-1+TIM-3+, CD8+PD-1+CD27hi, and CD8+PD-1+CD27−. The numbers of sorted cells were expanded in vitro for 15 days. On day 15, the cells were washed and co-cultured with target autologous dendritic cells electroporated with RNA encoding mutated tandem minigenes (TMGs 1-9; each encoding multiple 25mers containing a mutation flanked by the endogenous sequence) identified by exome sequencing of a tumor from patient 3784. The effector cells were co-cultured with the target cells at a ratio of 2×104 effector cells: to about 1×105 DCs. The effector cells were also co-cultured with autologous DCs electroporated with RNA encoding epitopes for cytomegalovirus (CMV), Epstein—Barr virus (EBV), FLU (CEF), or an irrelevant TMG. T cells were also co-cultured with the autologous tumor cell line (TC3784) or with an allogeneic tumor cell line (TC3903). Reactivity was assessed by quantifying IFN-gamma spots 16 h after the co-culture by IFN-γ ELISpot.
The results are shown in Tables 4A-4C. As shown in Tables 4A-4C, CD8+PD-1+, CD8+PD-1hi, CD8+PD-1+TIM-3+, CD8+PD-1+CD27hi, and CD8+PD-1+CD27− cell populations, but not bulk CD8+, CD8+ TIM-3−, CD8+ TIM-3+, or CD8+PD-1− cell populations, isolated from peripheral blood recognized target cells electroporated with RNA encoding unique, patient-specific mutated epitopes.
In this patient, the peripheral blood CD8+ lymphocytes expressing PD-1 were enriched in mutation-specific cells recognizing up to three antigens (TMG-3, TMG-5, and TMG-8). Peripheral blood CD8+PD-1+ and PD-1hi T cells also recognized gp100.
Further separation of peripheral blood CD8+PD-1+ lymphocytes into CD27hi or CD27− separated the lymphocytes recognizing TMG-3 from those recognizing TMG-5 and TMG-8.
The co-culture of the sorted cells with the autologous tumor cell line or the allogeneic tumor cell line revealed that peripheral blood CD8+ lymphocytes expressing PD-1 alone or in combination with TIM-3 or CD27 were enriched in tumor-reactive cells.
This example demonstrates that CD8+PD-1+ and CD8+PD-1hi cell populations, but not bulk CD8+ or CD8+PD-1− cell populations, isolated from peripheral blood recognize target cells electroporated with RNA encoding unique, patient-specific mutated epitopes.
Pheresis from a colorectal cancer patient 3971 was thawed and rested overnight in the absence of cytokines. CD8+ cells were enriched by bead separation and then sorted according to PD-1 expression into the following populations: CD8+ bulk, CD8+PD-1−, CD8+PD-1+, and CD8+PD-1hi. The numbers of sorted cells were expanded in vitro for 15 days. On day 15, the cells were washed and co-cultured with target autologous dendritic cells electroporated with RNA encoding mutated tandem minigenes (TMGs 1-9; each encoding multiple 25mers containing a mutation flanked by the endogenous sequence) identified by exome sequencing from the patient's tumor (at a ratio of 2×104 effector cells: about 1×105 DCs). TMG-1 encoded mutated CASP8 peptide. The cells were also co-cultured with cells electroporated with RNA encoding a mock (empty) control vector or irrelevant TMG. Reactivity was assessed by quantifying IFN-gamma spots 16 h after the co-culture by IFN-γ ELISpot. The results are shown in Table 5. As shown in Table 5, CD8+PD-1+ and CD8+PD-1hi cell populations, but not bulk CD8+ or CD8+PD-1− cell populations, isolated from peripheral blood recognized target cells electroporated with TMG-1 or TMG-3 RNA.
This example demonstrates the isolation of a nucleotide sequence encoding a TCR having antigenic specificity for target cells electroporated with RNA encoding unique, patient-specific mutated epitopes from a CD8+PD-1hi cell population.
The TMG-1 and TMG-3 reactive cells present in the CD8+PD-1hi cell population of Example 5 (colorectal cancer patient) were selected by FACS based on the upregulation of 4-1BB (CD137). On day 15, PD-1hi bulk cells, as well as CD137−, and CD137+ fractions, were co-cultured with target DCs electroporated with RNA encoding for TMG-1 or TMG-3, or plate-bound OKT3. Reactivity was assessed by CD137 upregulation after 20 h. The number of cells and the percentage of cells (with respect to bulk cells) having the indicated phenotype are shown in Table 6A.
The numbers of cells in Table 6A were expanded in vitro for 14 days. The cell yields obtained are shown in Table 6B.
A nucleotide sequence encoding a TCR was isolated from the TMG-1 and TMG-3 reactive cells that were selected on the basis of CD137 upregulation. The CD137+ TMG-1 reactive cells (>97% one clonotype) comprised an alpha chain CDR3 amino acid sequence of CAVRDRGTGGFKTIF (SEQ ID NO: 1) and a beta chain CDR3 amino acid sequence of CASITKDRAYEQYF (SEQ ID NO: 2). The CD137+ TMG-3 reactive cells (>93% one clonotype) comprised an alpha chain CDR3 amino acid sequence of CAYRSASDMRF (SEQ ID NO: 3) and a beta chain CDR3 amino acid sequence of CASSPETGGISEQYF (SEQ ID NO: 4).
