CN115461061A - Genetically modified T cells and uses thereof - Google Patents

Abstract

The disclosure herein includes T cells genetically modified to express recombinant Chimeric Antigen Receptors (CARs) and recombinant chimeric co-stimulatory receptors (CCR), pharmaceutical compositions comprising the T cells, methods of making the T cells, and methods of treating cancer or tumors using the T cells. The CAR may comprise an extracellular domain comprising an extracellular domain of the NKG2D receptor, a transmembrane domain, and an intracellular signaling domain, and not a costimulatory signaling domain. The CCR may comprise an extracellular domain comprising a PD-L1 specific single-chain variable fragment, a transmembrane domain, a costimulatory signaling domain, and no intracellular signaling domain.

Description

Genetically modified T cells and uses thereof

Technical Field

The present invention relates generally to the fields of biotechnology and cell therapy. In particular, the present invention relates to genetically modified T cells for cancer immunotherapy, methods for their preparation and use in patients in need thereof.

Background

Adoptive transfer of T lymphocytes bearing Chimeric Antigen Receptors (CARs) is very effective in eliminating the substantial burden of certain types of hematological cancers and has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of B cell leukemia. The CAR constructs combine the specific and high affinity binding functions of B cell secreted monoclonal antibodies with the intracellular signaling domain of the T Cell Receptor (TCR) and the cytotoxic ability of T cells to redirect T cells to cancer cells and achieve efficient killing of target cells. Activation of T cells by CARs is independent of TCR interaction with Major Histocompatibility Complex (MHC) -peptide complex, overcoming immune evasion mechanisms often used by tumor cells, such as down-regulation of MHC class I molecules and the development of antigen processing defects.

The first generation CARs consisted of an extracellular tumor-specific domain (e.g., a single chain variable fragment (scFv) from a monoclonal antibody), a transmembrane domain, and an intracellular signaling moiety for T cell activation. Despite the tremendous efforts, the first generation of CAR-T cell studies did not reach the expected clinical outcome, mainly because CAR-T cells are poorly proliferated, resulting in short in vivo life span and insufficient tumor killing.

Complete activation of T cells requires two signals. The first signal is antigen-specific and is provided by a T Cell Receptor (TCR) that interacts with an antigenic peptide-MHC complex on the surface of an antigen presenting cell. The second signal is antigen-nonspecific, being transmitted by the interaction between costimulatory molecules expressed on antigen presenting cells and costimulatory receptors on T cells. One such example is the interaction of B7 ligand with the co-stimulatory receptor CD28; the interaction between 4-1BB ligand (CD 137L) and 4-1BB (CD 137) receptor is another example. The costimulatory signals promote the synthesis and secretion of cytokines produced by the antigen presenting cells and/or the activated T cells themselves. The second signal is crucial for proper T cell activation without anergy, and can promote long-term proliferation of activated T cells. In view of the important role of dual signaling on T cell activation, second generation CARs include intracellular co-stimulatory moieties, such as those from CD28 and 4-1BB receptors, and significantly improve the function of modified CAR-T cells, particularly the in vivo proliferation and persistence of CAR-T cells, making these CAR-T cells a "true live drug" in patients.

Although adoptive transfer of CAR-T cells constitutes a very promising therapeutic strategy for leukemia, the transformation of this approach for non-hematologic malignancies is challenging due to the specific pathophysiological characteristics of solid tumors. Major limitations of clinical CAR-T cell technology against solid tumors include factors affecting target antigen selection (e.g., tumor heterogeneity and antigen-loss variants), intrinsic negative regulatory mechanisms of the Tumor Microenvironment (TME) (e.g., PD-L1/PD-1 signaling pathways), and off-tumor (on-target) toxicity at the target.

The programmed cell death 1 (PD-1) receptor (CD 279) is an inhibitory immune receptor that plays an important role as an immune checkpoint, especially in T cell co-suppression and depletion. The interaction between PD-1 and its cognate ligand, PD-L1, is one of the major processes used by many tumors for immunosuppression and evasion. PD-1 is expressed on activated lymphocytes and has been shown to negatively modulate antigen receptor signaling upon binding to PD-L1 expressed on tumor cells, resulting in impaired immune cell effector functions such as cell proliferation, cytokine secretion and reduced tumor cell lysis. Using PD-L1 detection antibodies and immunohistochemical staining, high PD-L1 expression in tumor cells has been found to correlate with poor prognosis in terms of overall survival of various human cancers. Thus, PD-L1 expression has been introduced into clinical practice as a biomarker for selecting PD-1/PD-L1 antibody therapy responders. The use of monoclonal antibodies (mabs) to modulate PD-1/PD-L1 interactions is a method known as "immune checkpoint blockade" that has produced a good clinical response and improved overall survival in melanoma, renal cell carcinoma, non-small cell lung cancer and other tumor patients. However, PD-1mAb treatment may lead to immune-related adverse events (irAE), which may be serious or even fatal. The most serious adverse event of PD-1mAb treatment was pneumonia, which led to the death of 3 people in an early study.

The present invention addresses some of the above-mentioned shortcomings in the field of Chimeric Antigen Receptor (CAR) modified T lymphocytes for the treatment of cancer, in particular solid tumors.

Disclosure of Invention

In one aspect, the invention discloses a T cell genetically modified to express: i) A recombinant Chimeric Antigen Receptor (CAR) comprising (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a costimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of the NKG2D receptor; and ii) a recombinant Chimeric Costimulatory Receptor (CCR) comprising (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, (c) a costimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-L1 specific single-chain variable fragment (scFv).

In another aspect, the invention discloses a pharmaceutical composition comprising a pharmaceutically effective amount of the T cells of the invention, and a pharmaceutically acceptable excipient.

In another aspect, the invention features a method of making a T cell of the invention, the method comprising: a) Obtaining or providing a T cell; b) Providing i) a recombinant nucleic acid encoding a recombinant CAR; and ii) a recombinant nucleic acid encoding a CCR; and c) transferring the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the CCR into a T cell.

In yet another aspect, the invention discloses a method of treating cancer or tumor in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a T cell or pharmaceutical composition of the invention.

In a further aspect, the present invention discloses a method of treating a cancer or tumor in a subject in need thereof, the method comprising: a) Obtaining T cells from the subject or a donor different from the subject to be treated; b) Providing i) a recombinant nucleic acid encoding a recombinant Chimeric Antigen Receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a costimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of the NKG2D receptor; and ii) a recombinant nucleic acid encoding a recombinant Chimeric Costimulatory Receptor (CCR), wherein the recombinant CCR comprises (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, and (c) a costimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-1L1 specific single-chain variable fragment (scFv); c) Transferring a recombinant nucleic acid encoding a recombinant CAR and a recombinant nucleic acid encoding a recombinant CCR into a T cell to obtain a genetically modified T cell; and d) administering to the subject a pharmaceutically effective amount of the T cells obtained from c).

Drawings

The invention will be better understood with reference to the detailed description when considered in connection with non-limiting embodiments and the accompanying drawings, wherein:

figure 1 shows a schematic of the chimeric receptors described in the present application. Figure 1A shows that the first generation NKG2D CARs were generated by fusing the extracellular domain of the NKG2D receptor with an activation domain. The activation domain is derived from CD3 ζ or DAP12. FIG. 1B shows that α PD-L1 CCR was generated by fusion of a PD-L1 specific scFv to a single costimulatory signaling domain. Figure 1C shows that the PD-1 Chimeric Switch Receptor (CSR) is generated by fusing the extracellular domain of the PD-1 inhibitory receptor with a single costimulatory signaling domain. In FIGS. 1B and 1C, the co-stimulatory signaling domain is derived from 4-1BB or CD28. FIG. 1D shows that two NKG2D CAR-based combinations described in this application were generated by pairing a first generation NKG2D CAR with either alpha PD-L1 CCR (CAR-T1) or PD-1 CSR (CAR-T2). CAR, chimeric antigen receptor; CCR, chimeric costimulatory receptor; CSR, chimeric switch receptor.

FIG. 2 shows bar graph results of cytotoxicity assays, demonstrating that CAR-T1 and CAR-T2 modified T cells exhibit dose-dependent cytotoxicity against a panel of cancer cell lines. HepG2 (NKG 2DL +), cav 3 (NKG 2DL +, PD-L1 +) and Detroit-562 (NKG 2DL +, PD-L1+, PD-L2 +) cancer cells were used as target cells in a standard 2-hour Delfia time-resolved cytotoxicity assay. CAR-T1 and CAR-T2 modified T cells were seeded with target cells at different E: T ratios of 20 and 10. Both CAR-T1 and CAR-T2 show a dose-dependent increase in cytotoxicity against target cells. No significant difference was observed in cytotoxicity of CAR-T1 and CAR-T2 on all three cell lines (NS, p > 0.05). T, effector and target. Data shown are mean ± SD of triplicates, representative of three independent experiments. Statistical analysis was performed by two-way analysis of variance followed by Tukey's multiple comparisons.

Figure 3 antigen-dependent expansion of nkg2d CAR-T cells was further enhanced by α PD-L1 CCR. CAR-T1 and CAR-T2 cells were expanded with K562 myeloid leukemia cells expressing NKG2D ligand and PD-1 ligand. A batch of fresh gamma irradiated K562 feeder cells was used every 10 days with an E: T ratio of 2. Data shown are the number of cells obtained from trypan blue exclusion assay on days 17, 27, and 37 after electroporation of DNA from one donor, representing three independent experiments.

FIG. 4. Incorporation of α PD-L1 CCR enhances the development of CCR7-/CD45 RA-responsive memory T cells. The CAR-T1 and CAR-T2 groups were characterized for memory T development by detection of CCR7 and CD45RA antigens. (A) The proportion of CCR7-CD45RA + effector T cells in CAR-T1 and CAR-T2 in the middle and end stages of the expansion phase. (B) The proportion of CCR7-CD45 RA-effector memory cells in CAR-T1 and CAR-T2 in the middle and end stages of expansion. Data shown are single measurements from one donor, representative of three independent experiments.

Figure 5. Incorporation of α PD-L1 CCR attenuated T cell depletion marker expression. (a) single endpoint measurement from three donors: at the end of the amplification phase depicted in FIG. 3, expression levels of PD-1, TIGIT, TIM3, and LAG3 were assessed on CAR-T1 and CAR-T2 cells. (B) Summary mean of expression levels of each of the depletion markers classified according to CAR panel.

car-T cell therapy eliminated established tumors in the mouse xenograft model. Four groups of mice (5 mice per group) received 2X 10 ⁶ Intraperitoneal injection of individual HCT116-Luc human colorectal cancer cells (day 0), followed by intraperitoneal injection of PBS, control CAR-T cells, T cells modified with NKG2D CAR + PD-1 CSR, or T cells modified with NKG2D CAR + α PD-L1 CCR on days 7 and 32 (1 × 10 injections per mouse per time) ⁷ Individual CAR-T cells). Growth of HCT116 was monitored by bioluminescence imaging on the indicated date. A bioluminescent image is displayed.

car-T cell treatment significantly prolonged survival of mice vaccinated with human cancer cells. In the mouse xenograft model described in fig. 6, animal survival was monitored up to 150 days after tumor inoculation and analyzed by Kaplan-Meier method.

Figure 8 shows a schematic of the design concept of constructs used to modify T cells as described in the present disclosure.

Definition of

"genetically modified cell" refers to any cell of any organism that is modified, transformed or manipulated by the addition or modification of a gene, DNA or RNA molecule, or protein or polypeptide.

