PD1-SPECIFIC CHIMERIC ANTIGEN RECEPTOR AS AN IMMUNOTHERAPY
Provided herein are methods and compositions useful for treating PDL1 and/or PDL2 positive cancers through adoptive cell transfer of T cells genetically engineered to express a PDI-specific chimeric antigen receptor, Co-stimulatory domains such as Dap 10 may be included to enhance efficacy.
The disclosure. generally relates to chimeric antigen receptors that target PDI ligands, and genetically engineered T cells enressing the same, for the treatment of cancer. In particular, the chimeric antigen receptor may include a co-stinmiatory domain, such as Dap10, to enhance efficacy of the treatment.
BACKGROUND OF THE INVENTIONEngineering cells with chimeric antigen receptors (CARs) is one approach to increase T-cell anti-tumor efficacy. CARs are used to redirect T-cell specificity and allow MHC-independent recognition of tumor-associated antigens, so enhancing tumor targeting. Advantages to using CAR-modified I cells for cancer therapy include the ability to recognize a broad range of tumor types, overcome the mechanisms that tumors use to escape immune detection, and enhance T-cell function. However, the up-regulation of inhibitory receptor expression on Tcells and expression of inhibitory ligands in the tumor microenvironment limit CAR T-cell responses and efficacy.
In cancer patients, negative regulation of immune responses often occurs after sustained activation of T cells. One such inhibitory receptor that plays an important role in inhibiting anti-tumor T-cell responses is the programmed death receptor 1 (PD1, CD279), which is up regulated shortly after T-cell activation and inhibits multiple T-cell functions downstream of T-cell receptor and CD28 signaling including proliferation, cytokine production and cytotoxicity. The PD1 receptor binds to two different ligands, programmed death ligind l (PDL1, B7-H1, CD274) and programmed death ligand 2 (PDL2, B7-DC, CD273), both of which are overexpressed on many types of solid tumors and haematological malignancies, including lymphoma. Effective therapies targeting the PD1 ligands are needed.
SUMMARY OF THE INVENTIONThe present disclosure provides CARs that target PD1 ligands and are suitable for adoptive T cell therapy.
One aspect of the disclosure provides a CAR polypeptide comprising an extracellular binding domain specific for at least one of programmed death ligand 1 (PDL 1) and programmed death ligand 2 (PDL2); a transmembrane domain; and a cytoplasmic signaling domain. In some embodiments, the extnweltular domain is a programmed death receptor 1 (PDI) extracellular domain. In some embodiments, the cytoplasmic signaling domain is a CD3ζ cytoplasmic domain. In some embodiments, the CAR polypeptide further comprises a DNAX-activating protein 10 (Dap10) co-sgimulatory domain. In some embodiments, the polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 3.
Another aspect of the disclosure provides T lymphocytes genetically modified to express a CAR according to the disclosure.
Another aspect of the disclosure provides a composition for adoptive cell transfer comprising T lymphocytes of the disclosure and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises one or more chemotherapeutic or radiotherapeutic agents.
Another aspect of the disclosure provides a method of treating cancer in a subject in need thereof, wherein cells of said cancer express at least one of PDL1 and PDL2, comprising administering to the subject a therapeutically effective amount of a composition for adoptive cell transfer according to the disclosure. In some embodiments, the cancer is selected from the group consisting of lymphoma, melanoma, myeloma, pancreatic cancer, breast cancer, and ovarian cancer.
Immunotherapy that harnesses the host immune system to fight cancer provides an important option for the treatment of cancer. T cells protect individuals from disease by targeting and eliminating diseased cells, Tumor-specific T cells can be isolated, followed by activation and expansion outside the body, and then re-infused back into the patient to mediate cancer regression, a process termed adoptive cell therapy.
Provided herein are CARs that target PDI ligands, i.e, that target at least one of PDL1 or PDL2. The chimeric PDI receptor (chPDI) polypeptide may comprise an extracellular binding domain specific for at least one of PDL1 and PDL2 a transmembrane domain; and a cytoplasmic signaling domain.
As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound having amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can include a protein's or peptide's 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, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types,
“Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of pulmo peptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
The extracellular binding domain specific for (i.e. that targets, recognizes, or binds) at least one of PDL1 and PDL2 may be the extracellular domain of the PD (CD279) receptor. The PDI receptor is a protein found on the surface of cells, such as T lymphocytes, that has a role in regulating the immune system's response to an individual's own cells. In humans, the PDI protein is encoded by the PDCDI gene. A representative amino acid sequence for PDI is provided in SEQ ID NO: 1. In some embodiments, the domain comprises or consists of amino acids 1-55 of PDI.
