Patent Application
20250195572
The contents of the electronic sequence listing (NYG-LIPP-158PCT.xml; Size: 304,622 bytes; and Date of Creation: Mar. 15, 2023) is herein incorporated by reference in its entirety.
Cellular immunotherapies with engineered autologous patient T cells redirected against a chosen tumor antigen have yielded great efficacy against blood cancers, resulting in five approvals for chimeric antigen receptors (CARs) by the US Food and Drug Administration (FDA) so far6. By contrast, CAR therapy for solid tumors has shown a much lower efficacy overall, owing to the suppression of T cell effector function in the tumor microenvironment. Even for blood malignancies, with the exception of B acute lymphoblastic leukemia, most patients do not experience a durable response, with resistance being primarily due to T cell dysfunction rather than antigen loss7. Considerable efforts have been devoted to identifying genes and pathways that contribute to T cell dysfunction8,9. However, comprehensive, genome-wide screens for modulators of T cell function thus far have been limited to loss-of-function screens2-4.
The advances in CRISPR genome engineering have made it possible to readily knock out every gene in the genome in a scalable and customizable manner. Although its large size makes it challenging (albeit not impossible10) to deliver Cas9 via lentivirus to primary T cells, alternative approaches have been developed, which rely on transient delivery of Cas9 protein2 or mRNA11, or on constitutive Cas9 expression in engineered isogenic mouse strains3. These approaches, however, are not amenable to gain-of-function screens in human cells, which require continuous expression of the transcriptional activator that drives target gene expression.
What is needed is improved compositions and methods for more effective immunotherapies.
Provided herein, in a first aspect, is a lymphocyte genetically modified to express a chimeric antigen receptor (CAR). The CAR includes an antigen binding domain; a transmembrane domain; and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain from a gene of Table 3. In certain embodiments, the LTBR domain is an LTBR intracellular domain, or fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.
In another embodiment, the CAR includes an antigen binding domain; a transmembrane domain; a co-stimulatory signaling domain; and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain from a gene of Table 3. In certain embodiments, the LTBR domain is an LTBR intracellular domain, or fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.
In another aspect, a nucleic acid molecule is provided. The molecule includes a sequence that encodes a chimeric antigen receptor (CAR). The CAR includes an antigen binding domain; a transmembrane domain; and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain from a gene of Table 3. In certain embodiments, the LTBR domain is an LTBR intracellular domain, or fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435. In another embodiment, the CAR includes an antigen binding domain; a transmembrane domain; a co-stimulatory signaling domain; and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain In other embodiments, at least one domain comprises a domain from a gene of Table 3. In certain embodiments, the LTBR domain is an LTBR intracellular domain, or fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.
In another aspect, an expression cassette is provided that includes a nucleic acid molecule that includes a sequence that encodes a chimeric antigen receptor (CAR).
In another aspect, a method treating cancer in a subject in need thereof is provided. The method includes administering a composition comprising a modified lymphocyte as described herein.
In another aspect, a method of treating a viral disease in a subject in need thereof is provided. The method includes administering a composition comprising a modified lymphocyte In another aspect, a method of treating an autoimmune in a subject in need thereof is provided. The method includes administering a composition comprising a modified lymphocyte In another aspect, a fusion protein comprising an LTBR domain and at least one domain from a second protein, that is not LTBR is provided.
In another aspect, a host cell comprising a nucleic acid molecule or expression cassette as described herein, is provided.
Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention.
The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However, further improvements are needed to increase response and cure rates. Provided herein are modified T cells that include nucleic acids encoding fusion proteins that include LTBR, or domains thereof. When overexpressed in T cells. LTBR induced profound transcriptional and epigenomic remodeling. leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies5. We provide improved CAR. TCR and related T cell therapies for treatment of cancer and other diseases.
Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.
As used throughout this specification and the claims, the terms “comprising”, “containing”. “including”, and its variants are inclusive of other components, elements, integers, steps and the like. Conversely, the term “consisting” and its variants are exclusive of other components, elements, integers, steps and the like.
It is to be noted that the term “a” or “an”, refers to one or more, for example, “T cell”, is understood to represent one or more T cell(s). As such, the terms “a” (or “an”), “one or more.” and “at least one” is used interchangeably herein.
As used herein, the term “about” means a variability of plus or minus 10% from the reference given, unless otherwise specified.
Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone). and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C: A, B, or C; A or C; A or B: B or C; A and C; A and B: B and C; A (alone): B (alone); and C (alone)
The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991): Qhtsuka et al. J. Biol. Chem. 260:2605-2608(1985); and Rossolim et af., Mol. Cell. Probes 8:91-98(1994))
The terms “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be cither single stranded or double stranded DNA or RNA, e.g., an mRNA.
Nucleic acids described herein can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScript, Life Technologies, Eurofins). The nucleic acid sequences encoding aspects of a CRISPR-Cas editing system described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and/or for delivery to a host cells in a subject. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. The methods used to make such engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).
“Variants” of proteins or peptides as defined in the context of the present invention may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s). Preferably, these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g., its specific inhibitory property. “Variants” of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e., non-mutated physiological, sequence. Substitutions in which amino acids, which originate from the same class, are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bonds, e.g., side chains which have a hydroxyl function. This means that e.g., an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g., using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (cd.), Elsevier, Amsterdam). A variant may also include a non-natural amino acid.
A “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75, 100 or more amino acids of such protein or peptide, or over the full length of the protein or peptide.
The term “gene” can refer to a segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
As used herein, the terms “coding region” and “region encoding” and grammatical variants thereof, refer to an open reading frame (ORF) in a polynucleotide that upon expression yields a polypeptide or protein.
“Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
The term “domain” refers to a region of the protein's polypeptide chain that is self-stabilizing and that folds independently from the rest of the protein. The protein domain need not be identical to the native protein from which it is derived, but may be a variant thereof, including a variant that has a deletion, truncation, etc.
The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
Unless otherwise specified, a “nucleic acid sequence encoding an amino acid sequence” includes all nucleic acid sequences that are degenerate versions of each other and that encode the same amino acid sequence. A nucleic acid sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
The term “expression” is used herein in its broadest meaning and comprises the production of RNA, of protein, or of both RNA and protein. Expression may be transient or may be stable.
The terms “expressing” and “overexpression” refer to increasing the expression of a gene or protein. The terms refer to an increase in expression, for example, in increase in the amount of mRNA or protein expressed in a T cell, other lymphocyte or host cell, of at least 10%, as compared to a reference control level, or an increase of least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300% or at least about 400%. Various methods for expression and/or overexpression are known to those of skill in the art, and include, but are not limited to, stably or transiently introducing a exogenous polynucleotide encoding a fusion protein. TCR, or CAR to be expressed and/or overexpressed in the cell or inducing expression or overexpression of an endogenous gene encoding the protein in the cell.
The term “autologous” refers to any material derived from the same subject to whom it is later to be re-introduced.
The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.
The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
As used herein, an “expression cassette” refers to a nucleic acid molecule which encodes one or more ORFs or genes, e.g., an effector-enhancing gene, or a CAR or TCR or component thereof. An expression cassette also contains a promoter and may contain additional regulatory elements that control expression of one or more elements of a gene editing system in a host cell. In one embodiment, the expression cassette may be packaged into the capsid of a viral vector (e.g., a viral particle). In one embodiment, such an expression cassette for generating a viral vector as described herein is flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.
The term “regulatory element” or “regulatory sequence” refers to expression control sequences which are contiguous with the nucleic acid sequence of interest and expression control sequences that act in trans or at a distance to control the nucleic acid sequence of interest. As described herein, regulatory elements comprise but are not limited to, promoter: enhancer. transcription factor: transcription terminator: efficient RNA processing signals such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE): sequences that enhance translation efficiency (i.e., Kozak consensus sequence): sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. Also, see Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleic acid sequence in many types of target cell and those which direct expression of the nucleic acid sequence only in certain target cells (e.g., tissue-specific regulatory sequences).
A “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The term “constitutive” when referring to a promoter specifies a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. The term “inducible” or “regulatable” when referring to a promoter specifies a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. In certain embodiments, the inducible promoter is activated in response to T cell stimulation In certain embodiments, the promoter is an NFAT, AP1, NFκB, or IRF4 promoter. The term “tissue-specific” when referring to a promoter specifies a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription Exemplary promoters include the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) promoters.
The term “operably linked” refers to functional linkage between one or more regulatory sequences and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, where necessary to join two protein coding regions, are in the same reading frame.
The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
In certain embodiments, one or more genes are encoded by a nucleic acid sequence that is delivered to a host cell by a vector or a viral vector, of which many are known and available in the art. In one embodiment, provided is a vector comprising an expression cassette as described herein. In one embodiment, a vector is a non-viral vector. In another embodiment, a vector is a viral vector. A “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a nucleic acid sequence of interest is packaged in a viral capsid or envelope. Examples of viral vectors include but are not limited to lentivirus, adenoviruses, retroviruses (y-retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAVs), baculoviruses, herpes simplex viruses. In one embodiment, the viral vector is replication defective. A “replication-defective virus” refers to a viral vector, wherein any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient, i.e., they cannot generate progeny virions but retain the ability to infect cells.
The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR& gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
Provided herein, in one aspect, is an engineered lentiviral vector comprising the sequence of SEQ ID NO: 132, or a sequence sharing at least 90% identity with SEQ ID NO: 132. In certain embodiments, the sequence shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 132. The engineered vector is useful for, inter alia, the ORF screens described herein. In certain embodiments, an open reading frame (ORF) for a gene of interest is inserted within the vector sequence. In certain embodiments, a barcode is inserted within the vector sequence SEQ ID NO: 133 provides an exemplary embodiment in which various primer binding sites, meganuclease recognition sites for restriction digests, and cloning recombination sites have been inserted in the sequence at the site that the ORF and/or barcode can be inserted. Primer binding sites, restriction sites, cloning sites and the like can be included in addition to the ORF and/or barcode. In certain embodiments, the ORF and/or barcode is/are inserted after nucleotide 3291 of SEQ ID NO: 132.
In certain embodiments, the vector is a non-viral plasmid that comprises an expression cassette described herein, e.g., naked DNA, naked plasmid DNA, RNA, and mRNA: coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid-nucleic acid compositions, poly-glycan compositions and other polymers, lipid and/or cholesterol-based-nucleic acid conjugates, and other constructs such as are described herein. See, e.g., X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: Mar. 21, 2011; WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which are incorporated herein by reference.
Plasmids, other cloning and expression vectors, properties thereof, and constructing/manipulating methods thereof that can be used in accordance with the present invention are readily apparent to those of skill in the art. In one embodiment, an expression cassette as described herein is engineered into a suitable genetic element (a vector) useful for generating viral vectors and/or for introduction to a host cell, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
The term “transfected” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” cell is one which has been transfected with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
As used herein, “transient” refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
RNA or DNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
As used herein, the term “subject” means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In one embodiment, the subject of these methods and compositions is a human. Still other suitable subjects include, without limitation, murine, rat, canine, feline, porcine, bovine, ovine, non-human primate and others. As used herein, the term “subject” is used interchangeably with “patient”.
Provided herein are compositions which include nucleic acids, expression cassettes, and/or lymphocytes which include coding sequences for certain genes (or fragments thereof). which have been shown to enhance T cell survival, proliferation and/or effector function (collectively referred to herein as an “effector-enhancing gene”). In certain embodiments, the effector-enhancing gene comprises any of the genes identified in Table 1, 2, or 3.
Provided herein are expression cassettes that include nucleic acid sequences that encode fusion proteins that include at least a fragment of one or more effector-enhancing genes. In certain embodiments, the gene comprises any of the genes identified in Table 1, above, or a fragment or variant thereof. In another embodiments, the gene comprises any of the genes identified in Table 2, below, or a fragment or variant thereof. In another embodiments, the gene comprises any of the genes identified in Table 3, below, or a fragment or variant thereof. Desirable fragments include protein domains, such as an intracellular signaling domain, a transmembrane domain, or extracellular domain. In certain embodiment, the expression cassette includes more than one effector-enhancing gene fragment.
As used herein, where reference to a specific gene of Table 1, Table 2, or Table 3 is mentioned, it is intended that the use of the coding sequence for the full-length protein, a fragment having a deletion or truncation, a domain, or a variant having one or more substitutions in the amino acid, is intended. For example, in certain embodiments, the nucleic acid encodes a protein sequence having a deletion in the N terminus. In certain embodiments, the nucleic acid encodes a protein sequence having a deletion in the C terminus. In one embodiment, the nucleic acid encodes a protein having of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, or at least 125 amino acids.
In one embodiment, the effector-enhancing gene is LTBR, LTBR, a receptor endogenously expressed by professional antigen presenting cells but not lymphocytes, was identified as a strong synthetic driver of both T-cell proliferation and secretion of key cytokines: IL-2 and IFNγ. Using a multimodal single-cell sequencing approach, it was shown that LTBR induces profound transcriptional changes when overexpressed in T-cells, activating cellular programs involved in antigen presentation and prevention of apoptosis. As described herein, a platform was developed for testing combinatorial perturbations in T-cells, by co-expressing a gene of interest (e.g. LTBR) together with CRISPR sgRNAs targeting other genes, to map signaling networks in T-cells. Also demonstrated herein is that mRNA delivery of LTBR as an alternative to constitutive lentiviral expression, highlighting the translational potential of our screening approach.
In a certain embodiment, the expression cassette comprises a nucleic acid encoding LTBR, or a fragment thereof. LTBR (lymphotoxin-beta receptor), which encodes for tumor necrosis factor receptor superfamily member 3, is essential for the development and organization of secondary lymphoid tissues and chemokine release. A representative nucleic acid sequence of LTBR can be found at Accession ID NM_002342.3.
The full-length amino acid sequence of LTBR is SEQ ID NO: 2 (Uniprot P36941):
The LTBR protein can be divided into three regions, or domains: the extracellular domain (amino acids 31-227 of SEQ ID NO: 2); the transmembrane (or helical) domain (amino acids 228-248 of SEQ ID NO: 2); and the cytoplasmic (or intracellular) domain (amino acids 249-435 of SEQ ID NO: 2). The signal peptide of the immature protein is at amino acids 1-30 of SEQ ID NO: 2. When a domain is referred to herein, it is intended that variants, including N-terminal or C-terminal truncated variants, are included.