A "T cell" is a lymphocyte that develops in the thymus and plays a central role in the immune response. T cells can be distinguished from other lymphocytes by the presence of T cell receptors on the cell surface. These immune cells originate from bone marrow-derived precursor cells, and once they migrate into the thymus, develop into several different types of T cells. T cells have been initially classified into a series of subpopulations according to their function, but have also been classified into subpopulations according to the expression pattern of the relevant gene or protein. The T Cell Receptor (TCR) is a molecule found on the surface of T cells, responsible for recognizing antigenic fragments as peptides bound to Major Histocompatibility Complex (MHC) molecules. The TCR consists of two distinct protein chains (i.e., it is a heterodimer). In humans, in most T cells, the TCR consists of an alpha chain and a beta chain (encoded by TRA and TRB, respectively), and is therefore referred to as an α β T cell. In a small fraction of T cells, the TCR consists of gamma and delta chains (encoded by TRG and TRD, respectively), and is therefore termed a gamma delta T cell. For different classes of T cells, the capacity to differentiate and proliferate increases in the following order: effector T cells, effector memory T cells, central memory T cells, stem cell memory T cells. Instead, their effector functions, such as cell-mediated cytotoxicity and cytokine release, decrease in the same order. The clinical efficacy of CAR-T cells is directly related to the ability to proliferate and persist in vivo. Thus, the higher memory order allows CAR-T cells to persist in the patient for a longer period of time, thereby exerting a more durable anti-tumor activity.

As used herein, the term "chimeric antigen receptor" or abbreviated form "CAR" refers to an artificial receptor protein or chimeric immune receptor and includes engineered receptors that specifically engraft the artificial onto specific immune effector cells. CARs can be used to provide T cells with the specificity of a monoclonal antibody, so that a large number of specific T cells can be generated, e.g., for adoptive cell therapy. The CAR typically comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. CARs can combine antibody-based specificity for a desired antigen with an intracellular domain that activates a T cell receptor to produce a chimeric protein with specific cellular immune activity (e.g., anti-tumor cellular immune activity). In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging, gene products that conditionally clear NK cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

As used herein, the term "chimeric co-stimulatory receptor" or abbreviated form "CCR" refers to an artificial receptor protein or chimeric immunoreceptor that contributes to the complete activation of a particular immune effector cell, such as a T cell. For example, T cells require two signals to be fully activated, the first being an antigen-specific signal provided by the T Cell Receptor (TCR), and the second being an antigen-non-specific costimulatory signal provided by the interaction between the costimulatory molecule and the costimulatory receptor. The CCR typically comprises an extracellular domain containing an antigen binding region, a transmembrane domain, a costimulatory signaling domain, but does not comprise an intracellular activation domain. Thus, CCR alone does not result in complete activation and production of immune effector cells. CCR must generally be used with another recombinant receptor containing an intracellular activation domain (e.g., CAR) in order to fully activate immune effector cells.

As used herein, the term "antigen" is a molecule capable of being bound by an antibody or cell surface receptor. Antigens are commonly used to induce a humoral immune response and/or a cellular immune response leading to the production of lymphocytes.

Natural killer group 2 member D, also known as Klrk1 (NKG 2D), is a C-type lectin-like receptor that was first identified in NK cells as activating immune receptors. NKG2D is a type II transmembrane glycoprotein that does not contain any known signaling elements in the intracellular domain. Like many activation receptors, NKG2D relies on adaptor molecules to initiate signal transduction and cell activation. In humans, NKG2D is expressed not only by all NK cells, but also by all CD8 ⁺ T cells and gamma delta ⁺ A subset of T cells express as co-stimulatory receptors. NKG2D expression and signaling can be regulated by cytokines and tumor-derived factors. Cytokines such as IL-2, IL-7, IL-12, IL-15 and type I Interferon (IFN) increase the cell surface expression of NKG 2D. Cytokines such as IL-21, IFN- γ, and TGF- β have been shown to reduce NKG2D expression. IL-21 reportedly reduces human CD8 ⁺ Expression of NKG2D in T cells and NK cells. In mice, IL-21 stimulation of NK cells is dependent on modulating the expression of NKG2D in mouse models of breast cancer.

Programmed death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD 274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene in humans. PD-L1 is a 40kDa type 1 transmembrane protein which acts as a ligand for PD-1. Binding of PD-L1 to its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. Binding of PD-L1 to the inhibitory checkpoint molecule PD-1 delivers an inhibitory signal based on interaction with a phosphatase (SHP-1 or SHP-2) through an immunoreceptor tyrosine-based switching motif (ITSM). This reduces the proliferation of antigen-specific T cells in the lymph nodes, while reducing apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells) -further mediated by lower regulation of the gene Bcl-2. Upregulation of PD-L1 may allow cancer to evade the host immune system.

Programmed cell death protein 1 in humans, also known as PD-1 and CD279 (cluster of differentiation 279), is encoded by the PDCD1 gene. PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily, expressed on T cells and pre-B cells. PD-1 binds two ligands, PD-L1 and PD-L2.PD-1 regulates the immune system's response to human cells by down-regulating the immune system and promotes self-tolerance by inhibiting T cell inflammatory activity. PD-1 is an immune checkpoint that prevents autoimmunity by two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells). Exemplary sequences of the extracellular domain of PD-1 are (from N-terminus to C-terminus): <xnotran> PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 1), : </xnotran> <xnotran> CCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTG (SEQ ID NO: 2). </xnotran>

As used herein, the term "single chain variable fragment" or "scFv" refers to the variable region of the heavy chain (V) of an immunoglobulin _H ) And light chain variable region (V) _L ) The fusion protein of (3), linked by a short linker peptide of 10 to about 25 amino acids. The linker is generally glycine rich for increased flexibility and serine or threonine rich for increased solubility, and V may be substituted _H N-terminal of (1) and V _L Are linked and vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original immunoglobulin.

The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, which may be deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, may be made before or after polynucleotide assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise specified or required, a polynucleotide includes both the double-stranded form and each of the two complementary single-stranded forms known or expected to constitute the double-stranded form.

As used herein, the term "recombinant nucleic acid" refers to a nucleic acid formed by the laboratory process of genetic recombination (e.g., molecular cloning) to bring together genetic material from multiple sources. The nucleic acid sequence used to construct the recombinant nucleic acid molecule may be derived from any species. For example, human nucleic acids can bind to bacterial nucleic acids. In addition, nucleic acid sequences not found in nature can be produced by chemical synthesis of nucleic acids and incorporated into recombinant molecules. Proteins that can be produced by the expression of recombinant nucleic acids in living cells are referred to as recombinant proteins. When a recombinant nucleic acid encoding a protein is introduced into a host organism, it is not necessary to produce the recombinant protein. Expression of foreign proteins requires the use of specialized expression vectors and often requires significant recombination by foreign coding sequences.

As used herein, the term "vector" refers to an extrachromosomal nucleic acid comprising an intact replicon, such that the vector can be replicated when placed (e.g., by a transformation process) within a permissive cell. A vector can replicate in one cell type (e.g., bacteria), but has a limited ability to replicate in another cell (e.g., mammalian cell). The vector may be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers (such as heterogeneous polylysines, fixed length oligopeptides, and polyethylene imines), in some cases in liposomes; and the use of ternary complexes comprising virus and polylysine-DNA.

As used herein, the term "transfer" or "transfection" refers to the general process of introducing an exogenous nucleic acid into a host cell, which may be mechanical transfection (including electroporation), chemical transfection or viral transduction. A "transfected" cell is a cell that has been transfected with exogenous nucleic acid using any of the methods described above. Cells include primary subject cells and their progeny.

As used herein, the term "autologous" means any material derived from the same individual that is subsequently reintroduced into the individual.

As used herein, the term "allogenic" refers to any material derived from an individual that is different from the individual into which it is subsequently introduced.

As used herein, "percent identity" refers to sequence identity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing the position in each sequence, and the sequences can be aligned for comparison purposes (align). When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to a compound having amino acid residues covalently linked by peptide bonds. The protein or peptide must comprise at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise the protein or peptide sequence. Polypeptides include any peptide or protein having two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains (also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (commonly referred to in the art as proteins), each of many types. "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

As used herein, the phrase "homologous" or "variant" nucleotide sequence, or "homologous" or "variant" amino acid sequence, refers to a sequence characterized by at least a specified percentage of identity at the nucleotide or amino acid level. Homologous nucleotide sequences include those sequences that encode naturally occurring allelic variants and mutations of the nucleotide sequences described herein. Homologous nucleotide sequences include nucleotide sequences encoding proteins of mammalian species other than humans. Homologous amino acid sequences include those amino acid sequences that contain conservative amino acid substitutions and the polypeptides have the same binding and/or activity. In some examples, a homologous nucleotide or amino acid sequence has at least 60% or more (e.g., at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% or at least 99%) identity to a comparative sequence. In some examples, a homologous nucleotide or amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a comparative sequence. In some examples, a homologous amino acid sequence has no more than 15, or no more than 10, or no more than 5, or no more than 3 conservative amino acid substitutions. Percent identity can be determined using default settings, for example, by the Gap program (Wisconsin sequence analysis software package, UNIX version 8, genetics Computer group, university Research park, madison Wis.) using the algorithms of Smith and Waterman. In some examples, recombinant nucleic acid molecules for modifying T cells according to the present disclosure are homologous to exemplary nucleotide sequences disclosed herein (e.g., sequences provided in any of SEQ ID NOs: 24, 26, 28, and 30). In some other examples, the Chimeric Antigen Receptor (CAR) and/or chimeric co-stimulatory receptor (CCR) expressed in the modified T cell is homologous to an exemplary amino acid sequence disclosed herein (e.g., a sequence provided in any of SEQ ID NOs: 23, 25, 27, and 29).

The term "expression" refers to the production of a gene product in a cell.

The term "transient" when referring to expression means that the polynucleotide is not incorporated into the genome of the cell. Conversely, when referring to expression, the term "stable" means that the polynucleotide is incorporated into the genome of the cell. Transient expression may occur from an introduced construct that contains expression signals that are functional in the host cell, but that does not replicate and integrate little into the host cell, or that does not proliferate. Transient expression can also be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest, although such inducible systems often exhibit low basal expression levels. Stable expression can be achieved by introducing a nucleic acid construct that can be integrated into the host genome or autonomously replicated in the host cell. Stable expression of a gene of interest can be selected by using a selectable marker located on or transfected with the expression construct, and then by selecting cells that express the marker. Where stable expression results from integration, integration of the construct may occur randomly within the host genome, or may be targeted by use of such constructs: the construct contains a region of homology to the host genome sufficient to target recombination with the host locus. When the construct is targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions may be provided by the endogenous locus. To achieve expression in a host cell, the transformed nucleic acid is operably associated with transcriptional and translational initiation and termination regulatory regions that function in the host cell.

As used herein, the term "tumor" refers to a lump in a part of the body caused by abnormal growth of tissue, usually without inflammation. Tumors can be benign or malignant (i.e., cancerous). Benign tumors do not infiltrate nearby tissue or spread to other parts of the body. Common types of benign tumors include adenomas, fibroids, hemangiomas, lipomas, meningiomas, myomas, neuromas and osteochondromas. Adenomas are benign tumors of epithelial tissue that begin in glandular or adenoid structures. A common type of adenoma is colonic polyps. Adenomas may also grow in the liver or in the adrenal gland, pituitary gland or thyroid gland. Fibroids are tumors of fibrous or connective tissue that can grow in any organ. Hemangiomas are accumulations of vascular cells in the skin or internal organs. Lipomas arise from the growth of adipocytes. They are the most common benign tumors in adults, often found in the neck, shoulders, back or arms. Meningiomas are tumors that develop from membranes surrounding the brain and spinal cord. Myomas are tumors that grow from muscle. Neuroma is a tumor that develops from a nerve. Osteochondrosis is a tumor that develops from bone.