The transmembrane domain includes a hydrophobic polypeptide that spans the cellular membrane. In particular, the transmembrane domain spans from one side of a cell membrane (extracellular) through to the other side of the cell membrane (intracellular or cytoplasmic). The transmembrane domain may be in the form of an alpha helix or a beta barrel, or combinations thereof. The transmemehrane domain may include a polytopic protein, which has many transmembrane segments, each alpha-helical, beta sheets, or combinations thereof. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In another embodiment, the transme ft) brane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane don tins of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
For example, a transmembrane domain includes a transinerobrane domain of a T-cell receptor α or β chain, a CD3 zeta chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.
An artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.
A cytoplasmic signaling domain of a CAR may be responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation or inhibition of the immune cell and immune response. In other words, the signal transducing domain may be responsible for the activation or inactivation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. Thus, the term “cytoplasmic signaling domain” meters to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function. Examples of signal transducing domain for use in CARs of the present disclosure can be the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. Cytoplasmic signaling sequences can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases. Examples of ITAM can include as non limiting examples those derived from TCRzeta, FcRgamma, FeRbeta, FeRepsilon, CD3gamma, CD3delta, CD3epsilon, CD3, CD22, CD79a, CD79b and CD66d. In some embodiments, the signaling transducing domain of the CAR comprises a CD3zeta signaling domain. In some embodiments, the signaling transducing domain comprises or consists of amino acids 52-164 of CD3zeta.
In some embodiments, the inhibitory domains of PDI are replaced with the activating domains of the cytoplasmic signaling domain, thus switching, the negative PDI signal to become an activating signal for the T cells. This should reduce the immune suppressive effects of PDI and instead induce anti-tumor immunity upon interaction with PDI ligands,
Inclusion of co-stimulatory domains in CAR T cells can enhance T-cell functions, including cytokine secretion, differentiation, cytotoxicity, proliferation and survival. Co-stimulatory receptors that are compatible with CARs of the present disclosure include, but are not, limited to, functional domains from Dap10. CD28, OX40, ICOS, 4-IBB (CD137), NKG2C, and NKG2D. and active fragments, functional derivatives, and combinations thereof. As shown in Example 1, CD28 and Dap10 activate many similar pathways including phospinaidylinositol-3 kinase, AKT/Protein Kinase B and mitogen-activate protein kinases, However, CD28 and Dap10 stimulation seem to have unique effects on effector T cells, including differential activation of signal transduction. pathways including β-catenifi, nuclear factor-κB and mammalian target of rapainyein (mTOR), leading to dissimilar cytokine secretion and T-cell differentiation. Specifically, when compared with CD28, co-stimulation through Dap10 induces CD8 T-cell memory differentiation and secretion of pro-inflammatory but not anti-inflammatory cytokines, both of which seem to be preferable characteristics for successful CAR T-cell therapy. Hence, in some embodiments, inclusion of the Dap10 co-stimulatory domain in CARs may be preferential to CD28.
Functional-conservative derivatives or variants of a polypeptide as disclosed herein may result from modifications and changes that may be made in the structure of the polypeptide (and in the DNA sequence encoding it), and still obtain a functional molecule with desirable characteristics (e.g. tumoricidal and/or immunostimulatory effects). Functional-conservative derivatives may also consist of a fragment of a polypeptide that retains its functionality.
Accordingly, functional-conservative derivatives or variants are those in which a given amino acid residue in a protein has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, front 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A functional-conservative derivative also includes a polypeptide which has at least 70%, 75%, 80%, 85%, 90%, or 95% or more amino acid identity as determined by BLAST or FASTA algorithms and which has the. saale Mii'Ataritially similar properties or functions as the native or parent protein to which it is compared. For example, certain amino, acids may be substituted for other amino acids in a protein structure without appreciable loss of tumoricidal effects. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA encoding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the polypeptide sequences of the disclosure, or corresponding DNA sequences which encode said polypeptides, without appreciable loss of their biological activity. Said nimoricidal activity and immunostimuolatory activity can be assessed by various techniques well-known in the art, such as for instance the assays referred to in the Examples.