Accordingly, the selection of CD137+ cells that were reactive against target cells electroporated with TMG-1 or TMG-3 from CD8+PD-1hi lymphocytes sorted from the peripheral blood led to the generation of highly enriched TMG-1 and TMG-3 specific populations, each encoding for one dominant TCR.
This example demonstrates the identification of the mutation recognized by TMG-1 reactive cells isolated from CD8+PD-1+ peripheral blood cells.
Following 15 days in culture, the sorted TMG-1-reactive, CD137− and CD137+ effector populations of Example 6 were co-cultured with autologous DCs that were electroporated with TMG-1 RNA or were pulsed with wild type or mutated CASP8 minimal epitopes. Reactivity was assessed by quantifying IFN-gamma spots 16 h after the co-culture by IFN-γ ELISpot. The resulting numbers of IFN-γ spots measured per 2×104 cells are shown in Table 7.
As shown in Table 7, the TMG-1 reactive cells enriched from peripheral blood recognized a unique mutation in CASP8 identified through exome sequencing of 3971 tumor.
This example demonstrates the identification of the mutation recognized by TMG-3 reactive cells isolated from CD8+PD-1+ peripheral blood cells.
Following 15 days in culture, the sorted TMG-3-reactive, CD137− and CD137+ effector cell populations of Example 6 were co-cultured with autologous DCs that were pulsed with mutated long peptides (μg/ml) derived from TMG-3 (Nos. 1-16 in Table 8). Reactivity was assessed by quantifying IFN-gamma spots 16 h after the co-culture by IFN-γ ELISpot. The resulting numbers of IFN-γ spots measured per 2×104 cells are shown in Table 8.
As shown in Table 8, the TMG-3 reactive cells enriched from CD8+PD-hi population selected from peripheral blood recognized long peptide TMG-3 number 1, which encoded a mutated HISTH3B peptide.
This example demonstrates the reactivity of PBL engineered to express the mutated CASP8 peptide specific T-cell receptor isolated in Example 6.
PBL were transduced with the nucleotide sequence encoding the TMG-1 specific TCR of Example 6 or an empty vector (control). Autologous B cells were pulsed for 2 h with either wild type or mutated CASP8 peptides. The pulsed cells were co-cultured with TCR transduced or vector transduced cells (at a ratio of 2×105 B cells: 2×104 effector cells). Reactivity was measured by 4-1BB upregulation 24 h later. The frequency of 4-1BB within the CD3+CD8+ cells is shown in
This example demonstrates that CD8+PD-1+ and CD8+PD-1hi cell populations, but not bulk CD8+ or CD8+PD-1− cell populations, isolated from peripheral blood recognize target cells pulsed with unique, patient-specific mutated epitopes. This example also demonstrates that CD4+PD-1+ and CD4+PD-1hi cell populations, but not bulk CD4+ or CD4+PD-1− cell populations, isolated from peripheral blood recognize target cells electroporated with RNA encoding NY-ESO-1.
Pheresis from a melanoma patient (3998) was thawed and rested overnight in the absence of cytokines. CD8+ cells were sorted according to PD-1 expression into the following populations: CD8+ bulk, CD8+PD-1−, CD8+PD-1+, and CD8+PD-1hi as described in Example 2. CD4+ cells were sorted according to PD-1 expression into the following populations: CD4+ bulk, CD4+PD-1−, CD4+PD-1+, and CD4+PD-1hi as described in Example 2. The numbers of the sorted cells were expanded in vitro for 15 days. On day 15, the cells were washed and co-cultured with target autologous DCs electroporated with RNA encoding mutated tandem minigenes (TMGs 1-7; each encoding multiple 25mers containing a mutation flanked by the endogenous sequence) identified by exome sequencing of a tumor from patient 3998, or RNA encoding MART-1, gp100, tyrosinase, NY-ESO-1, MAGE-A3, or SSX2. The cells were also co-cultured with autologous tumor cell line or allogeneic tumor cell line (3713). T cells were also co-cultured with anti-CD3 antibody as a control. Reactivity was assessed by quantifying IFN-gamma spots 16 hours (h) after the co-culture by IFN-γ ELISpot. The results are shown in Tables 9A and 9B.
As shown in Table 9A, the CD8+PD-1+ and CD8+PD-1hi cell populations, but not bulk CD8+ or CD8+PD-1− cell populations, isolated from peripheral blood recognized target cells electroporated with RNA encoding with unique, patient-specific mutated epitopes (TMG-1). As shown in Table 9A, the CD8+PD-1hi cell population, but not bulk CD8+, CD8+PD-1+, or CD8+PD-1− cell populations, isolated from peripheral blood recognized target cells electroporated with RNA encoding with unique, patient-specific mutated epitopes (TMG-3). The CD8+PD-1+ and CD8+PD-1hi cell populations isolated from peripheral blood recognized target cells electroporated with RNA encoding NY-ESO-1 (Table 9A).