As used herein, the term "cancer" refers to uncontrolled cellular proliferation due to loss of normal control, resulting in uncontrolled growth, lack of differentiation, local tissue infiltration, and often metastasis. There are several major types of cancer. Cancer is cancer that begins in the skin or tissues lining or covering internal organs. Sarcomas are cancers that begin in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that begins in hematopoietic tissues such as bone marrow, resulting in the production and entry of large numbers of abnormal blood cells into the blood. Lymphomas and multiple myeloma are cancers that begin with cells of the immune system. Central nervous system cancer is a cancer of tissues that begin in the brain and spinal cord.

Detailed Description

The inventors of the present application found that after genetic modification of T cells to express a recombinant CAR comprising the extracellular domain of the NKG2D receptor and a recombinant CCR specifically targeting PD-Ll, the genetically modified T cells facilitate more precise cancer targeting by exhibiting specificity for cancer cells expressing both PD-L1 and NKG2D ligands, thereby minimizing the risk of on-target tumor removal associated with CAR-T cell therapy. The inventors have also found that such genetically modified T cells can evade major immunosuppressive mechanisms within the Tumor Microenvironment (TME).

Thus, in one example, the invention relates to a T cell genetically modified to express: i) A recombinant Chimeric Antigen Receptor (CAR) comprising (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a costimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of the NKG2D receptor; and ii) a recombinant Chimeric Costimulatory Receptor (CCR) comprising (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, (c) a costimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-L1 specific single-chain variable fragment (scFv).

In some examples, the extracellular domain of the recombinant CAR is the extracellular domain of the NKG2D receptor. In one example, the extracellular domain of the NKG2D receptor has the following amino acid sequence (from N-terminus to C-terminus): FNQEVPLTESYCGPCPKNWICYKNNCYQFDFDESKNWYESQASCMSQNASSKLVYSKEDQDLLKLVGS YHWMGLVHIPTGQWQWGSILNLLTIIEMQKGDCALYASSFKGYIIENCSTPNTYICQRRAS (SEQ ID NO: 3) encoded by the following exemplary nucleotide sequences: <xnotran> TTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGTATGCAAAGGACTGTGGCTAGC (SEQ ID NO: 4). </xnotran> NKG2D ligands are structural homologues of MHC class I molecules. NKG2D ligands are absent or poorly expressed in normal tissues, but are widely expressed in various malignant and virally infected tissues. Examples of human NKG2D ligands include class I chain-associated molecules a and B (MICA and MICB) proteins and retinoic acid early transcript-1 (RAET 1), also known as UL-16 binding protein. Examples of mouse NKG2D ligands include five different RAET1 subtypes (RAET 1 α, RAET1 β, RAET1 γ, RAET1 δ, and RAET1 ∈), three different H60 subtypes (H60 a, b, and c), and UL16 binding protein 1 (encoded by the MULT1 gene). Although NKG2D ligands are structural homologues of MHC class I molecules, they do not present antigen to T cells or bind β 2-microglobulin. In some examples, the NKG2D ligand to which the extracellular domain of the chimeric antigen receptor binds is a membrane-bound ligand.

In some examples, the extracellular domain of the recombinant CCR is a PD-L1 specific scFv, referred to as an α PD-L1 scFv. In a specific example, the PD-L1 scFv has the following amino acid sequence (from N-terminus to C-terminus): <xnotran> QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGWITAYNGNTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCARDYFYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIKAS (SEQ ID NO: 5), : </xnotran> <xnotran> CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCGACTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCACCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGTCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGACTACTTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGTCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAGCTAGC (SEQ ID NO: 6). </xnotran> In a specific example, the heavy chain variable domain of the PD-L1 scFv has the followingAmino acid sequence (from N-terminus to C-terminus): QVQLVQSGAEVKKPGASVKSGYTFTDYGVFVRQACPGQGLEW WITAYNYNTTNYAQKLQGRVTMTTSTSTSTVYMRSLRSDAVYYCARDYFYGMDVWGQGTTVSS (SEQ ID NO: 7), consisting of the following exemplary nucleotide coding sequences: <xnotran> CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCGACTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCACCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGTCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGACTACTTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 8). </xnotran> In a particular example, the light chain variable domain of the PD-L1 scFv has the following amino acid sequence (from N-terminus to C-terminus): <xnotran> EIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK (SEQ ID NO: 9), : </xnotran> <xnotran> GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGTCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO: 10). </xnotran> The heavy chain variable domain and the light chain variable domain of the PD-L1 scFv may be linked by a linker peptide. In a specific example, the heavy chain variable domain and the light chain variable domain of the PD-L1 scFv are encoded by a sequence of amino acids (from N-terminus to C-terminus) (G4S) ₃ Linker peptide ligation: GGGGSGGGGSGGGGS (SEQ ID NO: 11), encoded by the following exemplary nucleotide sequence: GGTGGAGGGCGGTTCAGGCGGAGGGTGGCAGCGGCGGTGGCGGGTCG (SEQ ID NO: 12).

The extracellular domain of the recombinant Chimeric Antigen Receptor (CAR) and/or the recombinant Chimeric Costimulatory Receptor (CCR) may also comprise a hinge region. A hinge region is a sequence located, for example, between an antigen binding region and a transmembrane domain. The sequence of the hinge region may be obtained from any suitable sequence, for example from any species, including human or parts thereof. In some examples, the hinge region comprises a hinge region of a human protein comprising CD3, CD-8 α, CD28, 4-1BB, OX-40, a T cell receptor α or β chain, a CD3zeta chain, CD28, CD3 ε, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof. In some embodiments, the hinge region is a hinge region of CD3, CD8, or CD28. In some examples, the hinge region of the recombinant CAR and the hinge region of the recombinant CCR are the same. In some other examples, the hinge region of the recombinant CAR and the hinge region of the recombinant CCR are different. In some examples, the hinge region may be one selected from, but not limited to, an immunoglobulin (e.g., igG1, igG2, igG3, igG4, and IgD).

It will be appreciated that the antigen binding region may include some variability within its sequence and still be selective for the targets disclosed herein. Thus, it is contemplated that the polypeptides of the antigen binding region may be at least 95%, at least 90%, at least 80%, or at least 70% identical to the antigen binding region polypeptide sequences disclosed herein and still be selective for the targets disclosed herein and are within the scope of the present disclosure.

The transmembrane domain of the recombinant CAR and/or recombinant CCR includes a hydrophobic polypeptide that spans the cell membrane. In particular, the transmembrane domain spans from one side of the cell membrane (extracellular) to the other side of the cell membrane (intracellular or cytoplasmic).

In some examples, the transmembrane domain is artificially designed such that more than 25%, more than 50%, or more than 75% of the amino acid residues of the domain are hydrophobic residues, such as leucine and valine.

The transmembrane domain may be in the form of an alpha helix or a beta barrel, or a combination thereof. The transmembrane domain may comprise a polytypic (polytopic) protein having a number of transmembrane segments, each a-helix, a β -sheet, or a combination thereof.

In one example, a transmembrane domain that is naturally associated with one of the domains in the CAR or CCR is used. In another example, the transmembrane domains are selected or modified by amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

For example, transmembrane domains include transmembrane domains of T cell receptor alpha or beta chains, CD3zeta chains, CD3 epsilon, CD8, CD45, CD4, CD5, CD7, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD68, CD134, CD137, ICOS, CD41, CD154, functional derivatives thereof, and combinations thereof. In some examples, the transmembrane domain of the recombinant CAR and the transmembrane domain of the recombinant CCR are the same. In some other examples, the transmembrane domain of the recombinant CAR and the transmembrane domain of the recombinant CCR are different. In a specific example, the transmembrane domain of the recombinant CAR is a CD28 transmembrane domain. In a specific example, the transmembrane domain of the recombinant CAR has the following amino acid sequence (from N-terminus to C-terminus): ESKYGPPCPPCPFWVLVVGGVLLACYSLLVTVAFIIFWV (SEQ ID NO: 13), encoded by the following exemplary nucleotide sequence: <xnotran> GAATCTAAATATGGCCCCCCTTGCCCACCATGTCCCTTCTGGGTTTTGGTAGTTGTCGGCGGGGTGCTTGCTTGCTATTCATTGTTGGTTACGGTGGCTTTTATAATTTTCTGGGTA (SEQ ID NO: 14). </xnotran> In a specific example, the transmembrane domain of the recombinant CCR is a CD8 transmembrane domain. In one particular example, the transmembrane domain of the recombinant CCR has the following amino acid sequence (from N-terminus to C-terminus): <xnotran> FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 15), : </xnotran> <xnotran> TTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAAC (SEQ ID NO: 16). </xnotran>

The intracellular signaling domain of the recombinant CAR is responsible for activating at least one of the normal effector functions of the immune cell into which the recombinant CAR has been placed. The term "effector function" refers to a specialized function of a differentiated cell, such as a T cell. The intracellular signaling domain typically includes at least one domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). Each ITAM has two repeats of the consensus sequence Tyr-X-X-Leu/Ile (X is any amino acid) 6 to 8 amino acids apart. Tyrosine residues in ITAMs are phosphorylated upon interaction of the receptor molecule with its ligand and form docking sites for other proteins involved in the cell signaling pathway. In some examples, the intracellular signaling domain of the recombinant CAR comprises only one ITAM, or no more than one ITAM. In some examples, a T cell genetically modified to express a recombinant CAR comprising only one ITAM in the intracellular signaling domain releases a lower level of a cytokine as compared to a T cell expressing a recombinant CAR comprising more than one ITAM in the intracellular signaling domain. In some examples, the intracellular signaling domain in the CAR comprises part or all of CD3 ζ, or a DAP molecule such as DAP12, or a combination thereof. In a specific example, the intracellular signaling domain used is CD3 ζ. In one particular example, the intracellular signaling domain used is DAP12. In a specific example, the intracellular signaling domain of the CAR has the following amino acid sequence (from N-terminus to C-terminus): YFLRLVPRGGAAAATRKQRTETTESPTYQELQGQRQRSVYSDLNTQRPYK (SEQ ID NO: 17), encoded by the following exemplary nucleotide sequence: <xnotran> TACTTCCTGGGACGACTTGTTCCTCGGGGGCGGGGTGCTGCAGAAGCTGCAACGCGAAAACAAAGAATTACCGAGACTGAGAGTCCGTATCAAGAACTGCAGGGCCAAAGAAGCGACGTTTACTCCGATCTCAATACCCAACGGCCTTACTACAAA (SEQ ID NO: 18). </xnotran>

In some examples, the recombinant CARs disclosed herein do not comprise a costimulatory signaling domain, e.g., OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; natural killer group 2 member C (NKG 2C); natural killer group 2 member D (NKG 2D), B7-H3; a ligand that binds at least one of CD83, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS and 4-1BB (CD 137); CDS; ICAM-1; LFA-1 (CD 1a/CD 18); CD40; CD27; CD7; B7-H3; NKG2C; PD-1; ICOS; an active fragment thereof; a functional derivative thereof; and combinations thereof.

In some examples, a spacer domain is incorporated between the extracellular domain and the transmembrane domain of the recombinant CAR and/or recombinant CCR, or between the intracellular signaling domain and the transmembrane domain of the recombinant CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an extracellular domain or an intracellular domain in a polypeptide chain. The spacer domain may comprise up to about 300 amino acids, or about 10 to about 100 amino acids, or about 25 to about 50 amino acids.

In a specific example, a recombinant CAR desired in a genetically modified T cell comprises an NKG2D receptor, a CD28 transmembrane domain, and a DAP12 intracellular signaling domain. In a specific example, a desired recombinant CCR in a genetically modified T cell comprises an alpha PD-L1 scFv, a CD8 transmembrane domain, and a 4-1BB costimulatory signaling domain.

The terms "CD3zeta", "CD3z" are used interchangeably herein to refer to the same T cell surface glycoprotein CD3zeta chain.