As outlined above, conservative amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like, Exemplary substitutions which take several of the foregoing characteristics into consideration are well known to those of skill in the art and include; arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
A polynucleotide of the disclosure can be cloned into a vector. A “vector” is a composition of matter which includes am isolated polynucleotide and which can be used to deliver the isolated polynucleotide to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids phagemid, cosmid, and viruses, Viruses include phages, phage derivatives. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer or nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to adenoviral vectors, adeno associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
Tn one embodiment vectors include cloning vectors, expression vectors, replication, vectors, probe generation vectors, integration vectors, and sequencing vectors. In an embodiment, the vector is a viral vector, in an embodiment, the viral vector is a retroviral vector or a lentiviral vector. In an embodiment, an engineered cell is virally transduced to express the polynucleotide sequence.
Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endomiclease sites, and one or more selectable markers.
Expression of chimeric antigen receptor polynucleotide may be achieved using, for example, expression vectors including, but not limited to, at least one of a SFFV or human elongation factor 11a (EF) promoter, CAG (chicken beta-actin promoter with CMV enhancer) promoter human elongation factor 1a (EF) prtimoter. Examples of less-strong/lower-expressing promoters utilized may include, but is not limited to, the simian virus 40 (SV40) early promoter, cytomegalovirus (CMV) immediate-earl v promoter, Ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK.) promoter, or a part thereof. Inducible expression of chimeric antigen receptor may be achieved using, for example, a tetracycline responsive promoter, including, but not limited to, TRE3GV (Tet-response element, including all generations and preferably, the 3rd generation), inducible promoter (Clontech Laboratories, Mountain View, CA) or a part or a combination thereof.
Methods of introducing and expressing genes into a cell are known in the art, in the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.
Adoptive T cell therapy involves isolating T cel s from an individual's blood and genetically engineering those T cells to express a CAR of the disclosure. The engineered T cells are then grown ex vivo and infused'back into the individual. The CAR T cells can then bind the targeted antigen on cancer cells and kill them.
An embodiment of the disclosure provides an engineered cell, e.g. a T cell/lymphocyte, expressing a CAR polypeptide as described herein or polynucleotide encoding for the same, and described herein. An “engineered cell” means any cell of any organism that is modified, transformed, or manipulated by addition or modification of a gene, a DNA or RNA sequence, or protein or polypeptide. Isolated cells, host cells, and genetically engineered cells of the present disclosure include isolated immune cells, such as NK cells and T cells that contain the DNA or RNA sequences encoding a CAR or CAR complex and express the chimeric receptor on the cell surface. Isolated host cells and engineered cells may be used, for example, for enhancing an NK activity or a T lymphocyte activity, treatment of cancer, and treatment of infectious diseases.
Any cell capable of expressing and/or capable of integrating the chimeric antigen receptor polypeptide, as disclosed herein, into its membrane may be used. In an embodiment, the engineered cell includes immunoregulatory cells.
Immtmoregulatory cells include T-cells (or T lymphocytes), such as CD4 T-cells (Helper T-cells), CD8 (Cytotoxic T-cells, CTLs), and memory T cells or memory stem cell T cells. In another embodiment, T-cells include Natural Killer T-celIs (NK T-cells).
An embodiment of the disclosure provides a method of treating cancer in a subject in need thereof, wherein cells of said cancer express at least one of PDL1 and PDL2, comprising administering to the subject a therapeutically effective amount of a composition comprising T cells genetically engineered to express a CAR according to the disclosure.
Depending upon the nature of the cells, the cells may be introduced into a host organism, e.g. a mammal, in a wide variety of ways. The cells may be introduced at the site of the tumor, in specific embodiments, although in alternative embodiments the cells hone to the cancer or are modified to hone to the cancer. The number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, the stability of the recombinant construct, and the like. The cells may be applied as a dispersion, generally being injected at or near the site of interest. The cells may be in a physiologically-acceptable medium.
In particular embodiments, the route of administration may be intravenous, intraarterial, intraperitoneal, or subcutaneous, for example. Multiple. administrations may be by the same route or by different routes. In some embodiments, multiple doses, e.g. 2, 3, 4, 5, or more doses are given over a period of time, e.g. over 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days.