In certain embodiments, the expression cassette comprises a nucleic acid encoding a fragment of LTBR. In certain embodiments, the nucleic acid encodes a protein sequence having a deletion of amino acids 2-31, 32-41, 32-151, 32-180, 393-435, 377-435, 324-377, 297-435, or 262-435 as compared to the native protein (SEQ ID NO: 2). In certain embodiments, the nucleic acid encodes a protein sequence having a deletion in the N terminus. In certain embodiments, the nucleic acid encodes a protein sequence having a deletion in the C terminus. In one embodiment, the LTBR is has a deletion of residues 393-435. In certain embodiments, the LTBR has a deletion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, or at least 125 amino acids.
In certain embodiments, the expression cassette comprises a nucleic acid encoding a fragment that is a domain of LTBR. In certain embodiments, the nucleic acid encodes the extracellular domain of LTBR (amino acids 31-227 of SEQ ID NO: 2). In certain embodiments, the nucleic acid encodes the transmembrane domain of LTBR (amino acids 228-248 of SEQ ID NO. 2). In certain embodiments, the nucleic acid encodes the cytoplasmic (or intracellular) domain of LTBR (amino acids 249-435 of SEQ ID NO: 2). In other embodiments, the domain is a variant of one of the LTBR domains, including a variant that has a deletion. Desirable variants of the cytoplasmic domain include those with amino acids 249-378, 249-379, 249-380, 249-381, 249-382, 249-383, 249-384, 249-385, 249-386, 249-387, 249-388, 249-389, 249-390, 249-391, or 249-392 all of SEQ ID NO: 2. Further desirable variants include those with amino acids 249-378, 249-379, 249-380, 249-381, 249-382, 249-383, 249-384, 249-385, 249-386, 249-387, 249-388, 249-389, 249-390, 249-391, or 249-392 all of SEQ ID NO: 2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions as compared to SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 249-396 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 249-393 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 249-387 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 249-377 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 262-435 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 297-435 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 324-435 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 345-435 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 358-435 of SEQ ID NO: 2.
In other embodiments, the expression cassette comprises a nucleic acid encoding two or more domains of LTBR. In one embodiment the nucleic acid encodes the cytoplasmic domain (or variant thereof) and the transmembrane domain of LTBR. In another embodiment, the nucleic acid encodes the cytoplasmic domain (or variant thereof), transmembrane domain, and extracellular domain of LTBR.
The fusion proteins described herein also include at least a fragment, including a domain, of second protein, different from the first. In certain embodiments, the second protein is a protein that is a component of a T cell receptor. In other embodiments, the second protein is a protein that interacts with T cell receptor. Such proteins include CD4, CD8A, CD8B, CD3E, CD3D, CD3G. and CD3Z.
In one embodiment a fusion protein includes an LTBR domain, or variant thereof, and CD4. In another embodiment a fusion protein includes an LTBR domain, or variant thereof, and CD8A and/or CD8B. In another embodiment a fusion protein includes an LTBR domain, or variant thereof, and CD3E. In another embodiment a fusion protein includes an LTBR domain, or variant thereof, and CD3D. In another embodiment a fusion protein includes an LTBR domain, or variant thereof, and CD3G. In another embodiment a fusion protein includes an LTBR domain, or variant thereof, and CD3Z. In certain embodiments, the LTBR domain is an intracellular domain. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.
Various isoforms of the genes identified in Table 1 are known in the art. Some are described in Table 2 below. In another embodiment, an expression cassette is provided which includes the coding sequence for any of the alternative isoforms. Alternative coding sequences accounting to the degeneracy of the genetic code, including codon optimized coding sequences, for these genes can be identified by the person of skill in the art, and utilized as an alternative embodiment of the compositions and methods described herein.
Engineered T cell Receptors Containing LTBR Domains
The present disclosure provides nucleic acid sequences encoding engineered T cell receptors, e.g., T cell receptors (TCRs), that incorporate an LTBR domain. Also provided are chimeric antigen receptors (CAR) that incorporate an LTBR domain or variant thereof. Components of the TCR and CARs are further described herein. Also provided are the engineered TCRs and CARs, and modified T cells incorporating the same.
Also provided herein are nucleic acid sequences encoding engineered T cell receptors, e.g., T cell receptors (TCRs), that incorporate a domain or variant thereof from one of the genes of Table 3. Also provided are chimeric antigen receptors (CAR) that incorporate domain from one of the genes of Table 3. In certain embodiments, the domain is an intracellular domain from a gene selected from those of Table 3. Also provided are the engineered TCRs and CARs, and modified T cells incorporating the same. As described below, embodiments incorporating LTBR domains are set forth. However, for each embodiment described for LTBR, an embodiment is intended for each of the genes of Table 3.
Provided herein are nucleic acid molecules that comprise a coding sequence for any of the TCRs described herein modified to include a domain of LTBR or a gene of Table 3. Certain exemplary TCRs are provided in the sequence listing in SEQ ID Nos: 3-54. However, it is intended that nucleic acids encoding all the described TCRs, as well as the TCR proteins, are encompassed herewith.
The TCR is a disulfide-linked membrane-anchored heterodimer present on T cell lymphocytes, and the majority of T cells are αβ T cells having a TCR consisting of an alpha (a) chain and a beta (B) chain. Each chain comprises a variable (V) and a constant (C) domain, wherein the variable domain recognizes an antigen, or an MHC-presented peptide. TCRα and TCRβ chains with a known specificity or affinity for specific antigens, e.g., tumor antigens described herein, can be introduced to a T cell using the methods described herein. TCRα and TCRβ chains having a desired, e.g., increased, specificity or affinity for a particular antigen can be isolated using standard molecular cloning techniques known in the art. Other modifications that increase specificity, avidity, or function of the TCRs or the engineered T cells expressing the TCRs can be readily envisioned by the ordinarily skilled artisan, e.g., promoter selection for regulated expression, mutations in the antigen binding regions of the TCRα and TCRβ chains. Any isolated or modified TCRα and TCRβ chain can be operably linked to or can associate with one or more intracellular signaling domains described herein. Signaling can be mediated through interaction between the antigen-bound αβ heterodimer to CD3 chain molecules, e.g., CD3zeta (3).
A smaller subset of T cells expresses a TCR having a (γ) gamma chain and a delta (8) chain. Gamma-delta (γδ) T cells make up 3-10% of circulating lymphocytes in humans, and the V82+ subset can account for up to 95% of γδ T cells in blood. Vδ2+ cells recognize non-peptide epitopes and do not require antigen presentation by major histocompatibility complexes (“MHC”) or human leukocyte antigen (“HLA”). The majority of Vδ2+ T cells also express a Vγ9 chain and are stimulated by exposure to 5-carbon pyrophosphate compounds that are intermediates in mevalonate and non-mevalonate sterol/isoprenoid synthesis pathways. The response to isopentenyl pyrophosphate (5-carbon) is universal among healthy human beings. Another subset of γδ T cells, Volt, make up a much smaller percentage of the T cells circulating in the blood, but are commonly found in the epithelial mucosa and the skin, γδ T cells have several functions, including killing tumor cells and pathogen-infected cells. Stimulation through the γδ TCR improves the capacity for cellular cytotoxicity, cytokine secretion and other effector functions. The TCRs of γδ T cells have unique specificities and the cells themselves occur in high clonal frequencies, thus allowing rapid innate-like responses to tumors and pathogens. See, e.g., Park and Lee, Exp Mol Med. 2021 March; 53(3): 318-327., which is incorporated herein by reference.
In certain embodiments, a T cell comprises a nucleic acid sequence encoding a TCR, e.g., a TCR that targets a tumor antigen, that includes an LTBR domain. In certain embodiments, the TCR includes the LTBR intracellular domain, or variant thereof as described herein. In one embodiment, the variant has a deletion in at least amino acids 393 to 435 of SEQ ID NO: 2. In one embodiment, the variant of the intracellular domain includes amino acids 249-392 of SEQ ID NO: 2. However, additional residues may be deleted. Thus, desirable variants of the intracellular domain include those with a sequence of amino acids 249-378, 249-379, 249-380, 249-381, 249-382, 249-383, 249-384, 249-385, 249-386, 249-387, 249-388, 249-389, 249-390, 249-391, or 249-392 all of SEQ ID NO: 2. Further desirable variants include those with a sequence of amino acids 249-378, 249-379, 249-380, 249-381, 249-382, 249-383, 249-384, 249-385, 249-386, 249-387, 249-388, 249-389, 249-390, 249-391, or 249-392 all of SEQ ID NO: 2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions as compared to SEQ ID NO: 2.
In certain embodiments, a T cell comprises a nucleic acid sequence encoding a TCR, e.g., a modified TCR that targets a tumor antigen described herein, that includes a domain from the genes of Table 3. In certain embodiments, the TCR includes an intracellular domain from the genes of Table 3, or variant thereof as described herein
In one embodiment, the TCR comprises an LTBR intracellular domain fused to C-terminus of the TCR alpha chain. In another embodiment, the TCR comprises an LTBR intracellular domain fused to the C-terminus of the TCR beta chain.
In one embodiment, the TCR comprises an intracellular domain of a gene of Table 3 fused to C-terminus of the TCR alpha chain. In another embodiment, the TCR comprises an intracellular domain of a gene of Table 3 fused to the C-terminus of the TCR beta chain.
In another embodiment, the LTBR intracellular domain is fused to the C-terminal intracellular tail of CD4. In another embodiment, the LTBR intracellular domain is fused to the C-terminal intracellular tails of CD8α and/or CD8β. See
In another embodiment, an intracellular domain of a gene of Table 3 is fused to the C-terminal intracellular tail of CD4. In another embodiment, an intracellular domain of a gene of Table 3 is fused to the C-terminal intracellular tails of CD8α and CD8β.
In other embodiments, at least one domain of LTBR is delivered to a lymphocyte via direct modification of the endogenous genome. Various techniques for modification of the endogenous genome are known in the art, including CRISPR, zinc finger nucleases, TALENS, etc. See, e.g., Azangou-Khyavy et al. CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer, Front. Immunol., 29 Sep. 2020 | https://doi.org/10.3389/fimmu.2020.02062, which is incorporated herein by reference. In on embodiment, the LTBR intracellular domain, or fragment thereof, is inserted into the genome of a lymphocyte. In other embodiments, at least one domain of a gene of Table 3 is delivered to a lymphocyte via direct modification of the endogenous genome.
In certain embodiments, the TCR is a known TCR, such as those identified in
In any of the embodiments described herein, a modified TCR can be substituted for a CAR described herein to generate a T cell. An engineered TCR described herein can be substituted for a CAR in any of the embodiments described herein.
The term “chimeric antigen receptor” or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a stalk/hinge, a transmembrane domain, and a cytoplasmic signaling domain (also referred to as an intracellular signaling domain) comprising a functional signaling domain derived from a stimulatory molecule as defined below. See Jayaraman et al, CAR-T design: Elements and their synergistic function, eBioMedicine, 58:102931 (August 2020), which is incorporated herein by reference. In some embodiments, the stimulatory molecule is TCR zeta, FcR gamma, FcR beta, CD3 gamma. CD3 delta. CD3 epsilon, CD5, CD22, CD79a, CD79b. CD66d. 4-1BB, or CD3-zeta. In a particular embodiment, the stimulatory molecule is the zeta chain associated with the T cell receptor complex As used herein for ease of reference, when referring to a stimulatory molecule, the term CD3ζ (may be used. However, it is intended that a similar embodiment is provided in which the CD3ζ is swapped for another suitable stimulatory molecule. In one embodiment, the stimulatory molecule is 4-1BB. In one embodiment, the stimulatory molecule is CD28. In another embodiment, the stimulatory molecule is LTBR and the stimulatory signaling domain includes the LTBR intracellular domain, or variant thereof. In another embodiment, the stimulatory molecule is a gene of Table 3, and the stimulatory signaling domain includes an intracellular domain of a gene of Table 3.
In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below (also referred to as a “costimulatory signaling domain”). In one embodiment, the costimulatory molecule is chosen from a costimulatory molecule described herein, e.g., OX40, CD27, CD28, CD30, CD40, PD-1, CD2. CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD11a/CD18), ICOS and 4-1BB (CD137), or any combination thereof. In certain embodiments, the costimulatory molecule is LTBR, and the costimulatory signaling domain includes the LTBR intracellular domain, or variant thereof. In another embodiment, the costimulatory molecule is a gene of Table 3, and the costimulatory signaling domain includes an intracellular domain of a gene of Table 3.
As described herein, the CAR itself comprises one or more LTBR domains. The LTBR domain(s) may, in some embodiments, replace a domain from an existing CAR construct, such as those described in
As described herein, the CAR itself comprises one or more domains from a gene of Table 3. The domain(s) from the gene of Table 3 may, in some embodiments, replace a domain from an existing CAR construct, such as those described in
In certain embodiments, the stalk and the transmembrane are from the same molecule, e.g., LTBR, CD8, or CD28. In other embodiments, the stalk and the transmembrane are from different molecules, e.g., CD8 stalk and LTBR™, CD28 stalk and LTBR™, etc.
In one embodiment the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the scFv domain during cellular processing and localization of the CAR to the cellular membrane.
In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from LTBR, or variant thereof. Without wishing to be bound by theory, T cells engineered with CARs lacking a CD3zeta chain do not induce specific cytokine secretion in the presence of CD19+ leukemia cells. However, these cells induce NFκB-induced genes (as demonstrated by CD74 expression (see
In certain embodiments, in addition to the LTBR signaling domain, the transmembrane domain is also derived from LTBR. In certain embodiments, in addition to the LTBR signaling domain and (optionally) the transmembrane domain, the stalk is also derived from LTBR.
In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived LTBR, or variant thereof.
In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, an intracellular signaling domain comprising a functional cosignaling domain derived from LTBR, or variant thereof, and a functional signaling domain derived from a stimulatory molecule, e.g., CD3 zeta chain. The placement of the LTBR cosignaling domain may be varied depending on the desired function of the CAR. In certain embodiments, the LTBR cosignaling domain, or variant thereof is placed between the transmembrane domain and the signaling domain (e.g., CD3C). In other embodiments, the CD3C is placed between the transmembrane domain and the LTBR cosignaling domain, or variant thereof.