The costimulatory signaling domain of the recombinant CCR can enhance T cell proliferation, survival, and/or development. Examples of co-stimulatory signaling domains include, but are not limited to: 4-1BB, CD27, CD28, OX-40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, functional derivatives thereof, and combinations thereof. In a specific example, the co-stimulatory signaling domain is 4-1BB.4-1BB, also known as CD137, is a member of the Tumor Necrosis Factor (TNF) receptor family. In one particular example, the co-stimulatory signaling domain of the CCR has the following amino acid sequence (from N-terminus to C-terminus): RFSVVKRRKKLLYFKQPFMRPVQTTQEEDGCSCRAFEEEGGCEL (SEQ ID NO: 19), encoded by the following exemplary nucleotide sequence: <xnotran> CGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 20). </xnotran> In another specific example, the co-stimulatory signaling domain is CD28.

In some examples, to generate a recombinant CAR and/or recombinant CCR, a signal peptide is added to the 5' end of the recombinant CAR/CCR construct. In one particular example, the signal peptide is a GM-CSF signal peptide having the following sequence (from N-terminus to C-terminus): MLLLVTSLLLCELPHAPFLLIPGHAS (SEQ ID NO: 21), encoded by the following exemplary nucleotide sequence: ATGCTTCTCCTGGGGACAAGCCTTCTGTGAGTTACCACCCAGCATGCC (SEQ ID NO: 22).

In one example, the recombinant CAR without signal peptide has the following sequence (from N-terminus to C-terminus): <xnotran> FNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVASNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 23), : </xnotran> <xnotran> TTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGTATGCAAAGGACTGTGGCTAGCAACTGGTCACACCCCCAGTTTGAGAAAGGTGGGGGAGGGTCAGGTGGTGGGGGGTCAAATTGGTCTCATCCCCAGTTCGAGAAGGGCGGAGGAGGTTCAGGTGGGGGTGGGAGCAACTGGAGCCACCCTCAATTTGAAAAAGGAGGTGGAGGGAGTGGAGGTGGTGGATCTGAATCTAAATATGGCCCCCCTTGCCCACCATGTCCCTTCTGGGTTTTGGTAGTTGTCGGCGGGGTGCTTGCTTGCTATTCATTGTTGGTTACGGTGGCTTTTATAATTTTCTGGGTATACTTCCTGGGACGACTTGTTCCTCGGGGGCGGGGTGCTGCAGAAGCTGCAACGCGAAAACAAAGAATTACCGAGACTGAGAGTCCGTATCAAGAACTGCAGGGCCAAAGAAGCGACGTTTACTCCGATCTCAATACCCAACGGCCTTACTACAAA (SEQ ID NO: 24). </xnotran> In one example, the recombinant CAR with signal peptide has the following sequence (from N-terminus to C-terminus): <xnotran> MLLLVTSLLLCELPHPAFLLIPGAHAFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVASNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 25), : </xnotran> <xnotran> ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGGCGCGCATGCCTTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGTATGCAAAGGACTGTGGCTAGCAACTGGTCACACCCCCAGTTTGAGAAAGGTGGGGGAGGGTCAGGTGGTGGGGGGTCAAATTGGTCTCATCCCCAGTTCGAGAAGGGCGGAGGAGGTTCAGGTGGGGGTGGGAGCAACTGGAGCCACCCTCAATTTGAAAAAGGAGGTGGAGGGAGTGGAGGTGGTGGATCTGAATCTAAATATGGCCCCCCTTGCCCACCATGTCCCTTCTGGGTTTTGGTAGTTGTCGGCGGGGTGCTTGCTTGCTATTCATTGTTGGTTACGGTGGCTTTTATAATTTTCTGGGTATACTTCCTGGGACGACTTGTTCCTCGGGGGCGGGGTGCTGCAGAAGCTGCAACGCGAAAACAAAGAATTACCGAGACTGAGAGTCCGTATCAAGAACTGCAGGGCCAAAGAAGCGACGTTTACTCCGATCTCAATACCCAACGGCCTTACTACAAA (SEQ ID NO: 26). </xnotran>

In one example, the recombinant CCR without signal peptide has the following sequence (from N-terminus to C-terminus): <xnotran> QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGWITAYNGNTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCARDYFYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIKASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 27), : </xnotran> <xnotran> CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCGACTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCACCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGTCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGACTACTTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGTCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAGCTAGCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 28). </xnotran> In one example, a recombinant CCR with a signal peptide has the following sequence (from N-terminus to C-terminus): <xnotran> MLLLVTSLLLCELPHPAFLLIPGAHAQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGWITAYNGNTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCARDYFYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIKASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 29), : </xnotran> <xnotran> ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGGCGCGCATGCCCAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCGACTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCACCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGTCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGACTACTTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGTCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAGCTAGCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 30). </xnotran>

In some examples, to measure the surface expression level of the recombinant CAR, a peptide tag is added to the recombinant CAR construct. In one example, a peptide tag is added between the extracellular domain and the transmembrane domain. In one particular example, the peptide tag is an ST2 tag, having the sequence (from N-terminus to C-terminus): NWSHPQFEEKGGGGGGSGGGGSNWSHPQFEEKGGGGGGSGGGGS (SEQ ID NO: 31), encoded by the following exemplary nucleotide sequence: <xnotran> AACTGGTCACACCCCCAGTTTGAGAAAGGTGGGGGAGGGTCAGGTGGTGGGGGGTCAAATTGGTCTCATCCCCAGTTCGAGAAGGGCGGAGGAGGTTCAGGTGGGGGTGGGAGCAACTGGAGCCACCCTCAATTTGAAAAAGGAGGTGGAGGGAGTGGAGGTGGTGGATCT (SEQ ID NO: 32). </xnotran>

Expression of the recombinant CAR and/or recombinant CCR in a genetically modified T cell can be transient or stable. In some examples, the constructs in each of fig. 1A and 1B are transiently or stably expressed in the genetically modified T cell.

In some examples, the genetically modified T cells disclosed herein target cancer or tumor cells that express both NKG2D ligands and PD-Ll. Binding of the NKG2D ligand to the extracellular domain of the recombinant CAR activates the genetically modified T cells, which then continue to proliferate, synthesize, and secrete cytokines. Binding of PD-L1 expressed on tumor cells to the extracellular domain of recombinant CCR activates the costimulatory signaling domain of recombinant CCR, thereby enhancing proliferation, survival and/or development of T cells and promoting cytokine synthesis and secretion. Upon binding to both PD-L1 and NKG2D ligands expressed by target tumor cells, the genetically modified T cells are fully activated and function.

The genetically modified T cells described herein can be provided as a composition or pharmaceutical composition. Thus, in one example, there is provided a pharmaceutical composition comprising a pharmaceutically effective amount of T cells as disclosed herein and a pharmaceutically acceptable excipient. The compositions described herein may be administered in a variety of ways depending on whether local or systemic treatment is desired. Administration can be topical, pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal) or systemic (e.g., oral) and/or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. In some examples, the route of administration may be selected from the group consisting of systemic administration, oral administration, intravenous administration, and parenteral administration.

The compositions or pharmaceutical compositions described herein may be provided in unit dosage form, wherein each dosage unit (e.g., injection) contains a predetermined amount of T cells as disclosed herein, alone or in appropriate combination with other active agents. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of T cells as disclosed herein, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier or excipient, as appropriate. The specification for a unit dosage form depends on the particular pharmacodynamics associated with the pharmaceutical composition in a particular subject. The unit dosage forms may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid or semi-liquid carriers.

The compositions described herein may additionally comprise other auxiliary components commonly found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically active agents, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional agents used to physically formulate various dosage forms of the compositions of the present invention, such as buffers, dyes, preservatives, antioxidants, opacifiers, thickeners, and stabilizers, or combinations thereof, which are suitable for use with pharmacologically active agents that may be added to the solution in any concentration suitable for use in eye drops. However, these materials, when added, should not unduly interfere with the biological activity of the components of the disclosed compositions. The formulations can be sterilized and, if desired, mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors and/or aromatic substances and the like, which do not deleteriously interact with the T cells of the formulation.

In another example, there is provided a method of making a T cell genetically modified to express a recombinant CAR and a recombinant CCR as disclosed herein, the method comprising: a) Obtaining or providing a T cell; b) Providing i) a recombinant nucleic acid encoding a recombinant CAR; and ii) a recombinant nucleic acid encoding a CCR; and c) transferring the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the CCR into a T cell. In some examples, the method further comprises, after step a), culturing the T cells to expand the number of T cells. T cell expansion methods known in the art may be used. In some examples, the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the recombinant CCR are transferred into a T cell simultaneously. In some examples, the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the recombinant CCR are transferred into T cells simultaneously, and the recombinant nucleic acids encoding the recombinant CAR and the recombinant CCR are cloned into the same vector. In some other examples, the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the recombinant CCR are transferred into T cells sequentially.

Recombinant nucleic acids encoding recombinant CARs and/or recombinant CCR can be generated using methods known in the art. For the amino acid sequence of each domain, the base sequence encoding the amino acid sequence can be obtained from NCBI RefSeq ID or GenBenk accession number, and standard molecular biological and/or chemical procedures can be used to prepare nucleic acids as disclosed herein. For example, based on the base sequence, a polynucleotide may be synthesized, and a polynucleotide of the present disclosure may be prepared by combining DNA fragments obtained from a cDNA library using Polymerase Chain Reaction (PCR). The sequence of the open reading frame encoding the CAR and/or CCR can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as introns have been found to stabilize mRNA. In addition, it may be advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

The nucleotide sequence encoding the recombinant CAR and/or recombinant CCR can be inserted into an appropriate expression vector, i.e., a vector that contains the elements necessary for transcription and translation of the inserted protein coding sequence. A variety of host-vector systems are available for expression of protein coding sequences. These include, but are not limited to, mammalian cell systems infected with a virus (e.g., vaccinia virus, adenovirus, etc.), insect cell systems infected with a virus (e.g., baculovirus), microorganisms such as yeast containing yeast vectors, or bacteria transformed with phage, DNA, plasmid DNA, or cosmid DNA, transgenic plants, or transgenic non-human animals. The expression elements of the vectors vary in strength and specificity. Any of a variety of suitable transcription and translation elements may be used, depending on the host-vector system used.

Any known method for inserting a DNA fragment into a vector can be used to construct an expression vector containing a chimeric nucleotide sequence consisting of appropriate transcription/translation control signals and a protein coding sequence. Exemplary methods include in vitro recombinant DNA and synthetic techniques. Expression of the recombinant nucleic acid sequence encoding the recombinant CAR and/or the recombinant CCR can be regulated by the second nucleic acid sequence such that the recombinant CAR and/or the recombinant CCR is expressed in a host transformed with the recombinant DNA molecule. For example, expression of the recombinant CAR and/or recombinant CCR can be controlled by any promoter/enhancer element known in the art.

In some examples, the basic backbone of the recombinant expression vector is a commercially available vector into which each of the above elements is inserted. Exemplary expression vectors include, but are not limited to, pFastbac1, pALTER-Ex2, pCal-n-EK, pCal-c, pCal-Kc, pcDNA2.1, pDUAL, pET-3a-c, pET-9a-d, pET-11a-d, pET-12a-c, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b (+), pET-21a-d (+), pET-22b (+), pET-23a-d (+), pET-24a-d (+), pET-25b (+), pET-26b (+), pET-21a (+), and pET-27b (+), pET-28a-c (+), pET-29a-c (+), pET-30a-c (+), pET-31b (+), pET-32a-c (+), pET-33b (+), pET-34b (+), pET-35b (+), pET-36b (+), pET-37b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a-c (+), pET-42a-c (+), pET-43a-c (+), TBpET-1, TBpElue-2, TBpElue-3, pGEMEX-1, pGEMEX-2, pRSET-3, pET-43a-c (+), pET-42 a-36 b (+), and TBpET-37 b (+) pTriEx-1, and pTriEx-2 expression vectors.