One of the adverse effects following infusion of CAR T cells is the onset of immune activation, known as cytokine release syndrome (CRS). In some embodiments, a co-stimulatory domain that does not induce, elevation of one or more cytokines selected from IFN-γ, GM-CSF, IL-10 and IL-6 is used. In some embodiments, a CRS therapy such as tocilizumab an IL-6 antagonist) is administered concomitantly or sequentially with the CAR of the disclosure. In some embodiments, radiotherapy andA.ir chemotherapy is administered concomitantly or sequentially with the CAR of the disclosure.
As Used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth, Hyperproliferative and neopiastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a diselse state. The term is meant to include all types of cancerous growths or oneogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
The term “cancer metastasis” has its general meaning in the art and refers to the spread of a tumor from one organ or part to another non-adjacent organ or part.
Any cancer or metastatic cancer expressing at least one of PDL1 and PDL2 may be targeted using the inventive therapy including, but not limited to lymphoma, melanoma, myelomia, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, brain cancer, liver cancer, kidney cancer, lung cancer, spleen cancer, gall bladder cancer, anal cancer, testicular cancer, ovarian cancer, cervical cancer, skin cancer, bone cancer, and colon cancer.
The terms “subject” and “patient” are used interchangeably herein, and refer to an animal such as a mammal, which is afflicted with or suspected of having, at risk of, or being pre-disposed to cancer. The terms may refer to a human. The terms also include domestic animals bred for food, sport, or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals, goats, apes (e.g. gorilla or chimpanzee), and rodents such as rats and mice. Typical subjects include persons susceptible to, suffering from or that have suffered from cancer.
The term “treating” or “treatment”, as used here means reversing, alleviating, inhibiting the progress of, or ameliorating the disorder or condition to which such term applies, or one or more sympiiiros of such disorder or condition. For example, the treatment of the disclosure may slow the growth of said cancer, reduce the number of tumor cells in said cancer, reduce tumor load, or eliminate said cancer.
By a “therapeutically effective amount” is meant a sufficient amount of the molecule to treat a cancer, (for example, to limit tumor growth or to slow or block tumor metastasis) at a reasonable benefitirisk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the molecules and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific polypeptide employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the Specific polypeptide employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For ex-ample, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
The T cell therapy described herein may be combined with standard-of-care treatments (e.g., radiation therapy, hormonal therapy). In some embodiments, the T lymphocytes of the disclosure may be administered sequentially or oncomitandy with one or more chemotherapeutic or radiotherapeutic agents.
In one embodiment, said chemotherapeutic or radiotheraputic agents are a therapeutic active agent used as anticancer agent. For example. said anticancer agents include but are not limited to fludarabine, capecitabine, methotrexate, mercaptopurine, thioguaninc, hydroxyurea, cytarabine, eyelophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, epipodophyllotoxins such as etoposide and teniposide, camptothecins such as irinotecan and topotecan, bleomycin, doxoruhicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epirubicin, 5-fluorouracil and 5-fluorouracil combined with leucovorin, taxane such as docetaxel and paclitaxel, levamisole, estramustine, nitrogen mustards, nitrosoureas such as carmustine and lomustin, vinca alkaloids such as vinblastine, vincristine, vindesine and vinorelbine, imatinib inesylate, hexamethylmelamine, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbiroycin A, genistein, erbstatio, and lavridostin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxins, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycin, bleornycin, anthracyclines, MDR inhibitors and Ca2+ ATPase inhibitors.
Additional anticancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polythodies.
Further therapeutic active agents may be an antiemetic agent. Suitable antiemetic agents include, hut are not limited to, metoclopramide, domperidone, prochlorperazine, promethazine, ch orpromazine, trimethobenzamide, ondanse granisetron, hydroxyzine, acetylleucine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebripride, cyclizine, dimenhyddnate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, pipamazine, scopolamine, sulpiride, tehahydrocannabinols, thiethylperazine, thioproperazine and tropisetroil. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.
In still another embodiment, the other therapeutic active agent can be an opioid or non-opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, buprenorphine, meperidine, loperamide, ethoheptazine, betaprodine, diphenoxylate, fentanyl, sufentanil, alfentanil, remifemanil, levorphanol, dextromethorphan phenazone, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diciorenac, diflunisal, etodolac, fenoprofen, flurbiprolen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate mefenamic acid, nabumetone, naproxen, piroxicam and sulindac.
In yet another, embodiment, the further therapeutic active agent can bt an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such, as diazeparti, lorazepam, oxazapara, clorazepate, clonazepam, chlordiazepoxide and alprazolam.