In certain embodiments, in addition to the signaling domain, the transmembrane domain is also derived from a gene of Table 3. In certain embodiments, in addition to the signaling domain and (optionally) the transmembrane domain, the stalk is also derived from a gene of Table 3.
In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a gene of Table 3, or variant thereof.
In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, an intracellular signaling domain comprising a functional cosignaling domain derived from LTBR, or variant thereof, at least one other functional cosignaling domain, and a functional signaling domain derived from a stimulatory molecule, e.g., CD3zeta chain. The placement of the LTBR cosignaling domain may be varied depending on the desired function of the CAR. In certain embodiments, the LTBR cosignaling domain, or variant thereof is placed between the transmembrane domain and the other cosignaling domain(s) (e.g., CD28 or 4-1BB). In certain embodiments, the LTBR cosignaling domain, or variant thereof is placed between the other cosignaling domain(s) and the signaling domain (e.g., CD3ζ). In other embodiments, the LTBR cosignaling domain, or variant thereof is placed downstream of the signaling domain (e.g., CD3ζ). See, e.g.,
In certain embodiments, in addition to the LTBR signaling domain, the transmembrane domain is also derived from LTBR. In certain embodiments, in addition to the LTBR signaling domain and (optionally) the transmembrane domain, the stalk is also derived from LTBR.
In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, an intracellular signaling domain comprising a functional cosignaling domain derived from a gene of Table 3, or variant thereof, at least one other functional cosignaling domain, and a functional signaling domain derived from a stimulatory molecule, e.g., CD3zeta chain. The placement of the cosignaling domain may be varied depending on the desired function of the CAR. In certain embodiments, the cosignaling domain from a gene of Table 3, or variant thereof is placed between the transmembrane domain and the other cosignaling domain(s) (e g., CD28 or 4-1BB). In certain embodiments, the cosignaling domain from a gene of Table 3, or variant thereof is placed between the other cosignaling domain(s) and the signaling domain (e.g., CD3ζ). In other embodiments, the cosignaling domain from a gene of Table 3, or variant thereof is placed downstream of the signaling domain (e.g., CD3ζ).
The present disclosure provides nucleic acid sequences, e.g., a DNA or an RNA construct, that encode any of the CARs described herein. This also refers to nucleic acid sequences encoding a known CAR such as one of those shown in
The present disclosure provides nucleic acid sequences, e.g., a DNA or an RNA construct, that encode any of the CARs described herein. This also refers to nucleic acid sequences encoding a known CAR such as one of those shown in
In certain embodiments, the CAR targets CD19. In certain embodiments, the CAR is a known CAR, such as one of those shown in
Exemplary sequences for the CARs and TCRs used in the Examples are provided in the sequence listing in SEQ ID Nos: 3-54. Other exemplary antibody sequences, useful for the antigen recognition domain, are provided in the sequence listing in SEQ ID Nos: 63-83.
Other chimeric antigen receptors as modified herein, include those useful for treatment for autoimmune disease, such as are chimeric autoantigen receptors (CAAR). Such CAARs include DSG3-CAART and MuSK-CAART. Others may be known in the art or may be designed by the person of skill.
The present disclosure provides nucleic acid sequences, e.g., a DNA or an RNA construct, that encode any of the TCRs described herein. This also refers to nucleic acid sequences encoding a known TCR such as one of those shown in
The expression cassettes referred to herein include a nucleic acid molecule which encodes one or more biologically useful nucleic acid sequences (e.g., a gene cDNA encoding a fusion protein, CAR, TCR, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence(s) and its gene product(s). Operably linked sequences include both regulatory sequences that are contiguous with the nucleic acid sequence and regulatory sequences that act in trans or at a distance to control the sequence. Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The expression cassette may contain regulatory sequences upstream (5′ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3′ to) a gene sequence, e.g., 3′ untranslated region comprising a polyadenylation site, among other elements. Thus, in addition to the coding sequences for the fusion protein, CAR, or TCR the expression cassette may also include expression control sequences.
The expression control sequences include a promoter. In some embodiments, its it is desirable to utilize a promoter having high transcriptional activity. Certain strong constitutive promoters are known in the art and include, without limitation, the CMV promoter, the EF-la promoter, CBG promoter. CB7 promoter, etc. Alternatively, other promoters, such as regulatable (inducible) promoters [see, e.g., WO 2011/126808 and WO 2013/049493, incorporated by reference herein], or a promoter responsive to physiologic cues may be utilized. In certain embodiments, the inducible promoter is activated in response to T cell stimulation. In certain embodiments, the promoter is an NFAT, API. NFκB, or IRF4 promoter.
The expression cassette may also include, in certain embodiments, one or more IRES or 2A sequence(s) to allow for expression of multiple coding sequences from the same expression cassette. As exemplified herein, in one embodiment, a TCR directed to NY-ESO-1 is provided in which an LTBR intracellular domain is expressed contiguously with the TCRα (or TCRβ) chain. See,
Provided herein, in certain aspects, are compositions which include modified lymphocytes which comprise the nucleic acids and/or expression cassettes described herein. In one embodiment, the host lymphocyte is a T cell. In another embodiment, the host lymphocyte is a natural killer (NK) cell. In certain embodiments, the composition comprises a population of cells which includes a mixed population of lymphocytes (e.g., alpha beta T cells and NK T cells). In other embodiments, the composition comprises cells which includes a population which is enriched for a particular lymphocyte population.
As used herein, the phrase “T cell” refers to a lymphocyte that expresses a T cell receptor molecule. T cells include human alpha beta (αβ) T cells and human gamma delta (γδ) T cells. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof. T cells can be CD4+, CD8+, or CD4+ and CD8+. T cells can also be CD4−, CD8−, or CD4− and CD8−. T cells can be helper cells, for example helper cells of type TH1, TH2, TH3. TH9, TH17, or TFH. T cells can be cytotoxic T cells. T cells can also be regulatory T cells. Regulatory T cells (Tregs) can be FOXP3+ or FOXP3−. T cells can be alpha/beta T cells or gamma/delta T cells. In some cases, the T cell is a CD4+CD25hiCD127lo regulatory' T cell. In some cases, the T cell is a regulatory T cell selected from the group consisting of type 1 regulatory (Tr1). TH3, CD8+CD28−, Treg 17, and Qa−1 restricted T cells, or a combination or sub-population thereof. In some cases, the T cell is a FOXP3+ T cell. In some cases, the T cell is a CD4+CD251loCD127hi effector T cell. In some cases, the T cell is a CD4+CD25lo CD127hiCD45RAhiCD45RO-naive T cell. In some cases, the T cell is a Vγ9V82 T cell. In some embodiments, the T cell expresses a viral antigen. In other embodiments, the T cell expresses a cancer antigen. A T cell can be a recombinant T cell that has been genetically manipulated.
As used herein, the phrase “primary” in the context of a primary cell is a cell that has not been transformed or immortalized. Such primary cells can be cultured, sub-cultured, or passaged a limited number of times (e.g., cultured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, the primary cells are adapted to in vitro culture conditions. In some cases, the primary cells are isolated from an organism, system, organ, or tissue, optionally sorted, and utilized directly without culturing or sub-culturing. In some cases, the primary cells are stimulated, activated, or differentiated. For example, primary T cells can be activated by contact with (e.g., culturing in the presence of) CD3, CD28 agonists, IL-2, IFN-γ, or a combination thereof.
Also provided herein are methods of making the modified cells and compositions containing modified cells as described herein. Methods of modifying cells, e.g., lymphocytes, to introduce an exogenous sequence, such as an expression cassette or expression vector comprising a coding sequence for a fusion protein, a CAR, or TCR, or more than one of these sequences, are known in the art. For example, see, e.g., WO 2016/109410 A2, which is incorporated herein by reference. In certain embodiments, more than one exogenous sequence is introduced.
By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids. Modifying can refer to altering expression of a gene in a lymphocyte, for example, by introducing an exogeneous nucleic acid that encodes the gene.
The lymphocytes provided herein can be genetically modified, e.g., by transfection, transduction, or electroporation, to express a nucleic acid sequence encoding a fusion protein. TCR, or CAR, as described herein. Depending on the clinical context, e.g., patient's condition or condition to be treated, prolonged or permanent expression of the gene and/or, e.g., for robust and long-lasting CAR activity, e.g., anti-tumor activity, may be desirable. In such embodiments, the lymphocytes are genetically modified, e.g., transduced, e.g., virally transduced, using vectors comprising nucleic acid sequences encoding a gene disclosed herein to confer a desired effector function. In other embodiments, transient expression of the gene is desirable. In such embodiments, the use of, e.g., mRNA or a regulatable promoter to express the effector-enhancing gene, may be used.
Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any known in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.
Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012. MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A suitable method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, and the like.
Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system. In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Also contemplated are lipofectamine-nucleic acid complexes.
Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant nucleic acid sequence in the host cell, a variety of assays may be performed. Such assays include, for example. Southern and Northern blotting, RT-PCR and PCR, biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots)
In certain embodiments, an expression vector is provided which includes the coding sequence for a fusion protein comprising an LTBR domain and a domain from a protein that is not LTBR. In other embodiments, the expression vector includes the coding sequence for one or more components of a CAR or TCR. In other embodiments, the expression vector includes the coding sequence for one or more components of a CAR or TCR that includes one or more LTBR domains. In other embodiments, the expression vector includes the coding sequence for one or more components of a CAR or TCR that includes one or more domains of a gene of Table I Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. In one embodiment, the expression vector is a lentivirus. If more than one expression vector is utilized, each expression vector may be individually selected from amongst those known in the art.
Provided herein is a method of making a population of immune effector cells (e.g., T cells, NK cells) that are modified to express a fusion protein, TCR, or CAR as described herein. Methods for making such immune cells include introducing an exogenous nucleic acid encoding a LTBR-domain fusion protein into the cell. Also provided herein is a method of making a population of immune effector cells (e.g. T cells. NK cells) that are modified to express a CAR or TCR that includes one or more LTBR domains. Methods for making such immune cells include introducing an exogenous nucleic acid encoding the CAR or TCR into the cell. In certain embodiments, immune effector cells comprising the fusion proteins described herein are long lived and/or resistant to apoptosis.
In the examples described below, a method of making modified T cells is described for convenience. However, alternative embodiments are envisioned using other kinds of immune cells, e.g., NK T cells or NK cells. Suitable methods are known in the art.
Briefly, an exemplary method includes providing a population of immune effector cells (e.g., T cells), and optionally, removing T regulatory cells, e.g., CD25+ T cells, from the population. In one embodiment, the population of immune effector cells are autologous to the subject who the cells will be administered to for treatment. In one embodiment, the population of immune effector cells are allogeneic to the subject who the cells will be administered to for treatment. In one embodiment, the T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, e.g., IL-2. In one embodiment, the anti-CD25 antibody, or fragment thereof, or CD25-binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein. In one embodiment, the T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody molecule, or fragment thereof. In another embodiment, CD25+ cells are not removed.
Another exemplary method includes providing a population of immune effector cells (e.g., T cells), and enriching the population for CD8+ cells and subsequently, enriching the population for CD4+ cells. In one embodiment, population is enriched for CD8+ and CD4+ T cells using an anti-CDS and anti-CD4 antibody, or fragment thereof, or a CD8-binding ligand or CD4-binding ligand. In one embodiment, the anti-CD4 or anti-CD8 antibody, or fragment thereof, or CD4 or anti-CD8-binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD4 antibody or anti-CD8, or fragment thereof, is conjugated to a substrate as described herein.
In one embodiment, the method further comprises transducing a cell from the population with one or more vectors comprising a nucleic acid encoding a fusion protein. CAR, or TCR as described herein. In one embodiment, the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector. In one embodiment, the cell from the population of T cells is transduced with a vector once, e.g., within one day after population of immune effector cells are obtained from a blood sample from a subject, e.g., obtained by apheresis. In one embodiment, the method further comprises generating a population of RNA-engineered cells transiently expressing exogenous RNA from the population of T cells. The method comprises introducing an in vitro transcribed RNA or synthetic RNA into a cell from the population, where the RNA comprises a nucleic acid encoding an LTBR domain-containing fusion protein, a TCR, or CAR.
In another embodiment, the method further comprises transducing a cell from the population with one or more vectors comprising a nucleic acid encoding a CAR or TCR that includes one or more LTBR domains as described herein. In one embodiment, the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector. In one embodiment, the cell from the population of T cells is transduced with a vector once, e.g., within one day after population of immune effector cells are obtained from a blood sample from a subject, e.g., obtained by apheresis. In one embodiment, the method further comprises generating a population of RNA-engineered cells transiently expressing exogenous RNA from the population of T cells. The method comprises introducing an in vitro transcribed RNA or synthetic RNA into a cell from the population, where the RNA comprises a nucleic acid encoding a CAR or TCR that includes one or more LTBR domains as described herein.
In one embodiment, modified cells described herein are expanded. In one embodiment, the cells are expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment, the cells are expanded in culture for 3 or 4 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. Potency can be defined, e g, by various T cell functions, e.g., proliferation, target cell killing, cytokine production, activation, migration, or combinations thereof.
Also provided herein, in certain aspects, are methods of treating cancer in a subject. In certain embodiments, the method includes administering to the subject a cell that expresses a fusion protein as described herein such that the cancer is treated in the subject. In certain embodiments, the cell further expresses a CAR. In other embodiments, the cell further expresses a TCR. As used herein in describing the methods of treatment, LTBR is utilized as an exemplary effector-enhancing gene, for convenience. However, in alternative embodiments, the other genes of Table 3 are also utilized in modified CAR or TCR containing cells in the methods. In one embodiment, the method includes obtaining cells from a patient, modifying the cells as described herein, and administering the cells to the patient.
In certain embodiments, the method includes administering to the subject a cell that expresses a CAR or TCR that includes one or more LTBR domains as described herein such that the cancer is treated in the subject. In one embodiment, the method includes obtaining cells from a patient, modifying the cells as described herein, and administering the cells to the patient.