Methods generally known in the art can be used to transcribe mRNA from the recombinant expression vectors described above. For example, PCR can be performed using the recombinant expression vector as a DNA template to produce a linear DNA template. In vitro transcription of the generated linear DNA template can then be used to generate recombinant CARs and/or recombinant CCR mRNA. Optionally, the linear DNA template is purified prior to performing in vitro transcription.

Nucleic acids encoding recombinant CARs and/or recombinant CCR can be cloned into expression vectors using molecular cloning techniques well known in the art.

Expression vectors comprising recombinant nucleic acids encoding recombinant CARs and/or recombinant CCR can be transferred into T cells using any technique known in the art, such as electroporation, non-viral chemical transfection, and viral transduction. In a particular example, the T cell is genetically modified to express the recombinant CAR and the recombinant CCR by electroporation.

The genetically modified T cells of the invention provide improved T cell proliferation, reduced expression of T cell depletion markers, and enhanced in vivo tumor killing compared to T cells genetically modified to express recombinant NKG2D CARs and recombinant PD-1 Chimeric Switch Receptors (CSRs), as demonstrated by the data provided in the experimental section. In some examples, T cells genetically modified to express recombinant CARs and recombinant CCR are capable of replicating in vivo, resulting in long-term persistence that can lead to sustained tumor control.

In some examples, a medical disease or disorder can be treated by administering a population of T cells as disclosed herein. In some examples, the medical disease or disorder is a cancer or tumor. In another example, there is provided a method of treating cancer or tumor in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a T cell as disclosed herein or a pharmaceutical composition as disclosed herein. In another example, there is provided the use of a pharmaceutically effective amount of a T cell as disclosed herein in the manufacture of a medicament for the treatment of a cancer or tumor. In yet another example, a T cell or a pharmaceutical composition as disclosed herein is provided for use in the treatment of cancer or a tumor. In some examples, the genetically modified T cells for use in treating cancer or tumor are prepared during a course of therapy. Thus, in one example, there is provided a method of treating a cancer or tumor in a subject in need thereof, the method comprising: a) Obtaining T cells from the subject or from a donor different from the subject to be treated; b) Providing i) a recombinant nucleic acid encoding a recombinant Chimeric Antigen Receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a costimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of a NKG2D receptor; and ii) a recombinant nucleic acid encoding a recombinant Chimeric Costimulatory Receptor (CCR), wherein the recombinant CCR comprises (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, and (c) a costimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-L1 specific single-chain variable fragment (scFv); c) Transferring a recombinant nucleic acid encoding a recombinant CAR and a recombinant nucleic acid encoding a recombinant CCR into a T cell to obtain a genetically modified T cell; and d) administering to the subject a pharmaceutically effective amount of the T cells obtained from c). In some other examples, the method further comprises, after step a), culturing the T cells to expand the number of T cells. T cell expansion methods are known in the art and exemplified in the experimental section of the present application.

As used herein, the term "pharmaceutically effective amount" includes within its meaning a sufficient amount of the T cells described herein to provide the desired therapeutic effect, but without toxicity. Ideally, an effective or sufficient amount of the T cells described herein are present in the composition and introduced into the subject to establish a long-term, specific anti-tumor response, reducing the size of the tumor or eliminating the growth or regeneration of the tumor, as compared to what would otherwise result in the absence of such treatment. Ideally, the amount of T cells introduced into the subject results in a reduction in tumor size of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% as compared to otherwise identical conditions in the absence of T cells. Required thickness of TThe exact number of cells will vary from subject to subject, depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated (e.g., the stage and/or size of the tumor), the mode of administration, and the like. In general, the concentration of T cells should ideally be sufficient to provide at least about 1X 10 in the subject being treated ⁶ To about 1X 10 ⁹ (ii) a T cell, even more desirably, about 1X 10 ⁷ To about 5X 10 ⁸ A T cell, although, for example, 5X 10 can be used ⁸ More than one cell or, for example, 1X 10 ⁷ Any suitable amount below one cell. The dosing regimen may be based on mature cell-based therapy, or alternative continuous infusion strategies may be employed. These values provide general guidance to the practitioner in optimizing the T cell range to be used in the treatment methods disclosed herein. These ranges recited herein in no way preclude the use of higher or lower amounts of the components, which may be warranted in a particular application. For example, the actual dosage and schedule may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or depending on the inter-individual differences in pharmacokinetics, drug disposition and metabolism. An appropriate "effective amount" in any given case can be determined by one of ordinary skill in the art using only routine experimentation.

Cancers or tumors for which the present treatment methods are useful include any malignant cell type, such as those found in solid tumors or hematological tumors. In some embodiments, the malignant tumor is a solid tumor. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, caecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. In some embodiments, the malignant tumor is a hematological tumor. Exemplary hematological tumors include bone marrow tumors, T cell or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma. The cancer may particularly belong to the following histological types, although not limited to these: neoplasms, malignant; cancer; cancer, undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphatic epithelial cancer; basal cell carcinoma; hair matrix cancer; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; bile duct cancer; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; a solid cancer; carcinoid, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; a cancer of the chromophobe; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic granulosa cancer; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-enveloped sclerosing cancers; adrenocortical carcinoma; endometrial cancer; skin adnexal cancer; hyperhidrosis carcinoma; sebaceous gland cancer; cerumen adenocarcinoma; mucoepidermoid carcinoma; cystic carcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; paget's disease, of the mammary gland; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecocytoma (thecoma), malignant; granulosa cell tumor, malignant; male cell tumor (androblastoma), malignant; sertoli cell carcinoma; leydig cell tumors, malignant; lipocytoma, malignant; paraganglioma, malignant; external paraganglioma of the breast, malignant; pheochromocytoma; angiosarcoma (glomangiospora); malignant melanoma; non-pigmented melanoma; superficial diffuse melanoma; freckle-like malignant melanoma; acral freckle-like melanoma; nodular melanoma; malignant melanoma in giant pigmented nevi; epithelial-like cell melanoma; blue nevus, malignant; a sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; interstitial sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; a carcinosarcoma; stromal tumor, malignant; brenner tumor, malignant; phylloid tumors, malignant; synovial sarcoma; mesothelioma, malignant; clonal cell tumors; an embryonic carcinoma; teratoma, malignant; ovarian goiter (struma ovarii), malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; vascular endothelioma, malignant; kaposi's sarcoma; vascular endothelial cell tumor, malignant; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumors, malignant; amelogenic cell dental sarcoma; ameloblastoma, malignant; amelogenic cell fibrosarcoma; pineal tumor, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; a plasma astrocytoma; fibroastrocytoma; astrocytomas; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectoderm; cerebellar sarcoma; ganglionic neuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumors; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granulocytoma, malignant; malignant lymphoma; hodgkin's disease; of Hodgkin; granuloma paratuberis; malignant lymphoma, small lymphocytes; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-hodgkin lymphomas; b cell lymphoma; low grade/follicular non-hodgkin lymphoma (NHL); small Lymphocyte (SL) NHL; moderate/follicular NHL; intermediate diffuse NHL; higher immunoblastic NHL; higher lymphoblastic NHL; high-grade small non-lytic cell type NHL; the massive disease NHL (bulk disease NHL); mantle cell lymphoma; AIDS-related lymphoma; waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; red leukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; hairy cell leukemia; chronic Lymphocytic Leukemia (CLL); acute Lymphocytic Leukemia (ALL); acute Myeloid Leukemia (AML); and chronic myeloid leukemia. In some examples, cancers/tumors that can be treated using the genetically modified T cells provided herein include colorectal cancer, ovarian cancer, head and neck cancer, liver cancer, breast cancer, cervical cancer, and glioma. In a particular example, the cancer/tumor that can be treated is colorectal cancer.

In some examples, the cancer/tumor expresses both NKG2D ligand and PD-Ll. In some other examples, the cancer/tumor expresses NKG2D ligand and PD-L1 after treatment with another drug, radiation, or biologic agent.

The terms "treat," "treatment," and grammatical variations thereof refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease or to achieve a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; the degree of alleviation of the condition, disorder or disease; a stable state (i.e., not worsening) of the condition, disorder or disease; delay or slowing of progression of the condition, disorder or disease; amelioration, palliation (whether partial or total), whether detectable or undetectable, of a condition, disorder or disease state; or improvement or amelioration of a condition, disorder or disease. Treatment involves the initiation of clinically significant cellular responses without producing excessive levels of side effects. Treatment also includes extending survival as compared to expected survival without treatment.

The terms "reduce," "reduced," "reduce," "decrease," "remove," or "inhibit" are used generically herein to mean a statistically significant amount of reduction. However, for the avoidance of doubt, "reduced", "reduce" or "reduce", "remove" or "inhibit" means a reduction of at least 10% compared to a reference level, such as at 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 up to and including a reduction of 100% (e.g., no level present compared to a reference sample), or any reduction between 10-100% compared to a reference level (e.g., in the absence of a treatment as described herein).

In some examples, the subject or patient to be treated is an animal, mammal, human, including but not limited to animals classified as bovine, porcine, equine, canine, wolf, feline, murine, ovine, avian, fish, caprine, crow, acrine, or delphine. In one example, the patient is a human.

The source of T cells useful for treating a medical disease or disorder can be of any kind, but in specific examples, the cells are obtained from a cord blood bank, a peripheral blood bank, a human embryonic stem cell bank, or an induced pluripotent stem cell bank.

In some examples, T cells useful for treating a medical disease or disorder are autologous, i.e., obtained from the same individual to whom the treatment is to be administered. For example, an autologous source of T cells can be collected from a patient in need of treatment and the T cells activated and modified using methods described herein and known in the art, and then infused back into the patient. Some autologous sources of T cells include PBMCs, cord blood obtained at birth and subsequently stored by the patient, and induced pluripotent stem cells derived from cells obtained from the patient.

In some other examples, T cells useful for treating a medical disease or condition are allogeneic, i.e., derived from a different individual of the same species as the patient, such as a T cell donor. In some other examples, T cells useful for treating a medical disease or condition are xenogeneic, i.e., derived from an animal of a different species than the patient. Genetically modified T cells derived from allogeneic xenogeneic sources may provide a ready-to-use product.

Allogeneic or autologous T cells induce a rapid immune response, but disappear relatively rapidly from circulation due to their limited life span. Thus, concerns about persistent side effects are reduced using the treatment methods disclosed herein.

In certain examples, the T cells described herein are administered in combination with a second therapeutic agent. For example, the second therapeutic agent may include an immunomodulatory agent, a monoclonal antibody, or a chemotherapeutic agent. In a non-limiting example, the immunomodulator is lenalidomide (lenalidomide), the monoclonal antibody is alemtuzumab (alemtuzumab), rituximab (rituxumab), trastuzumab (trastuzumab), ibritumomab (ibritumomab), gemtuzumab (gemtuzumab), adotrantuzumab (brentuximab), adintrantuzumab, blinatunomab (daratumumab), or elozumab (elotuzumab), and the chemotherapeutic agent is fludarabine or cyclophosphamide.

Following administration of the genetically modified T cells disclosed herein for treating or preventing cancer/tumor, the efficacy of the treatment can be assessed in various ways well known to the skilled artisan. For example, one of ordinary skill in the art will appreciate that therapeutic genetically modified cells delivered with a chemical adjuvant are effective in treating or inhibiting cancer in a patient by observing that the therapeutic genetically modified cells reduce the cancer cell burden or prevent a further increase in the cancer cell burden. Cancer cell burden can be measured by methods known in the art, for example, using a polymerase chain reaction assay to detect the presence of certain cancer cell nucleic acids, or using, for example, an antibody assay to detect the presence of markers in a sample (e.g., without limitation, blood) from a subject or patient to identify certain cancer cell markers in the blood, or by measuring the level of circulating cancer cell antibody levels in the patient.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and without limitation. Furthermore, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

As used in this application, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a genetic marker" includes a plurality of genetic markers, including mixtures and combinations thereof.