The term “radiotherapeutic agent” as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation. For instance, the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.
Another aspect of the disclosure relates to a pharmaceutical composition comprising a T lymphocyte according to the disclosure and a pharmaceutically acceptable carrier. “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject, such as a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
The T lymphocytes of the disclosure may be contained in physiological saline, phosphate buffered saline (PBS), culture medium, or the like in order to maintain stability.
In some embodiments, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodiurn phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
The pharmaceutical forms suitablee for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
Solutions comprising compounds of the disclosure as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropyicellulose, Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils, Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms,
Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, Cartark, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylainine, trimethylamine, histidine, procaine and the like.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof', and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like, In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride, Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium frionostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the T lymphocytes in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into is sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as, is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug releaserules and the like can also be employed.
For parenteral administratio in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
In addition to the compositions of the disclosure formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.
Any of the compositions described herein may be comprised in a kit. In a non-limiting example, one or more cells for use in cell therapy that harbors recombinantly expressed CARs according to the disclosure and/or the reagents to generate one or more cells for use in cell therapy may be comprised in a kit. The kit components are provided in suitable container means. In specific embodiments, the kits comprises recombinant engineering reagents, such as vectors, primers, enzymes (restriction enzymes, ligase, polymerases, etc.), buffers, nucleotides, etc.
Some components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present disclosure also will typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context dearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element, As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual enthodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
The invention is further described by the following non-limiting examples which further illustrate the invention, and are. not intended, nor should they be interpreted to, limit the scope of the invention,
EXAMPLE 1. Adoptive transfer of murine T cells expressing a chimeric-PDI-Dap10 receptor as an immunotherapy for lymphoma.
SummaryAdoptive transfer of T cells is a promising cancer therapy and expression of chimeric antigen receptors can enhance tumor recognition and T-cell effector functions. Provided herein is a murine chimeric PDI receptor (chPD1) comprising the PDI extracellular domain fused to the cytoplasmic domain of CD3ζ Additionally, chimeric antigen receptor therapies use various co-stimulatory domains to enhance efficacy. Hence, the inclusion of a Dap10 or CD28 co-stimulatory domain in the chPDI receptor was compared to determine which domain induced optimal anti-tumor immunity in a mouse model of lymphoma, The chPDI T cells secreted pro-inflammatory cytokines and lysed RMA lymphoma cells. Adoptive transfer of chPDI T cells significantly reduced established tumors and led to tumor-free survival in lymphoma-bearing mice. When comparing chPDI receptors containing a Dap10 or CD28 domain, both receptors induced secretion of pro-inflammatory cytokines; however, chPDI-CD28 T cells also secreted anti-inflammatory cytokines whereas chPDI-Dap10 T cells did not. Additionally, chPDI-Dap10 induced a central memory T-cell phenotype compared with chPDI-CD28, which induced an effector memory phenotype. The chPDI-Dap10 T cells also had enhanced in vivo persistence and anti-tumor efficacy compared with chPDI-CD28 T cells. Therefore, adoptive transfer of chPDI T cells represents a novel therapy for lyMphorna and inclusion of the Dap10 co-stimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile and T-cell differentiation phenotype for anti-tumor therapies.
Materials and MethodsGeneration wrtPDI and chPDI constructs
Murine cDNA clones of CD3ζ PD1, CD28 and Dap 10 were purchased from OriGene (Rockville, MD). The chPD1-Dap10 and chPDI-CD28 receptors were created by overlapping PCR using Phusion® high fidelity DNA polymerase (New England BioLabs, Ipswich, MA). To create the chPD1-Dap10 receptor, the extracellular domain of the murine PDI receptor [amino acids (aa) 1-155] was fused in frame to the transmembrane region of CD28 (aa 141-177) and the cytoplasmic domains of Dap10 (an 57-79) and CD3ζ (aa 52-164). To create the chPDI-CD28 receptor, the extracellular domain of the murine PDI receptor (aa 1-155) was fused in frame to the transmembrarie (aa 141-177) and cytoplasmic (aa 178-218) domains of CD28 and CD3ζ (aa 52-164). To create the wild-type PDI (wtPDI) receptor, the extracellular and transmembrane domain of the PDI receptor (aa 1-190) was used. All constructs were cloned into the pQCXIN retroviral expression vector using NotI and EcoRI digestion of the plasmid and constructs and were subsequently ligated into the vector. Ecotropic retroviral supernatants were expressed using the EcoPack 2-293 cell line according to the manufacturer's instructions (Cloutech, Mountain View, CA). Xfect polymer was used to co-transfect EcoPack 2-293 cells, with the pEco envelope vector and, the pQCXIN retroviral expression vector from the RetroX-Q vector set (Clontech). RetroX Concentrator was used to concentrate the ecotropic retroviral supernatants before transduction of primary murine T cells.