In certain embodiments, the method includes administering to the subject a cell that expresses a CAR or TCR that includes one or more domains from a gene of Table 3 as described herein such that the cancer is treated in the subject. In one embodiment, the method includes obtaining cells from a patient, modifying the cells as described herein, and administering the cells to the patient.
The term “cancer” as used herein refers to any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication. In certain embodiments, administration of the compositions disclosed herein, e.g., according to the methods disclosed herein, treats a cancer. In certain embodiments, the cancer is selected from the group consisting of adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin. Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor, secondary cancers caused by cancer treatment, and any combination thereof. An example of a cancer that is treatable by the modified cell (e.g., LTBR CART or LTBR TCR-T cell, or LTBR-containing CART or TCR-T cell) is a hematological cancer. In one aspect, a hematologic cancer including but is not limited to leukemia (such as acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoid leukemia, chronic lymphoid leukemia and myelodysplastic syndrome) and malignant lymphoproliferative conditions, including lymphoma (such as multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and small cell- and large cell-follicular lymphoma). In other embodiments, a hematologic cancer can include minimal residual disease, MRD, e.g., of a leukemia, e.g., of AML or MDS. Other cancers include breast cancer, lung cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, kidney cancer, cervical cancer, liver cancer, ovarian cancer, and testicular cancer. In other embodiments, the cancer is a solid tumor cancer. In certain embodiments, the cancer is one of those listed in
In certain embodiments, the CAR is selected from Axicabtagene ciloleucel (Yescarta®), Brexucabtagene autoleucel (Tecartus™), Idecabtagene vicleucel (Abecma™), Lisocabtagene maraleucel (Breyanzi®), Tisagenlecleucel (Kyrmriah®).
Autoimmune diseases are conditions arising from abnormal immune attack to the body, and they substantially increase the morbidity, mortality and healthcare costs worldwide. As T cells play a key role in the process of autoimmune diseases, engineered T-cell therapy has emerged and is also regarded as a potential approach to overcome current roadblocks in the treatment of autoimmune diseases. Either self-reactive or autoantibodies play a key role in the process of autoimmune diseases. Thus, engineering T cells to express a chimeric autoantibody receptor (CAAR) is a strategy for treatment for autoimmune disease. In one embodiment, the CAR comprises a CAAR. See, e.g., Zhang et al, Chimeric antigen receptor T-cell therapy beyond cancer: current practice and future prospects, Immunotherapy, 2020 September; 12(13):1021-1034, doi: 10.2217/imt-2020-0009. Epub 2020 Jul. 30, which is incorporated herein by reference. Autoimmune diseases include Pemphigus vulgaris (PV) (e.g., DSG3-CAAR-T) and lupus (e.g., MuSK-CAAR-T)). Other autoimmune diseases include type 1 diabetes, autoimmune thyroid disease, rheumatoid arthritis (RA), inflammatory bowel disease, colitis, systemic lupus erythematosus, and multiple sclerosis (MS). Sec, e.g., Chen et al, Immunotherapy Deriving from CAR-T Cell Treatment in Autoimmune Diseases, Journal of Immunology Research Volume 2019 Dec. 31, 2019, which is incorporated herein by reference. Other conditions treatable with the compositions described herein include graft-versus-host disease (GVHD) and transplant rejection.
In another embodiment, the subject bas a virally-driven cancer. In certain embodiments, the virally-driven cancer is selected from the following:
In one aspect, the methods comprise administering to the subject in need thereof an effective amount of an effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) described herein in combination with an effective amount of another therapy. Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
An effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
The effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) can be administered before the other treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.
When administered in combination, the effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) and the additional agent (e.g., second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell), the additional agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy. In other embodiments, the amount or dosage of the effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell), the additional agent (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent used individually, e.g., as a monotherapy, required to achieve the same therapeutic effect.
In further aspects, a effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation, or a peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971.
In certain instances, an effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) as described herein are combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, anti-nausea agents (or anti-emetics), pain relievers, cytoprotective agents, and combinations thereof.
In one embodiment, an effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) as described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine), an mTOR inhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomyein A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide), or a checkpoint inhibitor (e.g., a PD-1 or PD-L1 inhibitor, e.g., Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo). Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi)).
General chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-UR), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin (R). Rubex®), etoposide (Vepesid (R), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®), Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar (R), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkcran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).
Treatment with a combination of a chemotherapeutic agent and an effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) described herein can be used to treat a hematologic cancer described herein, e.g., AML. In embodiments, the combination of a chemotherapeutic agent and an effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) is useful for targeting, e.g., killing, cancer stem cells, e.g., leukemic stem cells, e.g., in subjects with AML. In embodiments, the combination of a chemotherapeutic agent and an effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell) is useful for treating minimal residual disease (MRD) MRD refers to the small number of cancer cells that remain in a subject during treatment, e.g., chemotherapy, or after treatment. MRD is often a major cause for relapse. The present invention provides a method for treating cancer, e.g., MRD, comprising administering a chemotherapeutic agent in combination with an effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell), e.g., as described herein.
In an embodiment, the chemotherapeutic agent is administered prior to administration of the effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell). In chemotherapeutic regimens where more than one administration of the chemotherapeutic agent is desired, the chemotherapeutic regimen is initiated or completed prior to administration of the effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell). In embodiments, the chemotherapeutic agent is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, or 30 days prior to administration of the effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell). In embodiments, the chemotherapeutic regimen is initiated or completed at least 1 day, 2 days, 3 days, 4 days, 5 days. 6 days, 7 days, 8 days. 9 days, 10 days, 11 days, 12 days. 13 days, 14 days, 15 days. 20 days. 25 days, or 30 days prior to administration of the effector-enhancing gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) or LTBR-containing CAR/TCR expressing cell (LTBR-containing CART or TCR-T cell).
When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “effective amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104 to 109 cells/kg body weight, in some instances 105 to 106 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J, of Med. 319:1676, 1988). As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats a tumor, an effective amount of an agent is, for example, an amount sufficient to reduce or decrease a size of a tumor or to inhibit a tumor growth, as compared to the response obtained without administration of the agent. The term “effective amount” can be used interchangeably with “effective dose,” “therapeutically effective amount.” or “therapeutically effective dose.”
Also provided herein is a method of vaccinating a subject with a combination vaccine including at least two nucleic acid sequences encoding at least one effector-enhancing gene and at least one viral protein. In one embodiment, the effector-enhancing gene is LTBR. In another embodiment, the viral protein is a coronavirus spike protein. Some embodiments provide vaccines comprising an RNA polynucleotide having an open reading frame encoding an effector-enhancing gene, an RNA polynucleotide having an open reading frame encoding an effector-enhancing gene a viral protein, and a pharmaceutically acceptable carrier or excipient, formulated within a cationic lipid nanoparticle (LNP) The vaccines described herein (e.g., LNP-encapsulated mRNA vaccines) produce prophylactically- and/or therapeutically-efficacious levels, concentrations and/or titers of antigen-specific antibodies in the blood or serum of a vaccinated subject. Sec, e.g., US 2018/0311336A1, which is incorporated herein by reference in its entirety.
As used herein, the term “treatment,” and variations thereof such as “treat” or “treating.” refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing or reducing the occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In certain embodiments, compositions described herein are used to delay development of a disease or to slow the progression of a disease.
Likewise, as used herein, the term “treatment of cancer” or “treating cancer” can be described by a number of different parameters including, but not limited to, reduction in the size of a tumor in an animal having cancer, reduction in the growth or proliferation of a tumor in an animal having cancer, preventing metastasis or reducing the extent of metastasis, and/or extending the survival of an animal having cancer compared to control. In certain embodiments, treatment results in a reduced risk of distant recurrence or metastasis.
With regard to the description of the inventions provided herein, it is intended that each of the compositions described, is useful, in another embodiment, in the methods of the invention. In addition, it is also intended that each of the compositions herein described as useful in the methods, is, in another embodiment, itself an embodiment of the invention.
The following examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to this example but rather should be construed to encompass any and all variations that become evident as a result of the teachings provided herein.
Standard buffy coats containing peripheral blood from de-identified healthy donors were collected by and purchased from the New York Blood Center under an IRB-exempt protocol. All donors provided informed consent. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats using Lymphoprep (Stemcell) gradient centrifugation. For most assays, CD8+ and CD4+ were isolated sequentially from the same donor. First, CD8+ T cells were isolated by magnetic positive selection using the Easy Sep Human CD8 Positive Selection Kit II (Stemcell). Then, CD4+ T cells were isolated from the resulting flowthrough by negative magnetic selection using the Easy Sep Human CD4+ T cell Isolation Kit (Stemcell), γδ T cells were isolated by magnetic negative selection using the EasySep Human Gamma/Delta T cell Isolation Kit (Stemcell). Immediately after isolation. T cells were resuspended in T cell medium, which consisted of Immunocult-XF T cell Expansion Medium (Stemcell) supplemented with 10 ng ml−1 recombinant human IL-2 (Stemcell).
Activation of T cells was performed with Immunocult Human CD3/CD28 T cell Activator (Stemcell) using 25 μl per 106 cells per ml. Typically. T cells were transduced with concentrated lentivirus 24 h after isolation. For some experiments, T cells were electroporated with in-vitro-transcribed mRNA 24 h after isolation or with Cas9 protein 48 h after isolation. At 72 h after isolation, lentivirally transduced T cells were selected with 2 μg ml−1 puromycin.
Every 2-3 days, T cells were either split or had the medium replaced to maintain a cell density of 1×106−2×106 cells per ml. Lentivirally transduced T cells were maintained in medium containing 2 μg ml−1 puromycin for the duration of culture. T cells were used for phenotypic or functional assays between 14 and 21 days after isolation, or cryopreserved in Bambanker Cell Freezing Medium (Bulldog Bio), γδ T cells were further purified before functional assays using anti-Vγ9) PE antibody (Biolegend) and anti-PE microbeads (Miltenyi Biotec) according to the manufacturer's recommendations, in the presence of dasatinib, a protein kinase inhibitor, to prevent activation-induced cell death resulting from TCR cross-linking42. PBMCs from patients with diffuse large B cell lymphoma were obtained from the Perlmutter Cancer Center under a protocol approved by the Perlmutter Cancer Center Institutional Review Board (S14−02164).
All vectors used were cloned using Gibson Assembly (NEB). For the experiments shown in
After adding Gibson overhangs by PCR, ORFs and P2A-puro were inserted into XbaI- and EcoRI-cut lentiCRISPRv2. The sgRNA cassette was removed from JentiCRISPRv2 using PacI and NheI digest. For LTBR overexpression and knockout experiments, the sgRNA cassette was not removed. CARs were synthesized as gBlocks (IDT). For CAR-ORF cloning, CAR-P2A-puro-T2A (partial) were first inserted into XbaI- and EcoRI-cut lentiCRISPRv2. For subsequent ORF insertion, the plasmid was cut with HpaI located within the partial T2A and EcoRI The following vectors were deposited to Addgene: pOT_01 (lenti-EFS-LTBR-2A-puro, Addgene 181970), pOT_02 (lenti-EFS-tNGFR-2A-puro, Addgene 181971), pOT_03 (lenti-EFS-FMC6.3-28z-2A-puro-2A-LTBR, Addgene 181972), pOT_04 (lenti-EFS-FMC6.3-BBz-2A-puro-2A-LTBR, Addgene 181973), pOT_05 (lenti-EFS-FMC6.3-28z-2A-puro-2A-tNGFR, Addgene 181974) and pOT_06 (lenti-EFS-FMC6.3-BBz-2A-puro-2A-tNGFR, Addgene 181975).
All sgRNAs were designed using the GUIDES webtool43. We selected guides that target initial protein-coding exons (with the preference for targeting protein family domains enabled in GUIDES) as well as minimizing off-target and maximizing on-target scores. For Cas9 nuclease nucleofection, we used purified sNLS-SpCas9-sNLS nuclease (Aldevron).
Preparation of ORF library plasmids for paired-end sequencing We re-amplified a previously described genome-scale ORF library14 using Endura electrocompetent cells (Lucigen). The identity of ORFs and matched barcodes was confirmed by paired-end sequencing. In brief, the plasmid was first linearized with I-SceI meganuclease, which cuts downstream of the barcode. Then, the linearized plasmid was tagmented using TnY transposase44. Then, the fragmented plasmid was amplified in a PCR reaction, using a forward primer binding to a handle introduced by TnY and a reverse primer binding to a sequence downstream of the barcode. All transposons and PCR primer oligonucleotides were synthesized by IDT. The resulting amplicon was sequenced on a NextSeq 500. The forward read (containing the ORF) was mapped to GRCh38.101 CDS transcriptome annotations using STAR v.2.7.3a (map quality ≥10)45. Using the paired-end read, we also captured the 24 nucleotide barcode downstream of the constant plasmid sequence. We tabulated ORF-barcode combinations and further curated this table by eliminating any spurious pairs that might be due to sequencing or PCR error. Specifically, a permutation test was performed to identify the maximum number of ORF-barcode combinations expected by random chance, after which we only kept ORF-barcode combinations with a count that exceeded this maximum number We excluded all non-coding elements from the reference and then collapsed barcodes that were within a Levenshtein distance less than 2.
HEK293FT cells were obtained from Thermo Fisher Scientific and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Serum Plus-II (Thermo Fisher Scientific). Nalm6, Jurkat and BxPC3 cells were obtained from ATCC and cultured in RPMI-1640 supplemented with 10% Scrum Plus-II. Capan-2 cells were obtained from ATCC and cultured in McCoy's medium supplemented with 10% Serum Plus-II. For γδco-incubation experiments, cell lines were pre-treated with 50 μM zoledronic acid (Sigma) for 24 h. Cell lines were routinely tested for mycoplasma using MycoAlert PLUS (Lonza) and found to be negative. Cell lines were not authenticated in this study.