As used herein, the term "about" in the context of concentrations of ingredients of a formulation typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Certain embodiments may also be broadly and generically described herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the general description of embodiments wherein contingent or negative limitations remove any subject matter from the dependent claims, whether or not the excised material is specifically recited herein.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims and non-limiting examples. Further, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Experimental part

Method and material

Cell lines and culture conditions

Human K562 myeloid leukemia feeder cells engineered to express CD64, CD86 and CD137L were maintained in IMDM. HepG2 and Detroit-562 were maintained in DMEM. CAOV3 was maintained in RPMI. All types of media were supplemented with 10% FBS (Gibco).

CAR-T cell preparation

Buffy coats of healthy donors were obtained from the Health Sciences Authority (HSA, singapore) under IRB approval and PBMCs thereof were separated using density gradient centrifugation. PBMC were processed at 5X 10 ⁶ The individual cells/ml were inoculated in T cell medium (AIM-V +5% AB serum) and activated with 1. Mu.g/ml of soluble OKT3 (Ebioscience) or TransAct (Miltenyi Biotec, germany) for 2-3 days, every 1X 10 ⁷ Mu.l TransAct was added to each PBMC. Recombinant IL-2 (300 IU/ml; peprotech) was added at the beginning of the culture and supplemented every other day.

On day 2 or 3, activated T cells were collected and transduced by non-viral electroporation. The culture of the modified T cells was continued for 5 days in AIM-V supplemented with 5% AB serum or 1% human plasma and 300IU/ml IL-2. On day 8, the genetically modified T cells were counted and co-cultured with γ -irradiated K562 or K562#6 cells in AIM-V medium supplemented with 300IU/ml IL-2 at a ratio of 1. CAR T cells were re-stimulated every 10 days with the addition of new gamma irradiated K562 cells at a rate of 1. For the proliferation of unmodified T cells, T cells were co-cultured with gamma irradiated K562#6 at a ratio of 1. IL-2 was added at 300IU/ml at the beginning of the culture and supplemented every other day.

CD56+ cells were removed from CAR-T cell cultures on day 17 according to the manufacturer's protocol (Miltenyi Biotec, germany). In short, up to 1X 10 ⁷ Individual cells were suspended in 80. Mu.l of the AutoMACS running buffer and incubated with 20. Mu.l of CD56 microbeads for 15 minutes at 4 ℃ in the absence of light. Cells were centrifuged at 300g for 10 min and resuspended in 500. Mu.l of autoMACS running buffer. The LS column was placed in MACS MultiStand and equilibrated by washing twice with 1ml of autoMACS running buffer. The cell suspension was added to the LS column to capture labeled CD56+ cells. The CD 56-flow through fractions were collected at the bottom of a column in a 15ml conical centrifuge tube (BD Biosciences, USA).

Flow cytometry

For phenotypic analysis of T cells, the following labeled anti-human antibodies were used: CD3 (clone: OKT3; eBioscience), CD45RO (clone: UCHL1; BD Biosciences), CCR7 (clone: 3D12 eBioscience), PD-1 (clone: MIH4; BD Pharmingen), TIGIT (clone: MBSA43; eBioscience), TIM3 (clone: F38-2Ebioscience), and LAG3 (clone: 3DS223H. The circle gate was verified using the appropriate isotype control. THE USE OF THE ON THE 5 th day after electroporation and 12 th day after one week of coculture with gamma irradiated K562#6 ^TM The nwspqfek tag antibody (Genescript) measures the expression of NKG2D CAR (which includes Strep tag) on T cells. Flow cytometry analysis and use of BD Accuri ^TM C6 (BD Biosciences) analysis.

In vitro cytotoxicity assay

The cytolytic activity of CAR-modified T cells was examined using the DELFIA EuTDA cytoxicity Reagents kit (PerkinElmer). The ratio of effector to target (E: T) used ranged from 20 to 1. The control group was set to measure spontaneous release (target cells added only), maximum release (target cells added with 10 μ l lysis buffer) and media background (no cells added). The killing efficacy was calculated using the following formula:

% specific release = (experimental release (count) -spontaneous release (count))/(maximum release (count) -spontaneous release (count)) × 100

In vivo experiments

Animal experiments were conducted according to protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Biological Resources Center (BRC) of the science, technology and research institute of singapore (a STAR) (accession number BRC IACUC # 181324). Non-obese diabetic/severe combined immunodeficiency/IL-2R γ cnell (NSG) mice (8-10 weeks old, male, jackson laboratories, usa) were maintained and used in the current study. All luminescence signals and images were collected and analyzed using Xenogen in vivo imaging software v 3.2.

To establish a mouse colorectal cancer (CRC) xenograft model, mice were intraperitoneally injected 2X 10 on day 0 ⁶ Individual HCT116-luc human colorectal cancer cells. Tumor implantation was confirmed by in vivo bioluminescence Imaging (BLI) monitored using the IVIS Spectrum Imaging platform with Living Image software (PerkinElmer) on day 7 post tumor inoculation. Mice with similar BLI signal intensity were randomly divided into 4 different treatment groups of 5 mice each. On day 7, 100. Mu.l PBS or 100. Mu.l containing 1X 10 was injected intraperitoneally ⁷ Mice were treated with cell suspensions of three different groups of T cells: (1) control T cells, (2) NKG2D CAR + PD-1 CSR double CAR modified T cells, or (3) NKG2D CAR + alpha PD-L1 CCR double CAR modified T cells. Same dose (1X 10) at day 32 after tumor inoculation ⁷ Individual T cells/mouse) for a second injection. Tumor progression was monitored weekly by BLI. The mice were closely monitored and humanely euthanized after observing the development of an moribund state characterized by significant abdominal distension, palpable hypothermia, inability to walk, and/or lack of significant response to the procedure as induced by ascites.

Statistics of

For in vitro and in vivo experiments, 2 groups of continuous variables were evaluated using unpaired student t-test, and more than 2 groups of continuous variables were evaluated using one-way analysis of variance with post-test Bonferroni. Survival was analyzed by the Kaplan-Meier method and the log rank (Mantel-Cox) test to compare paired groups. Statistics were calculated using GraphPad Prism 7.0 (GraphPad software). When the P value is less than 0.05, the difference is considered significant.

Results

In order to improve the in vivo persistence of tumor-reactive T cells, the use of PD-1 based CSR in combination with tumor-reactive TCRs or CARs has been investigated and validated in several studies. However, PD-1 naturally recognizes both PD-L1 and PD-L2, with the expression pattern of PD-L2 being primarily restricted to myeloid lineage cells, while PD-L1 is more commonly found to be upregulated on various tumors. Therefore, there is a need to further improve the tumor specificity of this approach. In the present invention, a first generation CAR was constructed by fusing the extracellular domain of the NKG2D receptor with the DAP 12-derived activation domain (figure 1A). To specifically target PD-L1, an α PD-L1 CCR was generated that recognized only PD-L1 but not PD-L2 (fig. 1B). At the same time, a PD-1 based CSR was also generated for comparative studies (fig. 1C). In both constructs, the antigen targeting moiety was expressed in tandem with a single co-stimulatory domain derived from 4-1BB. Hereinafter, the combination of NKG2D CAR and α PD-L1 CCR will be referred to as CAR-T1, and the combination of NKG2D CAR and PD-1 CSR will be referred to as CAR-T2.

To assess the function of CAR-T1 and CAR-T2 cells, magnetic bead-activated T cells were co-electroporated with plasmids encoding piggyBac transposase, NKG2D CAR, and alpha PD-L1 CCR or PD-1 CSR. To expand and enrich for NKG2D CAR expressing T cells, these T cells were co-cultured with the human K562 myeloid leukemia cell line at an effector to target (E: T) ratio of 1. Expanded CAR-T1 and CAR-T2 cells were then used as effector cells and their cytotoxicity was assessed in a 2-hour cytotoxicity assay for cancer cells bearing NKG2D and PD-1 ligands. Three different cancer cell lines with different PD-1 ligand expression levels were used: hepG2 only expressed NKG2DL, but not any PD-1 ligand; CAOV3 expresses NGK2DL and PD-L1; detroit-562 expresses NKG2DL and two PD-1 ligands. As shown in FIG. 2, there were no observable statistical differences in cytotoxicity of CAR-T1 and CAR-T2 cells on each of the three target cell lines.

To assess the potential for large-scale production, CAR-T1 and CAR-T2 cells were expanded continuously with a batch of fresh gamma-irradiated K562 feeder cells every 10 days in the presence of IL-2 alone. The increase in cell number was counted over a 30 day period and robust expansion of these CAR-T cells was observed (figure 3). Although there were no observable differences in cytotoxicity between NKG2D CAR-T cells implanted with either alpha PD-L1 CCR or PD-1 CSR, the overall cell yield of CAR-T1 was improved when co-cultured with K562 feeder cells compared to CAR-T2 (figure 3). This trend of improvement was consistent in all three donors tested.

Next, it was evaluated how the incorporation of α PD-L1 CCR could further improve the development of memory T cell subsets. Co-expression of both markers on CAR-T1 and CAR-T2 cells at the mid and end of the expansion phase was assessed using CCR7 and CD45RA as markers for classifying various memory subpopulations. As shown in FIG. 4, memory T cell development differed between the two CAR-T groups. Comparing the T cell composition at the mid-and end-expansion stages, the proportion of effector T cells in CAR-T2 rose slightly from 33.2% to 34.5%, whereas in CAR-T1 this proportion fell from 26.6% to 15.2%. In contrast, the percentage of effector memory T cells decreased from 66.2% to 64.9% in CAR-T2, but increased from 73.3% to 84.6% in CAR-T1. Although there were no detectable levels of central memory T cells or stem cell memory T cells, figure 4 shows a clear difference in effector memory T development between α PD-L1 CCR and PD-1 CSR.

Chronic antigen activation often results in increased expression of typical T cell depletion markers. To investigate how α PD-L1 CCR mitigates expression of T cell depletion markers and possibly reverses T cell fate in an immunosuppressive setting, expression of PD-1, TIGIT, LAG3 and TIM3 on CAR-T1 and CAR-T2 cells was measured at the end of the expansion phase. Consistent with the results seen in figures 3 and 4, CAR-T1 cells exhibited reduced expression levels of these depletion markers compared to CAR-T2 cells (figure 5). The experimental setup did not distinguish between endogenous inhibitory PD-1 receptors and exogenous PD-1 CSR, therefore, higher levels of PD-1 on CAR-T2 may not indicate increased inhibition of PD-1. However, the expression levels of TIGIT, TIM3 and LAG3 were consistently higher on CAR-T2 cells in all three subjects. The single endpoint measurements (fig. 5A) and the summary mean (fig. 5B) from a single donor together indicate that incorporation of α PD-L1 CCR alleviated T cell depletion to a greater extent than PD-1 CSR.

It was further investigated whether there was a difference in tumor killing effect in vivo between CAR-T1 (NKG 2D CAR + α PD-L1 CCR) and CAR-T2 (NKG 2D CAR + PD-1 CSR). We established a mouse colorectal model in NSG mice by intraperitoneal (i.p.) injection of human HCT116-luc CRC cells. Tumor progression was monitored by whole body bioluminescence imaging (fig. 6). On day 7, when all mice had established tumors in the abdominal cavity, animals were randomized into 4 groups for treatment: group 1 was given two intraperitoneal injections of PBS; group 2 two intraperitoneal injections, 10 each ⁷ Individual control CAR-T cells; group 3 and group 4 received two intraperitoneal injections, 10 each ⁷ CAR-T1 cells and CAR-T2 cells. As shown in fig. 6, the 4 groups of mice exhibited similar tumor burden on day 7 prior to treatment, as evidenced by similar bioluminescence intensity. Established tumors continued to grow in PBS and control CAR-T cell treated mice, all mice died on day 28. With dual CAR-T treatment, the established tumors were completely eliminated and their bioluminescent signals became undetectable on day 14. By treatment, a significant reduction in disease is maintained>At 8 weeks, by day 62, 4 of 10 treated mice developed tumor regeneration, 3 of the CAR-T2 group, and 1 of the CAR-T1 group. By day 150, while 3 mice died and 2 survived in the CAR-T2 group, all mice in the CAR-T1 group survived well: of these 4 had no tumor and 1 mouse showed stable disease. The Kaplan-Meier curve and log rank test results are shown in fig. 7, indicating that there is a significant difference between the two sets of survival curves. Overall, this animal experiment shows that while both CAR-T1 and CAR-T2 are able to effectively eradicate established solid tumors in vivo, tumor killing efficacy is further significantly enhanced by replacing PD-1 CSR with alpha PD-L1 CCR for dual CAR construction.