Expression wtPDI and chPDI receptors in T cells
Male C57BL/6 (B6) and B6,SJL-Piprc60 (Ly5.1 congenic) mice were purchased from Taconic Biosciences (Hudson, NY). Mice were between 8 and 12 weeks of age at the start of each experiment. All animal work was performed in accordance and with approval from Longwood University's Institutional Animal Use and Care Committee. Splenocytes from B6 or Ly5.1 congenic mice were activated with concanavalin A (1 μg/ml ) for 18 hr. T cells (0.5×106 cells/ml) were transduced by centrifugation at 1000 g for 1 hr in the presence of 8 μg/ml polybrene and 25 U/ml recombinant human interleukin-2 (IL-2) and were subsequently cultured for 6 hr before retroviral supernatants were removed and replaced with fresh complete RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 streptomycin, 1 mM pyruvate, 10 mM HEPES, 0.1 mM non-essential amino acids and 50 μM 2-mercaptoethanol. Two days after infection, T cells were selected in complete RPMI-1640 medium containing G418 (0.5 mg/ml) plus 25 U/ml recombinant human IL-2 for an additional 3 days. Viable cells were isolated using Histopaque-1083 (Sigma, St Louis, MO) and expanded for an additional 2 days without G418 before functional analysis.
RT-PCRTotal RNA was isolated from RMA cells or T cells using the SV Total RNA isolation kit according to the manufacturer's instructions (Promega, Madison, WI), cDNA was created using RevertAid First Strand cDNA. synthesis kit using random hexamer primers (Fermentas, Waltham, MA). As a template for RT-PCR, 100 ng of cDNA was used to measure gene expression of PDL1, PDL2 and β-actin. Maxima SYBRGreen qPCR Master Mix (Thermo Scientific, Waltham, MA) and gene specific primers were used: β-actin F 5′-GTGTGATGGTGGGAATGGGTCAGA-3′ (SEQ ID NO: 4) β-actin R. 5′-TACGACCAGAGGCATACAGGGACA-3′ (SEQ ID NO: 5), PDL1 F 5′-GCTCCAAAGGACTTGTACGTG-3′ (SEQ ID NO: 6). PDL1 R 5′-TGATCTGAAGGGCAGCATTTC-3′ (SEQ ID NO: 7), PDL2 F 5′-CTGCCGATACTGAACCTGAGC-3′ (SEQ ID NO: 8), PDL2 R 5′GCGGTCAAAATCGCACTC-3′ (SEQ ID NO: 9). Gene-specific primers for T-cell differentiation genes T-bet, BLIMP1, Eomes and BCL6 were previously described. Primers were purchased from Integrated DNA Technologies (Coralville, IA).
Flow cytometry
The expression of PDLI and PDL2 on RMA and T cells and of PDI for T cells was tested using flow cytometry. Cells were stained with allophycocyanin-labelled anti-PDL1 (clone 10F.9G2), phycoerythrin-labelled ariti-PDL2 (clone TY25), or phycoerythrin-labelled anti-PDI (clone RMP1-30) antibodies or isotype controls. For T-cell differentiation studies, wtPDI or chPDI T cells (2×105 cells/well) were stimulated with RMA cells (2×105 cells/well) for 24 hr and were analysed for cell surface marker expression by flow cytometry. Cells were stained with phycoerythrin-conjugated ariti-CD127 (clone A7R34) or anti-KLRG1 (clone 2FI/KLRG1) and allophycocyanin-conjugated anti-CD621L (clone MEL-14) isotype controls. To analyse T-cell surface expression, RMA cells were labelled with CFSE before incubation and CFSE+ cells were gated out. All antibodies were purchased from BioLegend (San Diego, CA). Cell fluorescence was measured using an Accuri C6 flow cytometer.