As shown in Table 9B, CD4+PD-1+ and CD4+PD-1hi cell populations, but not bulk CD4+ or CD4+PD-1− cell populations, isolated from peripheral blood recognized target cells electroporated with RNA encoding NY-ESO-1.
The following materials and methods were employed for the experiments described in Examples 11-17.
Leukapheresis products, and tumor samples were obtained from individuals with stage IV melanoma enrolled on a clinical protocol (03-C-0277), approved by the institutional-review board (IRB) of the National Cancer Institute (NCI). Informed consent was obtained from all subjects, and they all had progressive disease at the time of sample acquisition. The 5 individuals studied in detail were chosen on the basis of availability of pre-treatment leukapheresis, and matched frozen fresh tumor to perform whole-exome sequencing and transcriptome analysis. Patients were either treatment naïve (NCI-3998), or had undergone prior therapies including surgery, chemotherapy, and immunotherapy (NCI-3713, 3784, 3903, and 3926). The patient characteristics are provided in Table 10. The patients that received prior therapies had been last treated from 7-55 months before the leukapheresis product was obtained. A summary of the individuals included in the phenotypic characterization of circulating and tumor-infiltrating lymphocytes is provided in Table 11. Melanoma specimens were surgically resected and digested into single cell suspensions using the GENTLEMACS Dissociator (Miltenyi Biotec, Gladbach, Germany) as described in Gros et al., J. Clin. Invest., 124: 2246-2259 (2014), and cryopreserved. Peripheral blood mononuclear cells (PBMC) were obtained by leukapheresis, prepared over a Ficoll-Hypaque gradient (LSMTM; MP Biomedicals, Santa Ana, CA), and cryopreserved until analysis. Melanoma cell lines were established from enzymatically separated tumor cells cultured in RPMI 1640 supplemented with 10% FBS (HyClone Defined, Logan, UT) at 37° C. and 5% CO2. Melanoma cell lines were mycoplasma negative, and were authenticated based on the identification of patient-specific somatic mutations, and HLA molecules.
n.d.f
aMelanoma;
bAdoptive transfer of autologous T cells that were gene-engineered to express a MART-1 HLA-A*0201-restricted T-cell receptor.
cChemotherapy patient 3926: dacarbazine and vinblastine.
dPutative non-synonymous mutations were defined by: >2 exome variant reads, ≥10% variant frequency in the exome, ≥10 normal reads, and tumor/normal variant frequency ≥5. Common single nucleotide polymorphisms were filtered.
eMutations evaluated were selected based on whole-exome and transcriptome analysis.
fNot deteremined.
gNot evaluated.
Tumor biopsies and normal PBMC were subjected to DNA extraction, library construction, exome capture of approximately 20,000 coding genes, and next-generation sequencing by Macrogen (Rockville, MD), Personal Genome Diagnostics (PGDX, Baltimore, MD), or the Broad Institute (Cambridge, MA). The average number of distinct high quality sequences at each base ranged between 100 and 150 for the individual exome libraries. Alignments and variant calling were performed, as described in Tran et al., Science, 344: 641-645 (2014). The total number of putative non-synonymous mutations (Table 10) was determined using filters consisting of >2 exome variant reads, ≥10% variant allele frequency (VAF) in the tumor exome, >10 normal reads, tumor/normal variant frequency ≥5, and filtering out single nucleotide polymorphisms in dbSNP build 138. An mRNA sequencing library was also prepared from a tumor biopsy using Illumina TRUSEQ RNA library prep kit. RNA alignment was performed using STAR (Dobin et al., Bioinformatics, 29: 15-21 (2013)) duplicates, were marked using picard's MARKDUPLICATE tools, and FPKM values were calculated using cufflinks (Trapnell et al., Nature Biotechnol., 8: 511-515 (2010)). The levels of transcripts encoding putative non-synonymous variants, calculated as fragments per kilobase per million mapped reads (FPKM), were used to assess expression of candidate mutations identified using whole exome data.
The following criteria were used to prioritize mutations for immunological screening (Table 12). Initially, mutations with a variant allele frequency (VAF)>10% in the tumor exome, as well as mutations that were identified in both transcriptome and exome analysis without any additional filters, were selected. For some samples (NCI-3903), the mutations selected based on exome only were prioritized by selecting those with >10 variant reads to increase the confidence of mutation calling. For each of the immunogenic antigens detected, the amino acid changes are specified.