We produced lentivirus by co-transfecting third-generation lentiviral transfer plasmids together with packaging plasmid psPAX2 (Addgene 12260) and envelope plasmid pMD2.G (Addgene 12259) into HEK293FT cells, using polyethyleneimine linear MW 25000 (Polysciences). After 72 h, we collected the supernatants, filtered them through a 0.45-μm Steriflip-HV filter (Millipore) and concentrated the virus using Lentivirus Precipitation Solution (Alstem). Concentrated lentivirus was resuspended in T cell medium containing IL-2 and stored at-80 Cc.
For pooled ORF library screening, CD4+ and CD8+ T cells were isolated from a minimum of 500×106 PBMCs from 3 healthy donors. The amount of lentivirus used for transduction was titrated to result in 20-30% transduction efficiency, to minimize the probability of multiple ORFs being introduced into a single cell. The cells were maintained in T cell medium containing 2 μg ml-1 puromycin and counted every 2-3 days to maintain a cell density of 1×106-2×106 cells per ml. On day 14 after isolation, T cells were collected, counted, labelled with 5 μM CFSE (Biolegend) and stimulated with CD3/CD28 Activator (Stemcell) at 1.56 μl per 1×106 cells. An aliquot of cells representing 1,000>coverage of the library was frozen down at this step to be used as a pre-stimulation control. After 4 days of stimulation, cells were collected and an aliquot of cells representing 1,000× coverage of the library was frozen down to be used as a pre-sort control. The remaining cells were stained with LIVE/DEAD Violet cell viability dye (Thermo Fisher Scientific), and CFSElow cells (corresponding to the bottom 15% of the distribution) were sorted using a Sony SH800S cell sorter. Genomic DNA was isolated, and two rounds of PCR to amplify ORF barcodes and add Illumina adaptors were performed46.
For most of the analyses, equal numbers of reads from all three donors were combined per bin before trimming and alignment. The barcodes were mapped to the reference library after adaptor trimming with Cutadapt v. 1.13 (-m 24-e 0.1—discard-untrimmed) using Bowtie v. 1.1.2 (-v 1-m 1—best—strata)+7.48. All subsequent analyses were performed in RStudio v.1.1.419 with R 4.0.0.2. To calculate individual barcode enrichment, barcode counts were normalized to the total number of reads per sample (with pseudocount added) and log2-transformed. To calculate ORF enrichment, raw barcode counts were first collapsed by genes before normalization and log2 transformation.
We performed enrichment analyses at both the barcode and gene level. Statistical analysis on barcode enrichment was performed using MAGeCK49, comparing CFSElow samples to corresponding inputs (pre-stimulation), using CD4+ and CD8+ as replicates. Statistical analysis on ORF enrichment was performed using DESeq250. We obtained raw gene counts by collapsing barcodes into corresponding genes. CFSElow samples were compared to corresponding inputs (both pre-stimulation and pre-sort), using CD4+ and CD8+ as replicates. GO enrichment (biological process) on genes passing DESeq2 criteria (log2-transformed fold change >0.5, Padj<0.05) was performed using the topGO package51. For the genes enriched in the CFSElow screen (DESeq2 analysis), we overlapped these genes with differentially expressed genes after CD3/CD28 stimulation using data from the Database of Immune Cell eQTLs. Expression, Epigenomics (DICE: https://dice-database.org/)41. For differentially expressed genes, we used the following DICE datasets: ‘T cell, CD4, naive’ versus ‘T cell, CD4, naive [activated]’, ‘T cell. CD8, naive’ versus ‘T cell, CD8, naïve [activated]’. Significant differential expression was as given in the DICE dataset (Padj<0.05).
Transduced T cells were collected at day 14 after isolation, counted and plated at 2.5×104 cells per well in a round bottom 96-well plate, in 2 sets of triplicate wells per transduction. One set of triplicate wells was cultured in Immunocult-XF T cell Expansion Medium supplemented with 10 ng ml−1 IL-2 and another set of triplicate wells was further supplemented with 1.56 μl CD3/CD28 Activator per 1 ml of medium. The cells were cultured for 4 days, and then were collected and stained with LIVE/DEAD Violet cell viability dye. Before flow cytometric acquisition, the cells were resuspended in D-PBS with 10% v/v Precision Counting Beads (Biolegend). For quantification, the number of viable cell events was normalized to the number of bead events per sample. Then, for each ORF the normalized number of viable cells in wells supplemented with CD3/CD28 Activator was divided by the mean number of viable cells in control wells to quantify T cell proliferation. To enable comparisons between donors and CD4+/CD8+ T cells, the proliferation of T cells transduced with a given ORF was finally normalized to the proliferation of a matched tNGFR control.
In addition to the counting beads assay, we also measured proliferation using a dye dilution assay. For this assay, transduced T cells were collected at day 14 after isolation, washed with D-PBS and then labelled with 5 μM CellTrace Yellow (CTY) in D-PBS for 20 min at room temperature. The excess dye was removed by washing with a fivefold excess of RPMI-1640 supplemented with 10% Serum Plus-II. The labelled cells were then plated at 2.5×104 cells per well on a round bottom 96-well plate. One set of triplicate wells was cultured in supplemented Immunocult-XF T cell Expansion Medium (that is, without IL-2) and another set of triplicate wells was supplemented with 10 ng ml−1 IL-2 and 1.56 μl CD3/CD28 Activator per 1 ml of medium. The cells were cultured for 4 days, and then were collected and stained with LIVE/DEAD Violet cell viability dye. For quantification of the proliferation index, events were first gated on viable T cells in FlowJo (Treestar) and exported for further analysis in R/RStudio using the flowFit and flow Core packages52. Unstimulated cells were used to determine the parent population size and position to account for differences in staining intensity between different samples. These fitted parent population parameters were then used to fit the CTY profiles of matched stimulated samples, modelled as Gaussian distributions assuming log2-distanced peaks as a result of cell division and dye dilution. Fitted CTY profiles were inspected visually for concordance with the original CTY profiles and used to calculate the proliferation index. The proliferation index is defined as the sum of cells in all generations divided by the computed number of parent cells present at the beginning of the assay.
For CD25 (IL2RA) and CD154 (CD40L) quantification, T cells were restimulated with CD3/CD28 Activator (6.25 μl per 106 cells) for 6 h (CD154 staining in CD8+) or for 24 h before staining (CD25 staining in both CD4+ and CD8+, and CD154 staining in CD4+). For Ki-67 and 7-amino-actinomycin D (7-AAD) staining, T cells were rested overnight in Immunocult-XF T cell Expansion Medium without IL-2 and then activated with CD3/CD28 Activator (25 μl per 106 cells) for 24 h. In other cases, T cells were stained without stimulation. For detection of secreted proteins. T cells were stimulated for 24 h with CD3/CD28 Activator (25 μl per 106 cells) (LTA. LIGHT), and protein transport inhibitors brefeldin A (5 μg ml−1) and monensin (2 μM) were included for the last 6 h of stimulation (IL12B, LTA, LIGHT).
First, the cells were collected, washed with D-PBS and stained with LIVE/DEAD Violet cell viability dye for 5 min at room temperature in the dark, followed by surface antibody staining for 20 min on ice. After surface antibody staining (where applicable) the cells were washed with PBS and acquired on a Sony SH800S cell sorter or taken for intracellular staining. For intracellular staining, the cells were resuspended in an appropriate fixation buffer. The following fixation buffers were used for specific protein detection: Fixation Buffer (Biolegend) for IL12B and MS4A3 staining: True-Nuclear Transcription Factor Fix (Biolegend) for BATF, TCF1 and FLAG staining; and FoxP3/Transcription Factor Fixation Reagent, (eBioscience) for Ki-67. After resuspension in the fixation buffer, cells were incubated at room temperature in the dark for 1 h. Following the incubation, the cells were washed twice in the appropriate permeabilization buffer. The following permeabilization buffers were used: Intracellular Staining Permeabilization Wash Buffer (Biolegend) for IL12B and MS4A3 staining: True-Nuclear Perm Buffer (Biolegend) for BATF, TCF1 and FLAG staining; and FoxP3/Transcription Factor Permeabilization Buffer (eBioscience) for Ki-67. After permeabilization, the cells were stained with the specific antibody or isotype control for 30 min in the dark at room temperature. Finally, the cells were washed twice in the appropriate permeabilization buffer and acquired on a Sony SH800S flow cytometer. For cell-cycle analysis, the cells were further stained with 0.5 μg ml−1 7-AAD for 5 min immediately before acquisition. Gating was performed using appropriate isotype, fluorescence minus one and biological controls. Typically, 5.000-10,000 live events were recorded per sample.
T cells were rested for 24 h in in Immunocult-XF T cell Expansion Medium without IL-2 before detection of phosphorylated proteins. The rested cells were stimulated with CD3/CD28 Activator (25 μl per 106 cells) for the times indicated in the corresponding figure. Immediately after the stimulation period, the cells were fixed with a 1:1 volume ratio of the pre-warmed Fixation Buffer (Biolegend) for 15 min at 37° C., and washed twice with the cell staining buffer (D-PBS+2% FBS). As per the manufacturer's protocol, the cells were resuspended in the residual volume and permeabilized in 1 ml of pre-chilled True-Phos Perm Buffer (Biolegend) while vortexing. The cells were incubated in the True-Phos Perm Buffer for 60 min at −20° C. After permeabilization the cells were washed twice with the cell staining buffer and stained with anti-CD4, anti-CD8, anti-RELA and anti-phospho-RELA antibodies (or isotype controls) for 30 min at room temperature. After staining, the cells were washed twice in the cell staining buffer and acquired on a Sony SH800S cell sorter. Gating was performed on CD4+ or CD8+ cells, and the levels of RELA and phospho-RELA were determined using appropriate isotype and biological controls.
T cells expressing tNGFR or LTBR, resting or stimulated for 15 min with CD3/CD28 Activator (25 μl per 106 cells), were collected, washed with 1×D-PBS and lysed with TNE buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) in the presence of a protease inhibitor cocktail (Bimake B14001) and a phosphatase inhibitor cocktail (Cell Signaling Technologies 5872S) for 1 h on ice. Cell lysates were spun for 10 min at 10,000 g, and the protein concentration was determined with the BCA assay (Thermo Fisher Scientific). Equal amounts of cell lysates (25 mg) were denatured in Tris-Glycine SDS Sample buffer (Thermo Fisher Scientific) and loaded on a Novex 4-12 or 4-20% Tris-Glycine gel (Thermo Fisher Scientific). The PageRuler pre-stained protein ladder (Thermo Fisher Scientific) was used to determine the protein size. The gel was run in Ix Tris-Glycine-SDS buffer (IBI Scientific) for about 120 min at 120 V. Proteins were transferred on a nitrocellulose membrane (BioRad) in the presence of prechilled 1× Tris-Glycine transfer buffer (Thermo Fisher Scientific) supplemented with 20% methanol for 100 min at 100 V.
Immunoblots were blocked with 5% skimmed milk dissolved in 1×PBS with 1% Tween-20 (PBST) and incubated overnight at 4° C.′ separately with the following primary antibodies: rabbit anti-GAPDH (0.1 mg ml−1, Cell Signaling, 2118S), mouse anti-IKKα (1:1,000 dilution, Cell Signaling. 3G12), rabbit anti-IKKβ (1:1,000 dilution, Cell Signaling, D30C6), rabbit anti-NF-κB p65 (1:1,000 dilution, Cell Signaling, D14E12), rabbit anti-phospho-NF-κB p65 Ser536 (1:1,000 dilution. Cell Signaling, 93H1), mouse anti-IκBα (1:1,000 dilution, Cell Signaling, L35A5), rabbit anti-phospho-IκBα Ser32 (1:1,000 dilution, Cell Signaling, 14D4), rabbit anti-NF-κB p100/p52 (1:1,000 dilution, Cell Signaling, 4882) and rabbit anti-RELB (1:1,000 dilution. Cell Signaling, C1E4). After the primary antibody, the blots were incubated with IRDye 680RD donkey anti-rabbit (0.2 mg ml−1, LI-COR 926-68073) or with IRDye 800CW donkey anti-mouse (0.2 mg ml−1, LI-COR 926-32212). The blots were imaged using Odyssey CLx (LI-COR) and quantified using ImageJ v. 1.52.
For measurement of secreted IFNγ and IL-2, T cells were first collected and rested for 24 h in medium without IL-2. Then, they were counted, plated at 2.5×104 cells per well in a round bottom 96-well plate and incubated in medium without IL-2, with or without CD3/CD28 Activator (25 μl per 106 cells) for 24 h. Then, cell supernatants were collected, diluted and used for cytokine quantification with an enzyme-linked immunosorbent assay (Human IL-2 or IFNγ DuoSet, R&D Systems), using an Infinite F200 Pro (Tecan) plate reader. Multiplexed quantification of secreted cytokines and chemokines in resting or stimulated T cells was performed using the Human Cytokine/Chemokine 48-Plex Discovery Assay Array (Eve Technologies).
CD19+ Nalm6 cells were first transduced with a lentiviral vector encoding EGFPd2PEST-NLS and a puromycin resistance gene53. The transduced cells were kept in puromycin selection throughout the culture, to maintain stable EGFP expression, and puromycin was only removed from the medium before the killing assay. T cells were transduced with a vector encoding a CAR specific for CD19, using either a CD28 stalk, CD28 transmembrane and CD28 signaling domain or CD8 stalk and CD8 transmembrane domain with 4-1BB signaling domain, and CD3ζ signaling domain54. Fourteen days after transduction, transduced T cells were combined with 5×104 Nalm6 GFP+ cells in triplicate at indicated effector: target ratios in a flat 96-well plate pre-coated with 0.01% poly-1-ornithine (EMD Millipore) in Immunocult medium without IL-2. The wells were then imaged using an Incucyte SX1, using 20× magnification and acquiring 4 images per well every 2 h for up to 120 h. For each well, the integrated GFP intensity was normalized to the 2 h time point, to allow the cells to fully settle after plating.
In Vitro mRNA Preparation
The template for in vitro transcription was generated by PCR from a plasmid encoding LTBR or tNGFR with the resulting amplicon including a T7 promoter upstream of the ORF. The purified template was then used for in vitro transcription with capping and poly-A tailing using the HiScribe T7 ARCA mRNA Kit with Capping (NEB).