Cytokine production and cytotxicity by chPDI T cells
The chPD1, wtPDI and non-transduced T cells (105 ) were cultured with RMA cells (105) or medium in a mund-bottom 96-wll plate. After 24 hr, cell-free supernatants were tested for the presence of interferon-γ(INF-γ), tumor necrosis factor-α (INF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2 and IL-10 by ELISA according to the manufacturer's instructions (BioLegend). Cytokine and chemokine secretion was also measured in cell-free supernatants using mouse T helper cytokine and mouse pro-inflammatory chemokine LEGENDPlex assays (BioLegend) according to the manufacturer's instructions.
To determine lysis of tumor cells, chPD1, wtPDI and nota-transduced T cells (105) were cultured with RMA at various effector to target ratios (E:T 25:1, 5:1, and 1:1). Specific lysis was measured after 5 hr using a lactate dehydrogenase cytotoxicity assay kit (Pierce, Waltham, MA) according to the manufacturer's instructions. To block PDI receptors, T cells were pre-incubated at 37° for 2 hr with anti-PDI monoclonal antibodies (clone RMP1-14, 20 μg/ml, Low Endotoxin, Azide-Free LEAP purified, BioLegend) or isotype control monoclonal antibodies before addition of target cells.
Treatment of mice with genetically modified T cells
RMA and RMA-GFP cells were grown in complete RPMI- 1640. RMA-GFP cells (2×106) were injected intravenously into B6 mice. For tumor burden experiments, mice were administered one dose of wtPDI of chPD1-modified T cells (5×106) intravenously 2 days or 5 days after tumor injection, or two doses of T cells 5 and 8 daysafter tumor injection. For determination of tumor burden, spleens and lymph nodes (axillary, brachial and inguinal) were collected 13 days after tumor injection. The lymphoid tissues were mechanically teased and red blood cells were lysed with ACK lysis buffer (0.15 mol/l NH4Cl, 1 mmol/l KHCO3, 0.1 mmol/l). Cells were counted and the percentage of GFP+ cells was determined via flow cytometry. The total number of tumor cells was determined by multiplying the percentage of GFP+ cells by the total number of cells. For survival studies, mice were treated with wtPDI or chPDI T cells (5×106) on day 5 and 8 after tumor cell injection. The health of the mice was monitored closely and mice were killed when signs of stress (laboured breathing, dragging legs, hunched back, or ruffled fur) were observed. For analysis of T-cell survival, RMA-bearing mice were treated 5 days after tumor cell injection with congenic Ly5.1+chPD1-Dap10 or chPD1-CD28 T cells (5×106) intravenously and mice were killed 1, 3, 7, 10, 14 or 18 days after T-cell injection. Spleen and lymph node cells wen incubated with FcR block and mouse γ-globulin (Jackson Immuno.Research, West Grove, PA) to prevent non-specific binding, and stained with phycoerythrin-coinagated anti-CD3 and allophycocyanin-conjugated anti-CD45.1 (clone A20) and analysed by flow cytometry.
Statistical analysis
Statistical analysis was conducted using an impaired, two-tailed Student's t-test or analysis of variance with a post-hoc Tukey test when comparing multiple groups, The data were determined to be normally distributed using the Shapiro-Wilk test. The program R was used for statistical analysis of the data. All experiments were run in triplicate on at least two independent sets of T cells and P values <0.05 were considered significant. For survival studies, Kaplan-Meier survival curves were plotted and analysed using the Log rank test and pRism software (GRAPHPAD Software, San Diego, CA).