Fluorescently labeled antibodies were purchased from BD Biosciences, San Jose, CA (UCHT1, 1.6:100, CD3 PE-CF594; SK7, 1:100, CD3 APC-Cy7; SK1, 0.5:100, CD8 PE-Cy7; 4B4-1, 1.25:100, CD137 APC; NK-1, 3:100, CD57 FITC; J168-540, 1.2:100, BTLA PE), eBioscience, San Diego, CA (H57-597, 0.5:100, mTCRB FITC; 0323, 2:100, CD27 BV605), Biolegend, San Diego, CA (EH12.2H7, 0.7:100, PD-1 BV421), R&D Systems, Minneapolis, MN (344823, 2.6:100, TIM-3 PE and APC), Enzo Life Sciences, Farmingdale, NY (17B4. 1:100, LAG-3 FITC) and Miltenyi Biotec (4B4-1, 2.6:100, 4-1BB PE). Anti-PD-1 antibody was from Amplimmune (Gaithersburg, MD, AMP-514, 1/300, PD-1 Alexa Fluor 647). Cell-sorting experiments were carried out using anti-PD-1 AMP-514 antibody.
To perform the phenotypic characterization, PBMC and tumor single cell suspensions were thawed into T-cell media (1:1 mix of AIMV media [Life Technologies, Waltham, MA] and RPMI 1640 media [Lonza, Walkersville, MD], 5% in-house human serum, 100 U/ml penicillin and 100 μg/ml streptomycin [Life Technologies], 2 mM L-glutamine [Life Technologies], 10 μg/ml gentamicin [Quality Biological Inc., Gaithersburg, MD], 12.5 mM HEPES [Life Technologies]) supplemented with DNAse (Genentech Inc. San Francisco, CA, 1:1000), centrifuged, and plated at 2e6 cells/well in a 24-well plate in the absence of cytokines. After resting the cells overnight at 37° C. and 5% CO2, cells were harvested, and 2e6 cells were resuspended in 50 μl of staining buffer (PBS, 0.5% BSA, 2 mM EDTA) containing antibodies. Cells were incubated for 30 minutes at 4° C. and washed twice prior to acquisition. Flow cytometry acquisition was carried out on a modified FORTESSA analyzer, equipped to detect 18 fluorescence parameters, or CANTO II flow cytometers (BD Biosciences). Flow cytometry data were analyzed using FLOWJO software (Ashland, OR). Data were gated on live cells (PI negative), single cells. Gates were set based on fluorescence minus one (FMO) controls.
Cell-sorting was carried out using the BD JAZZ cell sorter (BD Biosciences). For all experiments requiring cell-sorting from PBMC, CD8+ cells were first enriched using CD8 microbeads (Miltenyi), and stained as described above in “Antibodies, and phenotypic characterization of T cells.” When sorting T cells from fresh tumor single cell suspensions, this pre-enrichment step was not performed. Cells were gated on live (PI negative), single cells, CD3+, and CD8+ cells, and on the population of interest. Half of the T cells isolated were spun and snap frozen to perform TRB deep sequencing, and the other half were expanded in vitro. T-cell yields ranged from 3×103 to 3×105. A similar sorting strategy was used to sort the 4-1BB+ lymphocytes, following a 20 h co-culture.
T cells were expanded in vitro using an excess of irradiated allogeneic feeders cells (5000 rad) pooled from three donors in T-cell media supplemented with 30 ng/ml anti-CD3 (OKT3, Miltenyi Biotec) and 3000 IU of IL-2 (Aldesleukin, Chiron). After day 6, half of the media was replaced with fresh T-cell media containing IL-2 every other day. At day 15, T cells were either used in co-culture assays or cryopreserved, until future analysis. Of note, enrichment of mutation-specific T cells was consistent between replicate CD8+PD-1+ T cell cultures, but stochastic outgrowth or loss of T cell reactivities can be observed and become more apparent when starting with less than 3×103 CD8+PD-1+ T cells. The minimum material required to sort 3×103 CD8+PD-1+ cells is approximately 1×107 PBMC.
Immature dendritic cells (CD11c+, CD14−, CD80low, CD86+ and HLA-DR+) were generated from PBMC using the plastic adherence method, as described in Tran et al., Science, 344: 641-645 (2014). On day 3, DC media was added, and at day 5-6 DCs were harvested and used in electroporation experiments or cryopreserved. DC media comprised of RPMI supplemented with 5% human serum, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine (Life Technologies), 800 IU/ml GM-CSF and 200 U/ml IL-4 (Peprotech, Rocky Hill, NJ). When used after cryopreservation, cells were thawed into DC media, spun at 1000 RPM for 10 min, resuspended in DC media at 2×106 cells/ml, and incubated at 37° C. and 5% CO2 for 2 h, prior to electroporation or peptide pulsing.
Autologous B cells were isolated from autologous PBMC by positive selection using CD19+ microbeads (Miltenyi Biotec) and expanded using irradiated NIH3T3 CD40L cells and IL-4 (Peprotech), as described in Tran et al., Science, 344: 641-645 (2014). B cells were harvested at day 5-6 after the initial stimulation, and either re-stimulated, cryopreserved, or used in co-culture assays. When used after cryopreservation, B cells were thawed into B cell media 16-24 h prior to using them in co-culture assays. B cell media comprised of IMDM media (Quality Biological Inc., Gaithersburg, MD) supplemented with 10% human serum, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine, and 200 U/ml IL-4 (Peprotech, Rocky Hill, NJ).