Activated T cells were nucleofected with in-vitro-transcribed mRNA at 24 h after activation or with Cas9 protein at 48 h after activation. The cells were collected, washed twice in PBS and resuspended in P3 Primary Cell Nucleofector Solution (Lonza) at 5×105 cells per 20 μl. Immediately after resuspension, 1 μg mRNA or 10 μg Cas9 (Aldevron) were added (not exceeding 10% v/v of the reaction) and the cells were nucleofected using the E0-115 program on a 4D-Nucleofector (Lonza). After nucleofection the cells were resuspended in pre-warmed Immunocult medium with IL-2 and recovered at 37° C., with 5% CO2 for 20 min. After recovery, the cells were plated at 1×106 cells per ml and used in downstream assays.
For single-cell sequencing. CD8+ T cells were individually transduced with ORFs and kept, separately, under puromycin selection for 14 days. Then, transduced cells were combined and split into two conditions: one was cultured for 24 h only in the presence of IL-2; the other was further supplemented with 6.25 μl CD3/CD28 Activator per 106 cells. After stimulation, the cells were collected, counted and resuspended in staining buffer (2% BSA+0.01% Tween-20 in PBS) at 2×107 cells per ml. Then, 10% (v/v) Human TruStain FcX Fc Receptor Blocking Solution (Biolegend) was added, and the cells were incubated at 4° C., for 10 min. After Fc receptor blocking, the cell concentration was adjusted to 5×106 cells per ml and the stimulated and unstimulated cells were split into 4 conditions each. Each condition received a different oligonucleotide-conjugated (barcoded) cell hashing antibody to allow for pooling of different conditions in the same 10× Genomics Chromium lane23. After 20 min co-incubation on ice, the cells were washed 3 times with staining buffer and counted using Trypan blue exclusion. Cell viability was typically around 95%.
Then, cells stained with different hashing antibodies were pooled together at equal numbers and stained with the following oligonucleotide-conjugated (barcoded) antibodies for quantification of cell surface antigens: CD11c (0.1 μg), CD14 (0.2 μg), CD16 (0, 1 μg), CD19 (0.1 μg), CD56 (0.2 μg), CD3 (0.2 μg), CD45 (0.01 μg), CD45RA (0.2 μg), CD45RO (0.2 μg), CD4 (0.1 μg), CD8(0.1 μg), CD25 (0.25 μg), CD69 (0.25 μg) and NGFR (0.25 μg) (TotalSeq-C, Biolegend). The cells were stained for 30 min on ice, washed 3 times with staining buffer, resuspended in PBS and filtered through a 40-μm cell strainer. The cells were then counted and the concentration was adjusted to 1×106 ml−1. For loading into the 10× Genomics Chromium, 3× 104 cells were combined with Chromium Next GEM Single Cell 5′ v2 Master Mix (10× Genomics) supplemented with a custom reverse primer binding to the puromycin resistance cassette for boosting ORF transcript capture at the reverse transcription stage. The custom reverse primer was added at a 1:3 ratio to the poly-d′T primer included in the Master Mix.
For cDNA amplification, additive primers for amplification of sample hashing and surface antigen barcodes were included23, as well as a nested reverse primer binding to the puromycin resistance cassette downstream of the ORF. Following cDNA amplification, SPRI beads were used for size selection of resulting PCR products: small-size (fewer than 300 bp) sample hashing and surface antigen barcodes were physically separated from larger cDNA and ORF amplicons for downstream processing. Sample hashing and surface antigen barcodes were also processed22. Amplified cDNA was then separated into three conditions, for construction of the gene expression library, of TCR library and ORF library. The ORF library was processed similarly to the af TCR library, using nested reverse primers binding downstream of the ORF. The quality of produced libraries was verified on BioAnalyzer using the High Sensitivity DNA kit (Agilent). The libraries were sequenced on a NextSeq 500. For the gene expression library, more than 25,000 reads per cell were generated. For other libraries, more than 5,000 reads per cell were generated.
Gene expression unique molecular identifier (UMI) count matrices and TCR clonotypes were derived using 10× Genomics Cell Ranger 3.1.0. Hashtag oligo (HTO) and antibody UMI count matrices were generated using kallisto v.0.46.055 and bustools v.0.39.356. ORF reads were first aligned to plasmid references using Bowtie2 v.2.2.857 and indexed to the associated ORF, after which kallisto and bustools were used to generate UMI count matrices. All modalities were normalized using a centred log ratio (CLR) transformation. Cell doublets and negatives were identified using the HTODemux58 function and then excluded from downstream analysis. The UMI cut-off quantile for HTODemux was optimized to maximize singlet recovery using grid search with values between 0 and 1. ORF singlets were identified using MULTIseqDemux59. We then excluded cells with low-quality gene expression metrics and removed cells with fewer than 200 unique RNA features or greater than 5% of reads mapping to the mitochondrial transcriptome.
Count matrices were then loaded into and analyzed with Seurat v.4.0.160. Cell cycle correction and scaling of gene expression data was performed using the CellCycleScoring function with default genes, followed by scaling the data using the ScaleData function Principal component (PC) optimization of the scaled and corrected data was then performed using JackStraw61, in which we selected all PCs up to the first non-significant PC to use in clustering. Clustering of cells was performed using a shared nearest neighbor (SNN)-based clustering algorithm and visualized using UMAP dimensional reduction62 to project cluster PCs into 2D space. Cluster marker analysis was performed using the FindAllMarkers function with the hypothesis set defined as positive and negative markers present in at least 25% of cluster cells and with a log 2-transformed fold change threshold of 0.25 as compared to non-cluster cells. Differential expression analysis of ORFs was performed using DESeq250 to identify genes up and downregulated in ORF-expressing cells as compared to NGFR (control) cells, with differential expression defined as those with q<0.1 calculated using the Storey method63.
CD4+ and CD8+ LTBR-or tNGFR-transduced T cells were stimulated for 24 h with CD3/CD28 Activator (25 μl per 106 cells) or left unstimulated (n=3 biological replicates). Total RNA was extracted using the Direct-zol RNA purification kit (Zymo). The 3′-enriched RNA-seq library was prepared as described before64. In brief, RNA was reverse-transcribed using SMARTScribe Reverse Transcriptase (Takara Bio) and a poly (dT) oligo containing a partial Nextera handle. The resulting cDNA was then PCR-amplified for 3 cycles using One Taq polymerase (NEB) and tagmented for 5 min at 55° C., using homemade transposase To Y44. Immediately afterwards, the tagmented DNA was purified on a MinElute column (Qiagen) and PCR-amplified using OneTaq polymerase and barcoded primers for 12 cycles. The PCR product was purified using a dual (0.5×-0.8×) SPRI clean-up (Agencourt) and the size distribution was determined using Tapestation (Agilent). Samples were sequenced on a NextSeq 500 (Illumina) using a v2.5 75-cycle kit (paired end). Paired-end reads were aligned to the transcriptome (human Ensembl v.96 reference65) using kallisto v.0.46.055 and loaded into RStudio 1.1.419 with R 4.0.0.2 using the tximport packaged66. Differential gene expression analysis was performed using DESeq250. GO enrichment (biological process) on genes passing DESeq2 criteria (log:-transformed fold change >1. Padj <0.05) was performed using the topGO package51.
CD8+LTBR and tNGFR T cells were stimulated for 24 h with CD3/CD28 Activator (25 μl per 106 cells) or left unstimulated (n=2 biological replicates). We performed bulk ATAC-seq as previously described44. In brief, cell membranes were lysed in the RSB buffer (10 mM Tris-HCL pH 7.4, 3 mM MgCl2, 10 mM NaCl) with 0.1% IGEPAL freshly added. After pipetting up and down, nuclei were isolated by centrifugation at 500 g for 5 min at 4° C. After discarding the supernatant, the nuclei were resuspended in the Tagmentation DNA (TD) Buffer44 with homemade transposase TnY protein44 and incubated at 37° C., for 30 min. After purification on a MinElute column (Qiagen), the tagmented DNA was PCR-amplified using a homemade Pfu X7 DNA polymerase44 and barcoded primers for 12 cycles. The PCR product was purified via a 1.5/SPRI clean-up (Agencourt) and checked for a characteristic nucleosome banding pattern using TapeStation (Agilent). Samples were sequenced on a NextSeq 500 (Illumina) using the v2.5 75-cycle kit (single end).
Single-end reads were aligned to the Gencode hg38 primary assembly67 using Bowtie2 v.2.4.457. We then used SAMtools v. 1.968 to filter out alignments with low-mapping quality (MAPQ<30) and subsequently to sort and index the filtered BAM files68. Read duplicates were removed using Picard v.4.1.8.169. Peaks were called using MACS3 v.3.0.070 with default parameters (-g 2.7e9-q 0.05).
To construct the union feature space (‘union peaks’) used for much of the downstream analyses, we began by performing intersections on pairs of biological replicate narrowPeak files using BEDTools v.2.29.0 (using bedtools intersect), keeping only those peaks found in both replicates71. After marking the shared peaks between replicates, we used bedtools merge to consolidate the biological replicates at each shared peak (at least 1 bp overlap). In this new peak BED file, each shared peak includes all sequence found under the peak in either of the biological replicates. Next, we took the union of each of these peak files (LTBR resting. LTBR stimulated, tNGFR resting, tNGFR stimulation); we combined any peaks with at least 1 bp overlap. Using the union peaks, we generated a peak read count matrix (union peaks×ATAC samples), in which each entry in the matrix corresponds to the number of reads overlapping that peak in the specified sample—we term this the per-peak ATAC matrix. The overlapping reads are taken directly from the BAM files (converted to BED) that provide an alignment for each sample. Thus, the matrix includes a column for each biological replicate. Although samples had minimal differences in aligned reads, we normalized each entry in the matrix by the number of reads that overlapped the TSS regions in each sample. In this manner, any difference in read or alignment depth between samples would be normalized appropriately. In addition to the per-peak ATAC matrix, we also constructed a per-gene ATAC matrix as follows: we assigned a gene's total ATAC reads as the sum of normalized reads from the per-peak ATAC matrix for all peaks within 3 kb of a gene's start or end coordinates.
We imported these two ATAC matrices (per-peak and per-gene) into R v.4.1.1 for gene and peak enrichment analysis using DESeq2 v. 1.32.0. For comparison between ATAC-seq and RNA-seq, we used a statistical threshold of adjusted P value <0.05 and either log 2-transformed fold change >0 (for increases in ATAC or RNA) or logs-transformed fold change <0 (for decreases in ATAC or RNA). For transcription factor-motif analysis we used Chrom-VAR v. 1.14.072 as follows: For each of the test versus control conditions, we constructed Summarized Experiment objects using column and sample subsets of the per-peak matrix and the union feature space. We used the matchMotifs function to annotate transcription factormotifs. We computed enrichment deviations between test and control conditions using the computeDeviations function. To produce read pile-up tracks at specific genomic loci, we pooled de-duplicated reads from biological replicates (BAM) using samtools merge. We converted these pooled-replicate BAM files to big Wig files by using the bamCoverage function from deeptools v.3.4.2 and setting the scaleFactor to the relative number of TSSs found in the pooled biological replicates compared to all other sample aggregates73. Using the big Wig files, read pileups were plotted with pyGenomeTracks v.3.674.
Finally, we performed k-means clustering on ATAC peaks near genes with increased chromatin accessibility. First, using DEseq2 on the ATAC per-gene matrix, we identified genes with log 2-transformed fold change >1 and adjusted P value <0.05 (that is, genes with increased chromatin accessibility) in either of two comparisons: (1) LTBR stimulated versus tNGFR stimulated: (2) LTBR resting versus tNGFR resting. After identifying these genes, we isolated all accessibility peaks in the per-peak ATAC matrix within 3 kb of the gene body; this subset of peaks from the per-peak ATAC matrix was used as input for the clustering. Then, using deeptools (computeMatrix and plotHeatmap functions) on this subset of ATAC peaks, we performed k-means clustering with k=4 clusters and 6 kb read windows.
Data between two groups were compared using a two-tailed unpaired Student's t-test or the Mann-Whitney test as appropriate for the type of data (depending on the normality of the distribution). Unless otherwise indicated, a P value less than or equal to 0.05 was considered statistically significant for all analyses, and not corrected for multiple comparisons. In cases in which multiple comparison corrections were necessary, we adjusted the P value using the Benjamini-Hochberg method. All group results are represented as mean±s.e.m., if not stated otherwise. Statistical analyses were performed in Prism (GraphPad) and RStudio (Rstudio PBC). Flow cytometry data were analyzed using FlowJo v. 10.7.1 (Treestar).
We performed a genome-scale gain-of-function screen in primary human CD4+ and CD8+ T cells, using a lentiviral library of barcoded human ORFs. We show that T cells with the strongest proliferation phenotypes are enriched for both known and unknown regulators of the immune response, many of which are not typically expressed by peripheral T cells. We validate top-ranked ORFs in cells from screen-independent donors and further demonstrate that these ORFs not only drive T cell proliferation but also increase the expression of activation markers and the secretion of key proinflammatory cytokines. To gain more comprehensive insight into the mechanism of action of these genes, we develop a single-cell sequencing approach coupled with direct ORF capture. We identify LTBR—one of the top-ranked ORFs not expressed by lymphocytes—as a key driver of profound transcriptional and epigenetic remodeling through increased NF-κB signaling, which results in a marked increase in the secretion of proinflammatory cytokines and resistance to apoptosis. Finally, we show that top-ranked ORFs potentiate antigen-specific T cell functions, in the context of CD19-directed CAR T cells and broadly tumor-reactive γδ T cells from healthy donors and patients with blood cancer.