Conclusion

Due to their special biological properties, such as clonal heterogeneity and a suppressive Tumor Microenvironment (TME) that is unfavorable for T cells, eradication of large solid tumors remains challenging for most cancer patients.

PD-1 recognizes both PD-L1 and PD-L2. PD-L1 is overexpressed on tumor cells and at low levels on a wide range of non-hematopoietic cells. The expression pattern of PD-L2 is more restricted than PD-L1, and its expression is mainly restricted to antigen presenting cells such as dendritic cells, macrophages, B1B cells and mast cells. Thus, of the two PD-1 ligands, PD-L1 is considered to be the primary mediator of cancer immune evasion, while PD-L2 does not appear to be the primary mediator of T cell inhibition at the tumor site, although PD-L2 may play a role in some cases. Another difference is their affinity for PD-1: PD-L2 has a2 to 6-fold higher affinity for PD-1 than PD-L1 and PD-L2 shows different binding/dissociation kinetics compared to the interaction between PD-1 and PD-L1. These different binding properties of PD-L2 and PD-L1 may be responsible for the different contributions of these two PD-1 ligands to the immune response.

Previous studies have utilized PD-1 receptor-based CSR to protect transduced T cells from PD-L1-induced T cell suppression and to convert inhibitory signals into the required costimulatory signals to achieve optimal T cell function. When used in bispecific dual CAR constructions, the higher binding properties of PD-L2 to PD-1 may lead PD-1-based dual CAR-T cells to be more readily activated by PD-L2 positive cells. To improve cancer specificity and reduce side effects on antigen presenting cells and adverse events associated with PD-1 blockade, PD-L1 specific CSR should be used, which unfortunately is difficult to construct by using the extracellular structure of the PD-1 receptor. To avoid this problem, the present invention utilizes scFv fragments specific for PD-L1 to generate chimeric co-stimulatory receptors (CCR). When used with first generation NKG2D CARs, the use of such anti-PD-L1 CCR based bispecific dual CARs has been demonstrated to improve T cell proliferation, reduce expression of T cell depletion markers, and most importantly, enhance tumor killing in vivo.

As clinical trials based on CAR-T cell therapy continue to fight off on-target tumoricidal effects, immune evasion and immunosuppression, new strategies are needed to supplement ongoing clinical work. As a concluding remark, the bispecific dual CARs developed here offer a conceivably attractive opportunity in different therapeutic regimens targeting PD-L1 and NKG2D ligand-positive cancer types. When a tumor type shows selective overexpression of PD-L1, the use of the presently disclosed dual CARs allows T cells to overcome immunosuppressive TME while activating T cell cytotoxicity against NKG2G ligand-expressing cancer cells. Importantly, this dual specificity dual CAR is theoretically safe and does not pose any potential threat to the patient, thereby paving the way for a smoother transformation application.

Sequence listing

<110> Singapore national university

<120> genetically modified T cell and use thereof

<130> 10104SG1209

<160> 32

<170> PatentIn version 3.5

<210> 1

<211> 150

<212> PRT

<213> Artificial sequence

<220>

<223> extracellular Domain of PD-1

<400> 1

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 2

<211> 450

<212> DNA

<213> Artificial sequence

<220>

<223> extracellular Domain of PD-1

<400> 2

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 60

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 120

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 180

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 240

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 300

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 360

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 420

aggccagccg gccagttcca aaccctggtg 450

<210> 3

<211> 136

<212> PRT

<213> Artificial sequence

<220>

<223> extracellular domain of NKG2D receptor

<400> 3

Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro

1 5 10 15

Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe

20 25 30

Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln

35 40 45

Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu

50 55 60

Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr

65 70 75 80

Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu

85 90 95

Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser

100 105 110

Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile

115 120 125

Cys Met Gln Arg Thr Val Ala Ser

130 135

<210> 4

<211> 408

<212> DNA

<213> Artificial sequence

<220>

<223> extracellular domain of NKG2D receptor

<400> 4

ttcaaccaag aagttcaaat tcccttgacc gaaagttact gtggcccatg tcctaaaaac 60

tggatatgtt acaaaaataa ctgctaccaa ttttttgatg agagtaaaaa ctggtatgag 120

agccaggctt cttgtatgtc tcaaaatgcc agccttctga aagtatacag caaagaggac 180

caggatttac ttaaactggt gaagtcatat cattggatgg gactagtaca cattccaaca 240

aatggatctt ggcagtggga agatggctcc attctctcac ccaacctact aacaataatt 300

gaaatgcaga agggagactg tgcactctat gcctcgagct ttaaaggcta tatagaaaac 360

tgttcaactc caaatacgta catctgtatg caaaggactg tggctagc 408

<210> 5

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 scFv

<400> 5

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Gly Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Thr Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Phe Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

130 135 140

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

145 150 155 160

Val Ser Ser Tyr Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

165 170 175

Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser

210 215 220

Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala

225 230 235 240

Ser

<210> 6

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<223> PD-L1 scFv

<400> 6

caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggtta cacctttacc gactatggtt tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaccgctt acaatggtaa cacaaactat 180

gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagtctac 240

atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagactac 300

ttctacggta tggacgtctg gggccaaggg accacggtca ccgtctcctc aggtggaggc 360

ggttcaggcg gaggtggcag cggcggtggc gggtcggaaa ttgtgttgac acagtctcca 420

gccaccctgt ctttgtctcc aggggaaaga gccaccctct cctgcagggc cagtcagagt 480

gttagcagct acttagtctg gtaccaacag aaacctggcc aggctcccag gctcctcatc 540

tatgatgcat ccaacagggc cactggcatc ccagccaggt tcagtggcag tgggtctggg 600

acagacttca ctctcaccat cagcagccta gagcctgaag attttgcagt ttattactgt 660

cagcagcgta gcaactggcc tcggacgttc ggccaaggga ccaaggtgga aatcaaagct 720

agc 723

<210> 7

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> VH PD-L1 scFv

<400> 7

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Gly Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Thr Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Phe Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 8

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> VH PD-L1 scFv

<400> 8

caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggtta cacctttacc gactatggtt tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaccgctt acaatggtaa cacaaactat 180

gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagtctac 240

atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagactac 300

ttctacggta tggacgtctg gggccaaggg accacggtca ccgtctcctc a 351

<210> 9

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL PD-L1 scFv

<400> 9

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 10

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> VL PD-L1 scFv

<400> 10

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agctacttag tctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctcggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 11

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 scFv VH and VL linker

<400> 11

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 12

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> PD-L1 scFv VH and VL linker

<400> 12

ggtggaggcg gttcaggcgg aggtggcagc ggcggtggcg ggtcg 45

<210> 13

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> transmembrane domain of CAR

<400> 13

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Phe Trp Val Leu

1 5 10 15

Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val

20 25 30

Ala Phe Ile Ile Phe Trp Val

35

<210> 14

<211> 117

<212> DNA

<213> Artificial sequence

<220>

<223> transmembrane domain of CAR

<400> 14

gaatctaaat atggcccccc ttgcccacca tgtcccttct gggttttggt agttgtcggc 60

ggggtgcttg cttgctattc attgttggtt acggtggctt ttataatttt ctgggta 117

<210> 15

<211> 83

<212> PRT

<213> Artificial sequence

<220>

<223> transmembrane domain of CCR

<400> 15

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

50 55 60

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn

65 70 75 80

His Arg Asn

<210> 16

<211> 249

<212> DNA

<213> Artificial sequence

<220>

<223> transmembrane domain of CCR

<400> 16

ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc cagcgccgcg accaccaaca 60

ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 120

gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 180

cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaac 240

cacaggaac 249

<210> 17

<211> 52

<212> PRT

<213> Artificial sequence

<220>

<223> intracellular signaling domain of CAR

<400> 17

Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala

1 5 10 15

Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu

20 25 30

Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg

35 40 45

Pro Tyr Tyr Lys

50

<210> 18

<211> 156

<212> DNA

<213> Artificial sequence

<220>

<223> intracellular signaling domain of CAR

<400> 18

tacttcctgg gacgacttgt tcctcggggg cggggtgctg cagaagctgc aacgcgaaaa 60

caaagaatta ccgagactga gagtccgtat caagaactgc agggccaaag aagcgacgtt 120

tactccgatc tcaataccca acggccttac tacaaa 156

<210> 19

<211> 47

<212> PRT

<213> Artificial sequence

<220>

<223> Co-stimulatory signaling domains of CCR

<400> 19

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40 45

<210> 20

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Co-stimulatory signaling domains of CCR

<400> 20

cgtttctctg ttgttaaacg gggcagaaag aagctcctgt atatattcaa acaaccattt 60

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 120

gaagaaggag gatgtgaact g 141

<210> 21

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 21

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gly Ala His Ala

20 25

<210> 22

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 22

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccaggcg cgcatgcc 78

<210> 23

<211> 284

<212> PRT

<213> Artificial sequence

<220>

<223> CAR without signal peptide

<400> 23

Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro

1 5 10 15

Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe

20 25 30

Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln

35 40 45

Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu

50 55 60

Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr

65 70 75 80

Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu

85 90 95

Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser

100 105 110

Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile

115 120 125

Cys Met Gln Arg Thr Val Ala Ser Asn Trp Ser His Pro Gln Phe Glu

130 135 140

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Ser His Pro

145 150 155 160

Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp

165 170 175

Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Phe Trp Val

195 200 205

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

210 215 220

Val Ala Phe Ile Ile Phe Trp Val Tyr Phe Leu Gly Arg Leu Val Pro

225 230 235 240

Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr

245 250 255

Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val

260 265 270

Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys

275 280

<210> 24

<211> 852

<212> DNA

<213> Artificial sequence

<220>

<223> CAR without signal peptide

<400> 24

ttcaaccaag aagttcaaat tcccttgacc gaaagttact gtggcccatg tcctaaaaac 60

tggatatgtt acaaaaataa ctgctaccaa ttttttgatg agagtaaaaa ctggtatgag 120

agccaggctt cttgtatgtc tcaaaatgcc agccttctga aagtatacag caaagaggac 180

caggatttac ttaaactggt gaagtcatat cattggatgg gactagtaca cattccaaca 240

aatggatctt ggcagtggga agatggctcc attctctcac ccaacctact aacaataatt 300

gaaatgcaga agggagactg tgcactctat gcctcgagct ttaaaggcta tatagaaaac 360

tgttcaactc caaatacgta catctgtatg caaaggactg tggctagcaa ctggtcacac 420

ccccagtttg agaaaggtgg gggagggtca ggtggtgggg ggtcaaattg gtctcatccc 480

cagttcgaga agggcggagg aggttcaggt gggggtggga gcaactggag ccaccctcaa 540

tttgaaaaag gaggtggagg gagtggaggt ggtggatctg aatctaaata tggcccccct 600

tgcccaccat gtcccttctg ggttttggta gttgtcggcg gggtgcttgc ttgctattca 660

ttgttggtta cggtggcttt tataattttc tgggtatact tcctgggacg acttgttcct 720

cgggggcggg gtgctgcaga agctgcaacg cgaaaacaaa gaattaccga gactgagagt 780

ccgtatcaag aactgcaggg ccaaagaagc gacgtttact ccgatctcaa tacccaacgg 840

ccttactaca aa 852

<210> 25

<211> 310

<212> PRT

<213> Artificial sequence

<220>

<223> CAR with Signal peptide

<400> 25

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gly Ala His Ala Phe Asn Gln Glu Val Gln