Tandem minigenes (TMGs) were constructed as described in Lu et al., Clin. Cancer Res., 20: 3401-3410 (2014); Tran et al., Science, 344: 641-645 (2014). Briefly, a minigene was constructed for each non-synonymous variant identified, composed of the mutated amino acid flanked by 12 amino acids of the wild-type protein sequence. Up to 16 minigenes were strung together to generate a tandem minigene (TMG) construct. These TMG constructs were codon optimized and cloned in frame into pcRNA2SL using EcoRI and BamHI. pcRNA2SL is based on the pcDNA3.1, and was modified to include a signal sequence and a DC-LAMP trafficking sequence to enhance processing and presentation (Wu et al., PNAS, 92: 11671-11675 (1995)). The sequences were verified by sanger sequencing. Following linearization of the constructs, phenol chloroform extraction was performed and DNA was precipitated with sodium acetate and ethanol. Next, 1 μg of linearized DNA was used to generate in vitro transcribed RNA using the MMESSAGE MACHINE T7 Ultra kit (Life Technologies), as instructed by the manufacturer. RNA was precipitated using LiCl2, and resuspended at 1 μg/μl. To screen for recognition of cancer germline antigens NY-ESO-1, MAGEA3 and SSX2, and melanoma differentiation antigens MART′, GP100 and TYROSINASE, full-length amino acid sequences were cloned individually into pcRNA2SL using EcoRI and BamHI, and these constructs were used to generate IVT RNA.
DCs were resuspended in Opti-MEM (Life Technologies) at 10-40×106 cells/ml. 8 μg of IVT RNA was aliquoted into the bottom of a 2 mm gap electroporation cuvette, and 100 μl of DCs were added. DCs were electroporated with 150 V, 10 ms, and 1 pulse, using a BTX-830 square wave electroporator (Holliston, MA). Cells were gently resuspended into DC media and transferred into ultra-low attachment polysterene 24-well plate (corning) at approximately 1×106 DCs/ml, and rested overnight at 37° C., 5% CO2. Transfection efficiencies were routinely between 70-90% assessed with a control GFP RNA (not shown). In co-culture assays, the irrelevant TMG RNA control was a random TMG from a different patient.
HLA alleles were cloned into pcDNA3.1. To interrogate which HLA alleles presented the neo-antigens identified, COST cells were co-transfected with TMG DNA constructs and plasmids encoding the individual HLA molecules using LIPOFECTAMINE 2000 reagent (Life Technologies). After 16 h, cells were harvested and used as targets in co-culture assays.
HLA was determined from next generation sequencing data using the algorithm PHLAT (Bai et al., BMC Genomics, 15: 325 (2014)). (NCI-3713: HLA-A*02:01, A*29:02, B*44:03, B*51:01, C*15:02, C*16:01. NCI-3998: HLA-A*01:01, A*30:02, B*15:01, B*18:01, C*03:03, C*05:01. NCI-3784: HLA-A*01:01, A*03:01, B*07:02, C*07:02. NCI-3903: HLA-A*02:01, A*24:02, B*27:02, B*38:01, C*02:02, C*12:03. NCI-3926: HLA-A*01:01, A*02:01, B*08:01, B*13:02, C*06:02, C*07:01).
Candidate 8 to 11-mers containing the mutated residues that were predicted to bind with high affinity to the patients' HLA-I molecules were identified using the immune epitope database (IEDB) (Vita et al., Nucleic Acids Res., 43: D405-412 (2015)). Crude and HPLC peptides were synthesized by GenScript (Piscataway, NJ), and resuspended in DMSO at 10 mg/ml and stored at −20° C.
For experiments requiring peptide pulsing, DCs or B cells were resuspended in DC or B cell media, respectively, at 1e6 cells/ml. DCs were incubated overnight at 3TC and 5% CO2 with wild-type or mutated 25-mers at a concentration of 10 μg/ml in DC media. B cells were pulsed with 1 μg/ml or with 10-fold serial dilutions of minimal epitopes starting at 10 μg/ml for 2 h at 37° C. and 5% CO2. DCs or B cells were washed once with PBS prior to co-incubation with T cells.
Both IFN-γ enzyme-linked immunospot assay (ELISPOT) and 4-1BB upregulation at 20 h after the co-culture were used to measure target cell recognition by T cells. After 15 days of T-cell expansion, or following overnight rest of cryopreserved T cells in T cell media supplemented with 3000 IU/ml IL-2, T cells were washed to remove excess cytokines. In the ELISPOT assays, 2×104 T cells were added per well in a 96-well plate. When DCs electroporated with IVT RNA encoding for TMGs or shared antigens were used as targets, approximately 3-7×104 cells/well were used in a 96-well plate. When peptide-pulsed B cells were used, 8×104 to 1.5×105 cells were added per well. All co-cultures were carried out in T-cell media in the absence of exogenously added cytokines. T cells cultured alone or stimulated with plate bound anti-CD3 (OKT3) were used as controls in all the assays. CEF RNA encoding for epitopes derived from CMV, EBV, and Flu (CEF) were included as controls in all the immunological screening assays (Nielsen et al., J. Immunol., Meth., 360: 149-156 (2010)).