To avoid relying on constitutive expression of large bacterial proteins or chromatin accessibility in the vicinity of target genes13, we decided to use a lentiviral library of human ORFs; this library contains nearly 12,000 full-length genes, with around 6 barcodes per gene14 (
We transduced the lentiviral ORF library into CD4+ and CD8+ T cells from three healthy donors, and after a brief period in culture (14 days) we restimulated the cells to identify drivers of proliferation in response to TCR stimulation. We were able to capture the majority of individual ORF barcodes, and nearly all ORFs, including the largest ones (
Each ORF in the library is linked to an average of six DNA barcodes (
Overall, the enriched ORFs spanned a range of diverse biological processes. Among the top-enriched Gene Ontology (GO) biological processes were lymphocyte proliferation, interferon-γ (IFNγ) production and NF-κB signaling (
To validate the top-ranked ORFs and understand their effect on other relevant aspects of T cell function, we subcloned 33 ORFs from the library into a vector co-expressing a P2A-linked puromycin resistance gene from the same promoter. We chose a truncated nerve growth factor receptor (tNGFR), Jacking its intracellular domain, as a control that has no effect on T cell phenotype21, CD4+ and CD8+ populations were separately isolated from several screen-independent healthy donors and transduced with individual ORFs (
Fourteen days after isolation, we restimulated the cells and measured the relative increase in cell numbers. We found that 16 tested ORFs significantly improved cell proliferation compared with tNGFR, and that proliferation was well correlated between CD4+ and CD8+ cells (Spearman's r=0.61, P=0.002) (
Finally, we measured the secretion of the cytokines interleukin-2 (IL-2) and IFNγ after restimulation with CD3/CD28 (
Building on our quantification of how each ORF affects proliferation, activation and cytokine release, we next sought to better understand the underlying mechanisms that drive these changes in cell state. To gain a more comprehensive view of the mechanisms of action of individual ORFs, and to provide a multidimensional characterization of the phenotypic changes they induce, we developed a single-cell sequencing strategy with direct ORF capture. This approach, OverCITE-seq (Overexpression-compatible Cellular Indexing of Transcriptomes and Epitopes by Sequencing) extends previous approaches that we have developed for quantifying surface antigens22 and CRISPR perturbations23, and allows for high-throughput, single-cell analysis of a pool of T cells with different ORFs. In brief, mRNA from lentivirally integrated ORFs is reverse-transcribed by a primer binding to a constant sequence of the transcript downstream of the ORF and barcoded, along with the cell transcriptome, during template switching. The resulting cDNA pool is then split for separate construction of gene expression and ORF expression libraries (
We optimized and applied OverCITE-seq to a pool of around 30 ORFs transduced into CD8+ T cells from a healthy donor. The cell pool was cither left unstimulated (‘resting’) or stimulated with CD3/CD28 antibodies to mimic TCR activation. To gain confidence in how well ORFs are assigned to each single cell, we leveraged the fact that the protein produced by the control gene, tNGFR, is expressed on the cell surface and can thus be captured with a DNA-barcoded antibody23. The proportion of cells designated as tNGFR positive was consistent when measured by CITE-seq or flow cytometry (
Unsupervised clustering showed clear separation for stimulated and resting T cells. Within these activation-driven super-clusters we could observe individual clusters associated with a particular cell state or function, such as cell cycle (clusters 1 and 9), macromolecule biosynthesis (cluster 2), type I IFN signaling (cluster 3), cytotoxicity (cluster 6). T cell activation and proliferation (cluster 10), and stress response and apoptosis (cluster 11) (
To investigate the mechanisms of genetic perturbations with the strongest transcriptional changes, we looked at the transcriptomic profiles of CD3/CD28-stimulated ORF T cells compared to unstimulated control T cells (
Having identified LTBR as a strong driver of proinflammatory cytokine secretion (
LTBR signaling in its endogenous context (in myeloid cells) is triggered either by a heterotrimer of lymphotoxin-α (LTA) and lymphotoxin-B (LTB) or by LIGHT (encoded by the TNFSF14 gene). As LTA, LTB and LIGHT are expressed by activated T cells, we sought to elucidate whether addition of exogenous LTA or LIGHT could modulate the cytokine secretion, differentiation or proliferation of CD3/CD28-stimulated LTBR-overexpressing T cells: however, we found no effect of exogenous ligands on LTBR T cell function (
Finally, to identify the key domains of the LTBR protein that drive its activity in T cells, we designed a series of point or deletion mutants of LTBR (
LTBR overexpression was shown to induce broad transcriptomic changes in T cells, accompanied by changes in T cell function (
We then decided to investigate changes in protein expression and/or phosphorylation of the members of the NF-κB signaling pathway. We observed a more rapid phosphorylation of p65 (RELA) and a strong increase in phosphorylation of an NF-κB inhibitor, IκBα, targeting IκBα for degradation; both of these effects enhance NF-κB activation or transcription (
Having established that LTBR activates both the canonical and the non-canonical NF-κB pathways, we sought to determine the molecular basis of this phenomenon by perturbing key genes in the LTBR and NF-κB pathways by co-delivery of LTBR or tNGFR and CRISPR constructs that target II genes involved in the LTBR signaling pathway32 (
RELA had a stronger effect on control cells than on LTBR cells. The effect of LTB loss on T cell activation in LTBR cells supports the observation that alanine mutagenesis of key residues involved in LTA or LTB binding (
To investigate the potential roles of canonical versus non-canonical NF-κB signaling in LTBR T cells, we decided to analyze the global effects of RELA or RELB loss on the LTBR-driven gene expression profiles. Using bulk RNA-seq on T cells overexpressing LTBR or tNGFR, we discovered that only the loss of RELA significantly downregulated the expression of ‘core’ LTBR genes, whereas loss of RELB had no effect (
Thus far we have shown that top-ranked genes from the ORF screen improve T cell function using a non-specific, pan-TCR stimulation. We next sought to determine whether a similar improvement could be observed using antigen-specific stimulation (
Since both CARs use different costimulatory domains, from CD28 or 4-1BB, we wanted to determine whether top-ranked genes that were selected using CD28 co-stimulation could also work in the context of 4-1BB co-stimulation. Nearly all of the top-ranked genes tested, with the exception of AKR1C4, improved upregulation of CD25 and antigen-specific cytokine secretion, with no major differences in the differentiation or exhaustion phenotype (
Although production of IL-2 and IFNγ is crucial for the clonal expansion and antitumor activity of T cells, another vital component of tumor immunosurveillance is direct cytotoxicity. Top-ranked genes had an overall stronger effect on the cytotoxicity of CD28 CAR T cells than 4-1BB CAR T cells (
T cells from healthy donors are relatively easy to engineer and rarely show signs of dysfunction in culture, whereas autologous T cells in patients with cancer are often dysfunctional, showing limited proliferation and effector functions34. To investigate whether top-ranked genes can improve CAR T cell response not only in healthy T cells but also in potentially dysfunctional T cells derived from patients, we transduced CD19 CARs co-expressed with LTBR or a control gene into peripheral blood mononuclear cells (PBMCs) from patients with diffuse large B cell lymphoma. After co-incubation with CD19+ target cells, we observed a similar increase in the secretion of IL-2 and IFNγ from LTBR CAR T cells to that seen in healthy donors, indicating that identified ORFs can be successfully used to engineer T cells from patients with lymphoma ex vivo (
The screen and subsequent validations were performed in αβ T cells, the predominant subset of T cells in human peripheral blood. Although immunotherapy based on αβ T cells has shown considerable potential in the clinic, γδ T cells present an attractive alternative, owing to their lack of MHC restriction, ability to target broadly expressed stress markers in a cancer-type-agnostic manner and more innate-like characteristics5. We therefore sought to determine whether the top genes validated in αβ T cells translated to γδ T cells. After co-incubation with leukemia or pancreatic ductal adenocarcinoma cancer cells, we observed an increase in IL-2 and IFNγ secretion from γδ T cells that were transduced with top-ranked genes (
In summary, here we developed a genome-scale gain-of-function screen in primary human T cells, in which we examined the effects of nearly 12,000 full-length genes on TCR-driven proliferation in a massively parallel manner. The largest-to our knowledge-previously published gain-of-function study in primary T cells involved 36 constructs, including full-length genes and synthetic receptors35. That approach relied on construct delivery via donor DNA and Cas9-mediated targeted insertion. Although using donor DNA for target gene delivery allows for more flexibility in terms of construct design, especially for engineering synthetic receptors, that method is less scalable and less accessible in terms of cost and complexity than the lentiviral library that we used here. Thus. ORF-based gain-of-function screens are readily applicable to a plethora of T cell phenotypes and settings, and that they offer the opportunity for clinical translation. In fact, all FDA-approved CAR therapies already rely on lentiviral or retroviral integration of a CAR transgene, and therefore an addition of an ORF to this system should pose no major manufacturing or regulatory challenges. The use of ORF-encoding mRNA delivered to CAR T cells before infusion is another translational route, especially if there are safety concerns about the mode of action of a particular ORF.
Gain-of-function screens have the potential to uncover regulators that are tightly controlled, restricted to a specific developmental stage or expressed only in certain circumstances. As shown here, LTBR is canonically absent from cells of lymphoid origin, but, owing to the intact signaling pathway, it can have a synthetic role when introduced to T cells. Although constitutive activation of other TNFRSF members might result in a similar phenotype, one of the features that distinguishes LTBR (and plausibly led to its enrichment, but not that of other TNFRSF members, in the screen) is the formation of an autocrine loop whereby the receptor and its ligands are present in the same cell. It is particularly noteworthy that expression of LTBR boosts IL-2 secretion, as this cytokine is produced exclusively by T cells and not by cell types that endogenously express LTBR. In addition to boosting cytokine secretion, overexpression of LTBR promoted stemness (expression of TCF1) and decreased activation-induced apoptosis, as well as offered a level of protection against phenotypic and functional hallmarks of T cell exhaustion—all of which are features not recapitulated by cell types that endogenously express LTBR. Previous work using overexpression of LTBR in cell lines showed that LTBR has a pro-apoptotic role36, in direct contrast to the phenotype that we observed in primary T cells. Transcript- and protein-level analyses revealed that LTBR drives the constitutive activation of both canonical and non-canonical NF-κB pathways. However, using epigenomic profiling and CRISPR-based functional perturbations we showed that the phenotypic and functional changes resulting from LTBR expression are mediated primarily through activation of the canonical NF-κB pathway, whereas changes in the non-canonical pathway may not be essential for the observed phenotypes-in contrast to the well-established role of non-canonical NF-κB activation in cells that endogenously express LTBR37.
Gene overexpression has been used for pre-clinical enhancement of CAR T cell therapies in numerous studies. For example, armoring CAR T cells with cytokines such as IL-12 or IL-18, which are not typically produced by T cells but are known to improve T cell function when secreted by other cell types, was shown to improve their antitumor activity38,39. Notably, a previous study found that CAR T cell exhaustion can be alleviated by overexpression of e-JUN, a transcription factor identified by RNA-seq as specifically depleted in exhausted cells40. Future studies that adapt genome-wide gain-of-function screens to relevant models of immunotherapy will lead to advanced target selection for engineering synthetic cellular therapies that can overcome the immunosuppressive tumor microenvironment and eradicate established cancers.
We have shown that LTBR and several other top-ranked genes (ORFs, open reading frames) identified in the screen boost the antitumor response of anti-CD19 CARs in context of a B cell leukemia. Here we tested whether a similar improvement of activity can be seen in conjunction with two clinically-tested anti-mesothelin CARs (using either 4-1BB or CD28 costimulatory domains) in the context of pancreatic cancers. We tested T cells co-expressing a CAR and an ORF against Capan-2, a pancreatic cancer line expressing high levels of mesothelin, the CAR target, and BxPC3, a pancreatic cancer line expressing low levels of mesothelin (
Following overnight co-incubation, we determined that all but one ORF tested (that is. AHNAK, BATF, IFNL2, IL 12B, and LTBR) boosted antigen-specific secretion of IFNγ, when used with either CAR (cither 41BB or CD28) against a mesothelin-high cell line Capan-2 (
Cytokine secretion is one of the aspects of a productive antitumor response-another one is direct cytotoxicity Therefore, we tested the ability of CAR T cells co-expressing top genes to kill GFP+ Capan2 or BxPC3 cells (
T cell therapies can rely on redirecting the cells to a given tumor target using either a CAR or a TCR. The former has the advantage of being able to target tumors in different patients, regardless of their HLA haplotype, while the latter can also target antigens that are intracellular (since epitopes from all cellular proteins are sampled by and displayed on the HLA molecules). Here we used a clinically-tested TCR directed against an epitope from NY-ESO-1, commonly expressed in many cancer histologies, including but not limited to melanoma, multiple myeloma, sarcoma, lung cancer. Due to size restrictions we delivered the TCR and the gene (ORF, open reading frame) on two separate lentiviruses that were used to co-transduce T cells (
We then tested the engineered CD8+ T cells against an HLA-A2+NY-ESO-1+ melanoma line A375. Most genes tested increased the secretion one or both of cytokines IFNγ and IL2 (
We have demonstrated that LTBR overexpression in T cells, in conjunction with anti-CD19 CARs utilizing either CD28 or 4-1BB costimulatory domains in addition to CD3z signaling domain, increases cytokine (IFNγ and IL-2) secretion and target cell killing (B cell leukemia cell line Nalm6). We have also demonstrated that CAR T cells co-expressing LTBR have an increased expression of core genes that are activated by LTBR, for instance an adhesion molecule ICAM-1 (also known as CD54) and CD74, an invariant part of the MHC-II complex with proposed anti-apoptotic roles. Finally, we have shown that T cells overexpressing LTBR with or without a CAR, show a less differentiated. “younger” phenotype (predominantly central memory [CM], with reduced frequency of terminally differentiated effector cells) which is beneficial in context of cellular therapies.