20 25 30

Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile

35 40 45

Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp

50 55 60

Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys

65 70 75 80

Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr

85 90 95

His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp

100 105 110

Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met

115 120 125

Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile

130 135 140

Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

145 150 155 160

Ala Ser Asn Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser

165 170 175

Gly Gly Gly Gly Ser Asn Trp Ser His Pro Gln Phe Glu Lys Gly Gly

180 185 190

Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Ser His Pro Gln Phe Glu

195 200 205

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly

210 215 220

Pro Pro Cys Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly

225 230 235 240

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

245 250 255

Trp Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala

260 265 270

Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr

275 280 285

Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr

290 295 300

Gln Arg Pro Tyr Tyr Lys

305 310

<210> 26

<211> 930

<212> DNA

<213> Artificial sequence

<220>

<223> CAR with Signal peptide

<400> 26

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccaggcg cgcatgcctt caaccaagaa gttcaaattc ccttgaccga aagttactgt 120

ggcccatgtc ctaaaaactg gatatgttac aaaaataact gctaccaatt ttttgatgag 180

agtaaaaact ggtatgagag ccaggcttct tgtatgtctc aaaatgccag ccttctgaaa 240

gtatacagca aagaggacca ggatttactt aaactggtga agtcatatca ttggatggga 300

ctagtacaca ttccaacaaa tggatcttgg cagtgggaag atggctccat tctctcaccc 360

aacctactaa caataattga aatgcagaag ggagactgtg cactctatgc ctcgagcttt 420

aaaggctata tagaaaactg ttcaactcca aatacgtaca tctgtatgca aaggactgtg 480

gctagcaact ggtcacaccc ccagtttgag aaaggtgggg gagggtcagg tggtgggggg 540

tcaaattggt ctcatcccca gttcgagaag ggcggaggag gttcaggtgg gggtgggagc 600

aactggagcc accctcaatt tgaaaaagga ggtggaggga gtggaggtgg tggatctgaa 660

tctaaatatg gccccccttg cccaccatgt cccttctggg ttttggtagt tgtcggcggg 720

gtgcttgctt gctattcatt gttggttacg gtggctttta taattttctg ggtatacttc 780

ctgggacgac ttgttcctcg ggggcggggt gctgcagaag ctgcaacgcg aaaacaaaga 840

attaccgaga ctgagagtcc gtatcaagaa ctgcagggcc aaagaagcga cgtttactcc 900

gatctcaata cccaacggcc ttactacaaa 930

<210> 27

<211> 371

<212> PRT

<213> Artificial sequence

<220>

<223> CCR without signal peptide

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Gly Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Thr Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Phe Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

130 135 140

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

145 150 155 160

Val Ser Ser Tyr Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

165 170 175

Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser

210 215 220

Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala

225 230 235 240

Ser Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala

245 250 255

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

260 265 270

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

275 280 285

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

290 295 300

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

305 310 315 320

Asn His Arg Asn Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu

370

<210> 28

<211> 1113

<212> DNA

<213> Artificial sequence

<220>

<223> CCR without signal peptide

<400> 28

caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggtta cacctttacc gactatggtt tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaccgctt acaatggtaa cacaaactat 180

gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagtctac 240

atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagactac 300

ttctacggta tggacgtctg gggccaaggg accacggtca ccgtctcctc aggtggaggc 360

ggttcaggcg gaggtggcag cggcggtggc gggtcggaaa ttgtgttgac acagtctcca 420

gccaccctgt ctttgtctcc aggggaaaga gccaccctct cctgcagggc cagtcagagt 480

gttagcagct acttagtctg gtaccaacag aaacctggcc aggctcccag gctcctcatc 540

tatgatgcat ccaacagggc cactggcatc ccagccaggt tcagtggcag tgggtctggg 600

acagacttca ctctcaccat cagcagccta gagcctgaag attttgcagt ttattactgt 660

cagcagcgta gcaactggcc tcggacgttc ggccaaggga ccaaggtgga aatcaaagct 720

agcttcgtgc cggtcttcct gccagcgaag cccaccacga cgccagcgcc gcgaccacca 780

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 840

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 900

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 960

aaccacagga accgtttctc tgttgttaaa cggggcagaa agaagctcct gtatatattc 1020

aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1080

tttccagaag aagaagaagg aggatgtgaa ctg 1113

<210> 29

<211> 397

<212> PRT

<213> Artificial sequence

<220>

<223> CCR with signal peptide

<400> 29

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gly Ala His Ala Gln Val Gln Leu Val Gln

20 25 30

Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys

35 40 45

Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Gly Phe Ser Trp Val Arg

50 55 60

Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Thr Ala Tyr

65 70 75 80

Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln Gly Arg Val Thr Met

85 90 95

Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Arg Ser Leu

100 105 110

Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Phe Tyr

115 120 125

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile

145 150 155 160

Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg

165 170 175

Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Val

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp

195 200 205

Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp

225 230 235 240

Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg Thr Phe

245 250 255

Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Ser Phe Val Pro Val Phe

260 265 270

Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

275 280 285

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

290 295 300

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

305 310 315 320

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

325 330 335

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Phe

340 345 350

Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

355 360 365

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

370 375 380

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

385 390 395

<210> 30

<211> 1191

<212> DNA

<213> Artificial sequence

<220>

<223> CCR with Signal peptide

<400> 30

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccaggcg cgcatgccca ggttcagctg gtgcagtctg gagctgaggt gaagaagcct 120

ggggcctcag tgaaggtctc ctgcaaggct tctggttaca cctttaccga ctatggtttc 180

agctgggtgc gacaggcccc tggacaaggg cttgagtgga tgggatggat caccgcttac 240

aatggtaaca caaactatgc acagaagctc cagggcagag tcaccatgac cacagacaca 300

tccacgagca cagtctacat ggagctgagg agcctgagat ctgacgacac ggccgtgtat 360

tactgtgcga gagactactt ctacggtatg gacgtctggg gccaagggac cacggtcacc 420

gtctcctcag gtggaggcgg ttcaggcgga ggtggcagcg gcggtggcgg gtcggaaatt 480

gtgttgacac agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 540

tgcagggcca gtcagagtgt tagcagctac ttagtctggt accaacagaa acctggccag 600

gctcccaggc tcctcatcta tgatgcatcc aacagggcca ctggcatccc agccaggttc 660

agtggcagtg ggtctgggac agacttcact ctcaccatca gcagcctaga gcctgaagat 720

tttgcagttt attactgtca gcagcgtagc aactggcctc ggacgttcgg ccaagggacc 780

aaggtggaaa tcaaagctag cttcgtgccg gtcttcctgc cagcgaagcc caccacgacg 840

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 900

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 960

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1020

gttatcaccc tttactgcaa ccacaggaac cgtttctctg ttgttaaacg gggcagaaag 1080

aagctcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1140

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact g 1191

<210> 31

<211> 57

<212> PRT

<213> Artificial sequence

<220>

<223> ST2 tag

<400> 31

Asn Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Asn Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly

20 25 30

Ser Gly Gly Gly Gly Ser Asn Trp Ser His Pro Gln Phe Glu Lys Gly

35 40 45

Gly Gly Gly Ser Gly Gly Gly Gly Ser

50 55

<210> 32

<211> 171

<212> DNA

<213> Artificial sequence

<220>

<223> ST2 tag

<400> 32

aactggtcac acccccagtt tgagaaaggt gggggagggt caggtggtgg ggggtcaaat 60

tggtctcatc cccagttcga gaagggcgga ggaggttcag gtgggggtgg gagcaactgg 120

agccaccctc aatttgaaaa aggaggtgga gggagtggag gtggtggatc t 171

ctacggtatg gacgtctggg gccaagggac cacggtcacc 420

gtctcctcag gtggaggcgg ttcaggcgga ggtggcagcg gcggtggcgg gtcggaaatt 480

gtgttgacac agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 540

tgcagggcca gtcagagtgt tagcagctac ttagtctggt accaacagaa acctggccag 600

gctcccaggc tcctcatcta tgatgcatcc aacagggcca ctggcatccc agccaggttc 660

agtggcagtg ggtctgggac agacttcact ctcaccatca gcagcctaga gcctgaagat 720

tttgcagttt attactgtca gcagcgtagc aactggcctc ggacgttcgg ccaagggacc 780

aaggtggaaa tcaaagctag cttcgtgccg gtcttcctgc cagcgaagcc caccacgacg 840

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 900

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 960

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1020

gttatcaccc tttactgcaa ccacaggaac cgtttctctg ttgttaaacg gggcagaaag 1080

aagctcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1140

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact g 1191

<210> 31

<211> 57

<212> PRT

<213> Artificial sequence

<220>

<223> ST2 tag

<400> 31

Asn

Claims (14)

1. A T cell genetically modified to express:

i) A recombinant Chimeric Antigen Receptor (CAR) comprising (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a costimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of the NKG2D receptor; and

ii) a recombinant chimeric co-stimulatory receptor (CCR) comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) a co-stimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-L1 specific single chain variable fragment (scFv).

2. The T cell of claim 1, wherein the extracellular domain of the recombinant CAR is the extracellular domain of NKG2D receptor, and/or wherein the extracellular domain of the recombinant CCR is a PD-L1 specific scFv.

3. The T cell of claim 1 or 2, wherein the intracellular signaling domain of the recombinant CAR is CD3 ζ or DAP12.

4. The T cell of any one of claims 1 to 3, wherein the co-stimulatory signaling domain of the recombinant CCR is 4-1BB or CD28.

5. The T cell of any one of claims 1 to 4, wherein the transmembrane domain of the recombinant CAR and the transmembrane domain of the CCR are different.

6. A pharmaceutical composition comprising a pharmaceutically effective amount of T cells according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

7. A method of making the T cell of any one of claims 1 to 5, the method comprising:

a) Obtaining or providing a T cell;

b) Providing i) a recombinant nucleic acid encoding a recombinant CAR; and ii) a recombinant nucleic acid encoding a CCR; and

c) Transferring a recombinant nucleic acid encoding the recombinant CAR and a recombinant nucleic acid encoding the CCR into the T cell.

8. A method of treating cancer or tumor in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the T cell of any one of claims 1 to 5 or the pharmaceutical composition of claim 6.

9. The method of claim 8, wherein the T cells are derived from allogeneic cells or autologous cells.

10. A method of treating a cancer or tumor in a subject in need thereof, the method comprising:

a) Obtaining T cells from the subject or a donor different from the subject to be treated;

b) Providing i) a recombinant nucleic acid encoding a recombinant Chimeric Antigen Receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen-binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a costimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of a NKG2D receptor; and ii) a recombinant nucleic acid encoding a recombinant chimeric co-stimulatory receptor (CCR), wherein the recombinant CCR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) a co-stimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-1L1 specific single-chain variable fragment (scFv);

c) Transferring a recombinant nucleic acid encoding the recombinant CAR and a recombinant nucleic acid encoding the recombinant CCR into the T cell to obtain a genetically modified T cell; and

d) Administering to the subject a pharmaceutically effective amount of the T cells obtained from c).

11. The method of any one of claims 8 to 10, wherein the cancer or tumor is a solid tumor.

12. The method of any one of claims 8 to 11, wherein the cancer or tumor expresses both NKG2D ligand and PD-L1.

13. The method of any one of claims 8 to 12, wherein the cancer or tumor is colorectal cancer or colorectal tumor.

14. The method of any one of claims 7 and 10-13, wherein transferring the recombinant nucleic acid in step c) is performed using electroporation.

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