IFN-γ ELISPOT assays were carried out as described in Tran et al., Science, 344: 641-645 (2014). The raw data were plotted without subtracting the background. Greater than 40 spots, and greater than twice the background was considered positive T cell reactivity. Prior to processing the ELISPOT assay, cells were harvested for flow-cytometry detection of 4-1BB upregulation, as described in Tran et al., Science, 344: 641-645 (2014).
TCR-α (TRA) and TCR-β (TRB) deep sequencing were performed on genomic DNA by Adaptive Biotechnologies (Seattle, WA). For the enriched populations of TMG-reactive cells, DNA was extracted from 1e6 lymphocytes. The number of circulating and tumor-resident CD8+ lymphocytes that were sequenced ranged from 3×103 to 3×105. The coverage per sample was >10×. Only productive TCR rearrangements were used in the calculations of TCR frequencies and TRB overlap. Analysis of TRB overlap of CDR3 nucleotide sequences between two given populations was calculated using an IMMUNOSEQ analyzer (Adaptive Biotechnologies, Seattle, WA) using the following formula: sample TRB overlap=[shared sequence reads in A+shared sequence reads in B]/Σsequence reads in A+B). Weighing in the frequency of the shared sequences rather than the total number of shared sequences helped account for potentially different sized samples. A TRB overlap of 1 represents 100% overlap between two populations.
For NCI-3998, TCRs were constructed by pairing the dominant TRA and TRB chains, and for each population the TCRs were designated based on the rank of the TRA and TRB (TCR A rank #/B rank #) within the population sequenced. In total, 2 TCRs were assembled from the TMG1 (MAGEA6E>K)-reactive population (TCR A1/B1, TCR A1/B2), and 4 TCRs from the TMG3 (PDS5AY>F;H>Y)-reactive, as well as the TMGS (MED13P>S)-reactive populations (TCR Al/B1, TCR A1/B2, TCR A2B1, TCR A2/B2). Briefly, TRA V-J regions and TRB V-D-J regions were fused to the mouse constant TCR-alpha and beta chains (Cohen et al., Cancer Res., 66: 8878-8886 (2006)), respectively. Mouse constant regions were modified, as described in (Cohen et al., Cancer Res., 67: 3898-3903 (2007); Haga-Friedman et al., J. Immunol., 188: 5538-5546 (2012). The full-length TCRB and TCRA chains were cloned, in this orientation, into pMSGV1 retroviral vector separated by a furin SGSG P2A linker (GenScript). For all TCRs, the amino acid residue at position 2 of the beta chain was changed from a glycine to an alanine in order to facilitate cloning into the vector.
Transient retroviral supernatants were generated, and autologous PBMCs were transduced as described in Tran et al., Science, 344: 641-645 (2014). Transduced T cells were used at day 15 or cryopreserved until used. GFP and mock transduced T cells were used as controls in all transduction experiments.
Data were reported as the median, mean±SEM, or mean±SD, as specified. The Mann-Whitney test was used to compare the percentage of PD-1 expression between PBMC and fresh tumor single cell suspensions. Dunn's test for multiple comparisons was used to analyze the statistical differences in TRB overlap. Statistical analysis was carried out using PRISM program 6.0 (GRAPHPAD Software Inc., La Jolla, CA). Unless otherwise specified, experiments were performed without duplicates. All data are representative of at least 2 experiments.
This example demonstrates the expression of PD-1 on peripheral blood and tumor-infiltrating CD8+ T cells in patients with melanoma.
The expression of PD-1 on peripheral blood and tumor-infiltrating CD8+ T cells was compared. PD-1 expression accounted for approximately 36% of the CD8+ TIL population, but matched peripheral blood samples from the same individuals contained only a median of 4.1% CD8+PD-1+ cells. Moreover, circulating CD8+ lymphocytes had limited co-expression of the inhibitory and co-stimulatory cell surface receptors PD-1, TIM-3, LAG-3 and 4-1BB compared to tumor-resident CD8+ lymphocytes. Thus, few PD-1-expressing circulating CD8+ lymphocytes are present in patients with melanoma.
This example demonstrates the screening of circulating in vitro expanded CD8+ cells from melanoma patients for recognition of mutations.
It was next examined whether selection of circulating CD8+PD-1+ lymphocytes was able to prospectively identify neoantigen-specific CD8+ T cells in the blood of four individuals with melanoma. A high-throughput personalized screening strategy capable of evaluating T cell reactivity to neoantigens presented on all of the HLA restriction elements of the individual was used. Briefly, mutations selected on the basis of tumor-exome and transcriptome analyses were incorporated into oligonucleotides (minigenes) that encoded a 25-residue peptide (25-mer), and these oligonucleotides were then concatenated to yield tandem minigenes (TMGs; designated in numerical order and for each patient). Each TMG encoded up to 16 minigenes, and the requisite number of TMGs that allowed for the expression of all of the mutant 25-mers that were identified were constructed.