Here, we inserted the intracellular part of LTBR (https://www.uniprot.org/uniprot/P36941, amino acids 249-435) within the intracellular domains of most commonly used CAR constructs to see if: 1) this insertion does not disrupt the CAR: 2) this insertion bestows similar phenotypic and functional benefit as expressing a full length LTBR as a separate gene in a multicistronic construct, and 3) this insertion boosts CAR activity above that of the CAR and LTBR delivered together but as separate proteins. To that effect we first engineered 3 constructs whereby the intracellular domain of LTBR is inserted into various position in a CAR construct identical to the one used in the FDA-approved axicabragene ciloleucel (here denoted as 19-28-z), specifically: 1) downstream of the CD28 stalk and transmembrane domains but upstream of CD28 signaling and CD3z signaling domains; 2) downstream of the CD28 signaling domain but upstream of CD3z signaling domain; and 3) downstream of both CD28 and CD3z signaling domains (
We first verified that the surface CAR expression is similar between these constructs, thus indicating that inserting LTBR intracellular domain did not have a profound effect on expression or protein stability of the CAR, and that the observed functional effects were not due to the expression level of the CAR (
Having established that some of the tested constructs not only phenotypically replicate the effects of full-length LTBR but are in fact superior, we then sought to determine if they also improve antigen-specific response of CAR T cells against CD19+ cancer cells. In terms of cytokine secretion, we observed that the 19-LTBR-28-z construct generally performed worse than the control (“regular” CAR T cells, i.e. 19-28-z+tNGFR) while 19-28-LTBR-z showed similar performance to the control. Most importantly, 19-28-zLTBR outperformed T cells expressing (separately) the CAR and full-length LTBR in terms of IFNg secretion and showed an improvement over control CAR T cells in terms of IL2 secretion (
While cytokine secretion (IFNg, IL2) is crucial for T cells to remodel the tumor microenvironment and stimulate T cell proliferation, another key aspect of anticancer activity is direct cytotoxicity, performed predominantly by CD8+ T cells. Therefore, we tested the ability of engineered T cells to kill CD19+ cancer cells. Interestingly, all CD28 LTBR constructs showed an improvement over control CAR at high T cell dose. More importantly, the 19-28-z-LTBR construct was also able to efficiently kill tumor cells at a low T cell dose, where T cells expressing 19-28-z CAR and full-length LTBR provided only a slim benefit over a regular CAR (
1st generation CAR constructs contained only the CD3z signaling domain attached to the target recognition modality. These 1st generation receptors were not efficient in vitro and especially in vivo because of lack of a costimulatory signal, thus necessitating the provision of costimulation via inclusion of CD28 or 4-1BB signaling domains in 2nd generation CARs (currently FDA approved). We wanted to assess if the presence of LTBR could improve the response of 1st generation CARs.
Thus, we designed constructs that possessed only the target recognition domain and transmembrane part of a CAR (generation 0)) as well as constructs that also contained the intracellular CD3z signaling domain (1st generation) and compared them to 2nd generation CARs utilizing the 4-1BB costimulatory domain (
19 + LTBR
19 + LTBR
When co-incubated with CD19+ leukemia cell line Nalm6, generation 0 CARs showed no response in terms of cytokine (IFNγ, IL2) secretion (
We have previously shown that LTBR overexpression improves T cell phenotype and function via ligand dependent and independent mechanisms. We wanted to assess whether the extracellular domains of LTBR can provide additional T cell activation via the same mechanisms (ligand binding or tonic signaling) when linked to signaling domains derived from 2nd generation CAR T cells.
To that effect we generated constructs that express the full-length extracellular domain of LTBR (LTBR: https://www.uniprot.org/uniprot/P36941, amino acids 1-248) including the transmembrane part, or utilize the stalk and transmembrane part from FDA-approved CARs-that is, CD28 stalk and transmembrane or CD8 stalk and transmembrane (LTBR: https://www.uniprot.org/uniprot/P36941, amino acids 1-227). In some cases, the extracellular domain of LTBR was linked to CD28 and CD3z or 4-1BB and CD3z signaling domains, as in FDA-approved CARs. In all iterations of the constructs, a matched NGFR fragment was used as a negative control (
We observed no cytokine (IFNγ, IL2) secretion when the T cells were incubated alone, indicating that ligand availability or tonic signaling mediated by LTBR were not sufficient to activate the cells. However, when the T cells were activated via their T cell receptor (CD3 and CD28 antibodies), we observed a strong increase in secretion of both IFNg and IL2 by T cells expressing a construct that contains the extracellular part of LTBR fused to the CD8 stalk and transmembrane domain, and 4-1BB and CD3z signaling domains (LTBR-CD8-BB-z) (
We have previously shown that LTBR overexpression improves T cell phenotype and function via ligand dependent and independent mechanisms. We wanted to assess whether the extracellular domains of LTBR can provide additional T cell activation via the same mechanisms (ligand binding or tonic signaling) when linked to signaling domains derived from 2nd generation CAR T cells.
To that effect we generated constructs that express the full-length extracellular domain of LTBR (LTBR: https://www.uniprot.org/uniprot/P3694 1, amino acids 1-248) including the transmembrane part, or utilize the stalk and transmembrane part from FDA-approved CARs-that is, CD28 stalk and transmembrane or CD8 stalk and transmembrane (LTBR: https://www.uniprot.org/uniprot/P36941, amino acids 1-227). In some cases, the extracellular domain of LTBR was linked to CD28 and CD3z or 4-1BB and CD3z signaling domains, as in FDA-approved CARs. In all iterations of the constructs, a matched NGFR fragment was used as a negative control (
We observed no cytokine (IFNg, IL2) secretion when the T cells were incubated alone, indicating that ligand availability or tonic signaling mediated by LTBR were not sufficient to activate the cells. However, when the T cells were activated via their T cell receptor (CD3 and CD28 antibodies), we observed a strong increase in secretion of both IFNg and IL2 by T cells expressing a construct that contains the extracellular part of LTBR fused to the CD8 stalk and transmembrane domain, and 4-1BB and CD3z signaling domains (LTBR-CD8-BB-z) (
The intracellular signaling domain of LTBR (https://www.uniprot.org/uniprot/P36941, amino acids 249-435*) fused to different components of the TCR complex.
In a similar manner as with using LTBR intracellular domain to modify αβ TCR complex. γδTCR complex can be modified too (
αβ T cells use CD4 to co-receive antigens presented by MHC-II (HLA-DR. HLA-DP, HLA-DQ) or CD8 (composed of CD8α and CD8b chains) to co-receive antigens presented by MHC-I (HLA-A, HLA-B, HLA-C). Thus, intracellular LTBR can be fused to the C-terminal intracellular tail of CD4 or to C-terminal intracellular tails of CD8a or CD8b to improve T cell function (
We have previously shown that co-delivery of LTBR with an anti-CD19 CARs into primary T cells boosts their activity against hematological cancers. See, e.g., Legut et al, Nature, (2022). We have shown similar results with co-delivery of LTBR with an anti-mesothelin CARs to boost T cell activity against a range of solid tumors. We have now expanded this application of LTBR co-delivery to an anti-B7-H3 CAR which is being tested clinically against a range of solid tumors, including but not limited to pediatric cancers, brain tumors, sarcomas and melanoma. See, e.g., Theruvath et al, Nature Medicine (2020).
When LTBR was co-delivered to primary human T cells together with an anti-B7-H3 CAR (MGA271-28-z), we observed (as before in cases of CD19 and mesothelin CARs) that LTBR had a positive impact on CAR T cell phenotype. Specifically. LTBR CAR T cells showed a predominantly central memory phenotype with practically no terminally differentiated effector cells, in contrast with CAR T cells co-expressing a control gene tNGFR (
In terms of antitumor efficacy. LTBR CAR T cells, both CD4 and CD8, showed a much stronger response to cancer cells expressing B7-H3 (including two atypical teratoid rhabdoid tumor lines BT12 and BT16, as well as a melanoma line A375) than control CAR T cells while remaining inert to a B7-H3-negative leukemia line Nalm6 (
Finally, we assessed the efficacy and safety of LTBR CAR T cells in a mouse xenograft models. We implanted A375 tumor into the flank of NSG mice and allowed it to grow for two weeks to be reminiscent of advanced solid tumors in human (
We have previously shown that fusion of the intracellular tail of LTBR to the C-terminus of CD19-targeting CARs, either utilizing CD28 or 4-1BB design (FMC6.3-28-z-LTBR or FMC6.3-BB-z-LTBR) results in CAR T cells that show superior antitumor potency and LTBR-driven phenotype than when the full-length CAR and full-length LTBR are co-expressed as separate molecules. Here we wanted to determine if truncations (either N- or C-terminal) of the LTBR signaling tail could result in a smaller but equally or more potent CAR construct than one incorporating the full-length LTBR signaling tail.
We began by annotating the LTBR intracellular tail based on the literature to identify key functional domains (
All CAR-LTBR fusions resulted in similar levels of CAR surface expression (
In terms of differentiation status of T cells, the LTBR truncation fusions showed a range of phenotypes, with certain variants (e.g., V7) resembling full length LTBR very closely while certain other variants (e.g., V4) more reminiscent of regular CAR T cells not expressing LTBR (
In addition to phenotypic measurements, we also assayed the antigen-specific antitumor response by co-incubating T cells expressing anti-CD19 CARs, with or without LTBR fusion, with CD19+ leukemia cells. To quantify that response, we measured the secretion of key antitumor cytokines IFNγ and IL2 (
We have previously shown that co-expression of full-length LTBR together with mesothelin-targeting CARs, utilizing both the 4-1BB and CD28 designs, boosts antitumor reactivity of CAR T cells as well as that fusing the intracellular tail of LTBR to CD19-targeting CARs, utilizing both the 4-1BB and CD28 designs, results in superior phenotype and function than co-expression of a regular CD19-targeting CAR and LTBR as two independent molecules.
To extend that observation, we fused the intracellular tail of LTBR to mesothelin-targeting CARs (see
When looking at the surface expression of the CAR, we observed, in contrast to our data in the CD19 system, that fusing the LTBR tail in any position within the mesothelin CAR resulted in a markedly decreased surface expression of the CAR (
To assess the functional impact of the reduced CAR expression upon fusion with LTBR, we measured key functional outputs of CAR-driven response to mesothelin-positive cancer cells: T cell proliferation, direct cancer cell killing, and secretion of key cytokines. In terms of antigen-driven proliferation, co-expression of the full-length LTBR resulted in the strongest expansion but CAR-LTBR fusions still outperformed the regular, unmodified CAR (
Given that CAR-specific functions were reduced upon fusion with the LTBR tail, we were wondering about the LTBR-specific functions of those constructs. When looking at key markers of LTBR activity, that is, surface expression of CD54 and CD74 (
To demonstrate the utility of using CAR-LTBR fusion as a superior LTBR-like molecule to be used in conjunction with an independent antigen receptor, we co-delivered natural LTBR or Meso-z-LTBR together with Meso-28-z CAR T to T cells (
In this co-delivery setting, CAR+Meso-z-LTBR T cells upregulated LTBR marker genes CD54 and CD74 to the similar extent as CAR+LTBR T cells (
We have previously shown that co-expression of full-length LTBR together with an NY-ESO-1 targeting αβ TCR boosts antitumor reactivity of engineered T cells as well as that fusing the intracellular tail of LTBR to CD19-targeting CARs, utilizing both the 4-1BB and CD28 designs, results in superior phenotype and function than co-expression of a regular CD19-targeting CAR and LTBR as two independent molecules.
To extend this observation, we fused the intracellular tail of LTBR to a or B chains of the TCR heterodimer, or δ, ε, γ or ζ chains of the CD3 complex. CD3 constitutively associates with the TCR and is required for its surface expression and downstream signaling (https://www.pnas.org/doi/10.1073/pnas. 1420936111).
To first test whether LTBR signaling tail can boost TCR activity as a direct fusion, we generated constructs that included LTBR intracellular tail attached to the C-terminus of the short intracellular tail of TCRα or TCRβ (
We first looked at whether LTBR fusion affects surface expression of the TCR. We noticed that LTBR fusion to TCR-β, but not TCR-α, strongly reduced its surface expression (
CD3 molecules are necessary for mediating TCR signaling. Therefore, we engineered fusions of all four CD3 polypeptides with LTBR signaling tail (
We have shown that co-expression of full-length LTBR together with an NY-ESO-1 targeting αβ TCR boosts antitumor reactivity of engineered T cells (WO 2023/279049) as well as that fusing the intracellular tail of LTBR to CD19-targeting CARs, utilizing both the 4-1BB and CD28 designs, results in superior phenotype and function than co-expression of a regular CD19-targeting CAR and LTBR as two independent molecules.
To extend this observation, we fused the intracellular tail of LTBR to CD8α or CD8β molecules (
In this experiment, CD8 T cells, expressing an endogenous CD8α-CD8β heterodimer, were sequentially transduced with an NY-ESO-1 targeting TCR as well as a modifier gene to be overexpressed: a control gene tNGFR, a full-length LTBR. CD8 (wild-type or fused with the LTBR signaling tail) or CD8β (wild-type or fused with the LTBR signaling tail) (
We first measured the surface expression of markers of LTBR activity, CD54 and CD74, as well as intracellular expression of a key transcription factor TCF1. As expected, NY-ESO-1 TCR-transduced T cells co-expressing LTBR exhibited a marked increase of expression of both surface markers and the transcription factor, compared with the control gene tNGFR (
Finally, we tested the impact of the CD8-LTBR fusion on the functional response of engineered T cells. NY-ESO-1 antigen is expressed on a variety of tumor types (Thomas et al, NY-ESO-1 Based Immunotherapy of Cancer Current Perspectives, Front. Immunol., 1 May 2018, Sec. Cancer Immunity and Immunotherapy, Volume 9-2018). To test the sensitivity and magnitude of response of LTBR engineered T cells to this antigen, we pulsed an HLA-A2+ cell line with different concentrations of the immunodominant peptide epitope derived from the NY-ESO-1 protein, SLLMWITQC. T cell response was measured as secretion of two key cytokines, IFNγ and IL2, after 24 h co-incubation of T cells with the peptide-presenting cell line (
We have previously shown that a pooled, barcoded lentiviral library containing >12,000 human genes can be used to discover new modulators of key T cell functions (Sec, e.g., Legut et al, Nature 2022). Here we show that engineering of the lentiviral vector backbone drastically boosts the functional titer of the library virus, improving the scalability of the approach. The improved ORF library vector (SEQ ID NO: 132) showed >5× increase over the original vector in terms of the transduction efficiency, making it possible to reach ˜30% transduction rate (which is desirable for pooled screening) with as little as 50 μl lentivirus (
All publications cited in this specification are incorporated herein by reference. U.S. Provisional Patent Application No. 63/320,100 filed Mar. 15, 2022 is incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.
This invention was made with government support under R00HG008171, DP2HG010099, and R01CA218668 awarded by the National Institutes of Health and D18AP00053 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/064452 | 3/15/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63320100 | Mar 2022 | US |