In parallel, CD8+ lymphocytes were separated from pre-treatment peripheral blood mononuclear cells (PBMCs) into CD8+, CD8+PD-1−, CD8+PD-1+, and CD8+PD-1hi (defined as the top 20% of PD-1-expressing CD8+ T cells), and expanded for 15 days. In vitro transcribed TMG RNA was electroporated into immature autologous dendritic cells (DCs) that were employed as targets in a T cell co-culture assay. Using this approach, the circulating in vitro expanded CD8+ subsets from 4 individuals with metastatic melanoma (patients NCI-3998, NCI-3784, NCI-3903, and NCI-3926, see Table 10) were screened for recognition of 115, 140, 308, and 128 mutations, respectively.
This example demonstrates the detection of mutation-reactive lymphocytes within the CD8+PD-1+ subset of Example 12.
Although the unseparated peripheral blood CD8+ cells, as well as the CD8+PD-1− lymphocytes, from NCI-3998 showed limited recognition of the mutant 25-mers encoded by TMG1 (hereafter referred to as recognition of TMG1 or TMG1 reactive), the circulating CD8+PD-1+ lymphocyte subset showed enhanced TMG1 reactivity and low, but reproducible, reactivity to TMG3 and TMGS. Based on upregulation of the activation marker 4-1BB, the frequency of CD8+PD-1+ cells that were reactive to DCs expressing these TMG-encoded peptides was 1.8% for TMG1, 0.5% for TMG3 and 0.3% for TMGS. Additionally, recognition of TMG1 and TMG3 by the CD8+PD-1hi subset was also observed. Similarly, CD8+PD-1+ and CD8+PD-1hi, but not CD8+ or CD8+PD-1−, lymphocytes from the peripheral blood of subjects NCI-3784 and NCI-3903 showed T cell reactivity to neoantigens. Circulating CD8+PD-1+ cells from NCI-3784 recognized at least three neo-antigens encoded by TMG3, TMGS and TMGS, and peripheral blood CD8+PD-1+ lymphocytes isolated from NCI-3903 detected at least one neo-antigen expressed by TMGS. NCI-3926 peripheral blood lymphocytes did not show T-cell reactivity to any of the neo-antigens screened. Overall, circulating mutation-reactive lymphocytes were prospectively identified in 3 of 4 melanoma patients evaluated, and these cells were consistently detected within the CD8+PD-1+ lymphocytes. Notably, with the exception of NCI-3998, who displayed low level recognition of TMG1 in the unseparated population of circulating CD8+ T cells, selection of CD8+PD-1+ or PD-1hi lymphocytes from the blood of the patients was necessary to expose CD8+ T cell reactivity to neoantigens.
This example demonstrates the isolation of TCRs from the mutation-reactive lymphocytes of Example 13.
The specific neo-antigens targeted by the mutation-reactive lymphocytes were next analyzed. Given the low frequency of some of the reactivities, and the polyclonal nature of the circulating PD-1+ subset, TMG-reactive cells were enriched by selecting 4-1BB+ lymphocytes following a co-culture with specific TMGs, expanding them in vitro, and co-incubating them with DCs individually pulsed with the mutated 25-mers encoded by the corresponding TMG (Table 12). In a representative example, TMG1-, TMG3- and TMG5-reactive cells isolated from the circulating CD8+PD-1+ subset of subject NCI-3998 showed reactivity to neoantigens derived from mutations in the genes MAGE family member A6 (MAGEA6), PDS5 cohesin-associated factor A (PDS5A) and mediator complex subunit 13 (MED13) (which are referred to as MAGEA6E>K, PDS5AY>F;H>Y and MED13P>S, respectively). The minimal predicted epitopes were determined, synthesized, and tested, and the TMG-reactive cells demonstrated specific recognition of the mutated neo-epitopes over the wild-type counterparts. The HLA alleles presenting the neo-antigens were also identified. Although MAGEA6E>K and PDS5AY>F;H>Y were presented by the alleles encoding HLA-A*30:02 and HLA-C*03:03, respectively, recognition of the MED13P>S neo-epitope was restricted to alleles encoding HLA-A*30:02 and HLA-B*15:01. Deep-sequencing analyses of the variable V-J or V-D-J region of the TRA and TRB genes (which encode the hypervariable regions of the TCR-α and TCR-β chains that are important for peptide recognition by the TCR) of the enriched populations of neoantigen-specific CD8+ T cells revealed multiple dominant TRA and TRB sequences that were unique for each of the T cell populations. To study the specificity of the mutation-specific cells at the clonal level, TCRs were constructed by pairing the sequences encoding the 2 most dominant TRA and TRB CDR3 sequences (Linnemann et al., Nature Med., 19: 1534-1541 (2013)) from the MAGEA6E>K, PDS5AY>F;H>Y, or the MED13P>S neo-antigen specific lymphocytes, and cloning them into retroviral vectors used to transduce autologous PBMCs. The full-length alpha and beta chain amino acid sequences encoded by the vectors are shown in Table 13. The two TCRs constructed by pairing the most dominant and the second most dominant TRA and TRB sequences (which are referred to as TCR A1/B1 and TCR A2/B2) from the MAGEA6E>K-reactive population displayed MAGEA6E>K recognition, based on 4-1BB upregulation against the mutated MAGEA6E>K minimal epitope. Four TCRs (TCR A1/B1, TCR A1/B2, TCR A2/B1, TCR A2/B2) were assembled for each of the remaining MED13P>S and PDS5AY>F;H>Y-specific lymphocyte populations. Two of the four potential MED13P>S-specific TCR-expressing lymphocytes tested, TCRA1/B1 and TCRA2/B2, recognized the MED13P>S mutated 25-mer peptide and recognition of MED13P>S was restricted to HLA-B*15:01 and HLA-A*30:02, respectively. Finally, out of four PDS5AY>F;H>Y TCRs constructed and screened, one single TCR displayed specific recognition of TMG3 and the PDS5AY>F H>Y neo-epitope.
In NCI-3784, peripheral blood neo-antigen specific responses were identified for three mutated antigens FLNAR>C, KIF16BL>P, and SONR>C presented by HLA-B*07:02. Moreover, circulating CD8+PD-1+ lymphocytes reactive against TMG9 from NCI-3903 displayed mutation-specific recognition of KIF1BPP>S 8-mer presented by HLA-B*38:01, and this population contained 3 dominant TRB clonotypes. Thus, selection of circulating CD8+PD-1+ lymphocytes led to the prospective identification of a diverse mutation-specific T-cell response in 3 of 4 melanoma patients tested, with 3, 3, and 1 unique, patient-specific neo-antigens recognized, respectively.
This example demonstrates that selection of circulating CD8+PD-1+ lymphocytes reveals that the T-cell response to mutated antigens derived from TIL also existed in the blood of Patient 3713 prior to TIL therapy.
Patient NCI-3713 experienced a complete tumor regression following administration of TIL-3713. Previous studies showed that TIL-3713 derived from a lung metastasis recognized multiple mutated neo-epitopes including WDR46T>I, SRPXP>L, AFMIDA>V, HELZ2D>N, CENPLP>L, AHNAKS>F, and PRDX3P>L. Analysis of the pre-treatment PBMCs from this patient demonstrated recognition of 6 of 7 neo-epitopes tested (recognizing WDR46T>I, SRPXP>L, AFMIDA>V, HELZ2D>N, CENPLP>L, and PRDX3P>L, but not AHNAKS>F). Reactivity was uniquely identified within the circulating CD8+PD-1+ and CD8+PD-1hi, but not the CD8+ or the CD8+PD-1− lymphocytes. T-cell reactivities observed were mutation-specific, as they displayed preferential recognition of the mutated over the wild-type peptides, and the percentage of neo-antigen-specific cells based on 4-1BB up-regulation ranged from 0.5% to up to 21% of the CD8+PD-1hi cells. Thus, selection of circulating CD8+PD-1+ lymphocytes revealed that the T-cell response to mutated antigens derived from TIL also existed in the blood of this patient prior to TIL therapy.
This example demonstrates the recognition of autologous tumor by the enriched populations of mutation-specific T cells and T-cell receptors isolated in Example 14.
In view of their potential use to treat cancer, the recognition of autologous tumor by the enriched populations of mutation-specific T cells and T-cell receptors isolated was next examined. MAGEA6E>K, PDS5AY>F;H>Y, or the MED13P>S TCR-transduced T cells from NCI-3998, and mutation-specific CD8+ T cells derived from the blood of NCI-3784, and 3903 recognized their corresponding autologous tumor cell line at variable levels (
3.3
13.8
3.4
10.3
4.9
40.2
20.7
86.2
29.7
89.7
7.3
59.8
This example demonstrates the characteristics of the CD8+PD-1+ lymphocytes of Examples 11-16.
The findings in Examples 11-16 indicated that circulating CD8+PD-1+ lymphocytes were enriched in cancer mutation-specific cells as well as other tumor-specific T cells. Additionally, simultaneous screening of matched circulating and tumor-resident CD8+PD-1+ lymphocytes in 4 patients revealed a high degree of similarity in the tumor antigens targeted by both populations. In concordance, TRB deep sequencing of matched tumor-resident and circulating lymphocytes in the absence of in vitro expansion manifested a relatively high degree of overlap between TRB repertoires of the tumor-infiltrating and circulating CD8+PD-1+ subsets, but far less with the circulating CD8+ or CD8+PD-1− (
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This patent application is a continuation of U.S. application Ser. No. 16/710,287, filed Dec. 11, 2019, which is a continuation of U.S. application Ser. No. 15/567,157, filed Oct. 17, 2017, now U.S. Pat. No. 10,544,392, which is the U.S. national stage of PCT/US2016/030137, filed Apr. 29, 2016, which claims the benefit of U.S. Provisional Patent Application No. 62/155,830, filed May 1, 2015, each of which is incorporated by reference in its entirety herein.
This invention was made with Government support under project number ZIABC010984 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62155830 | May 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16710287 | Dec 2019 | US |
| Child | 18174928 | US | |
| Parent | 15567157 | Oct 2017 | US |
| Child | 16710287 | US |