ResultschPDI T secrete pro-inflanmatory cytokines and lyse PDL-expressing RMA cells in a PD1-dependent manner
To target PDI ligands expressed on tumor cells, a chPDI receptor was created by fusing the extracellular region of the PDI receptor with the intracellular regions of the Dap10 co -stimulatory receptor and CD3ζ (
Sometimes activated T cells express PDL1, which could potentially cause chPDI T cells to kill each other.9Therefore, the expression of PDL1 and PDL2 was assessed on chPD T cells, The number of chPDl T cells obtained was similar to the number of wtPDI T cells for all T-cell batches tested. In addition, significant PDL1 or PDL2 expression on chPDI and wtPDI T cells, was not observed (
To determine if the murine lymphoma cell line RMA was a potential target of chPDI T cells, the expression of PDL1 and PDL2 was measured. RMA cells expressed cell surface PDL1 and PDL2, as determined by flow cytometry (
In addition to tumor cell lysis. T cells secrete pro-inflammatory cytokines to enhance antitumor immunity.15,30Compared with non-transduced or wtPDI T cells, chPDI T cells secreted significant amounts of pies-inflammatory cytokines INF-γ, TNF-α, GM-CSF and IL-2 but did not secrete anti-inflammatory cytokine IL-10 when cultured with RMA (
Treatment with chPDI T cells leads to a reduction in tumor burden and an increse in survival of RMA-GFP-bearing mice
When injected intravenously into mice, RMA tumor cells traffic to the spleen and lymph nodes; hence, this model recapitulates features of human lymphoma in syngenete, immunocompetent mice.31Therefore, the potential of using chPDI T cells in vivo as a therapy for lymphoma was investigated. Because ligands for PDI may also be expressed on healthy issues, the safety of chPDI T cells was first tested. The chPDI T cells did not lyse or secrete IFN-γ when cultured with splenocytes, liver cells or lung cells isolated from a naive mouse. In addition, after injection caf chPDI T cells, naive mice did not show any adverse symptoms or increased levels of serum IFN-γ, suggesting that chPDI T cells did not target healthy tissues. Next, to test, the anti-tumor efficacy of cliPDI cells, lymphoma-bearing mice were treated with a single dose of chPDI T cells 2 days after tumor cell injection and tumor burden was measured in the spleen and lymph nodes (
To test the in vieo therapeutic efficacy of chPDI T cells against a more established tumor burden, mice were treated with wtPDI sar chPDI T cells 5 days after tumor cell injection. Treatment with chPDI T cells significantly reduced these established tumors, although there was a low yet detectable level of tumor cells in the spleens and lymph nodes of the chPDI T-cell-treated mice (
chPDI-Dap10 T cells secrete increased levels of pro-inflammatory cytokines and decreased levels anti-inflammatory cytokines compared with chPDI-CD28 T cells
The inclusion of co-stimulatory domains in CARs enhances T-cell anti-tumor effector functions and each co-stimulatory receptor has a unique effect on T cells.15,16Therefore to compare the inclusion of the Dap10 domain with another co-stimulatory receptor, a chPDI receptor was made that contained the cytoplasmic domain of CD28 instead of the Dap10 cytoplasmic domain (
Inclusion of Dap10 co-stimulatory domain incduces a central memory phenotype in chPDI T cells
Another characteristic that is important for CAR T-cell efficacy is the differentiation phenotype of the T cells. CD28-containing CARs often induce an effector memory or effector cell phenotype and do not live as long in vivo whereas CARs that induce a central memory phenotype usually persist. longer in vivo and often have stronger anti-tumor efficacy.3 Stimulation of natural killer group 2D (NKG2D)/Dap10 has recently been shown to induce a central triemory phenotype in marine effector CD8 cells, so the differentiation phenotype of chPDI-Dap10 and chPDI-CD28 T cells was compared.29 When cultured with RMA cells, chPD -CD28 T cells increased the gene expression of transcription factors involved in effector cell differentiation. T-bet and BLIMP-I, whereas chPDI-Dap10 T cells increased the expression of transcription factors that support central memory differentiation, Eomes and BCL-6 (
Treatment with chPDI-Dap10 T cells leads to a greater reduction in tumor burden and increased survival of RMA-GFP-bearing mice compared with treatment with chPDI-CD28 T cell
To compare the in vivo therapeutic efficacy of chPDI-Dap10 and chPDI-CD28 T cells, lymphoma beating mice were treated with two doses of wtPD , chPDI-Dap10 or chPDI-CD28 T cells. Treatment with chPDI-Dap10 or chPDI-CD28 T cells significantly reduced tumor burden, but chPDI-Dap10 T cells reduced tumor burden significantly more than chPDI-CD28 T cells (
The introduction of CARs has dramatically increased the potential efficacy of T-cell therapy for cancer.1,2,22However, the up-regulation of inhibitory receptor expression on T cells, including expression of the PDI receptor, and expression of inhibitory ligands in the tumor microenvironment limit CAR T-cell responses and efticacy.9,34-36 This study demonstrates that expression of the novel chPDI receptor enhances T-cell anti-tumor efficacy in a mouse model of lymphoma. Our results suggest that chPDI receptor-transduced T cells target PDL expression on tumors and that interaction with PDL induces activation instead of inhibition of the T cells. The chPDI-expressing T cells secreted pro-inflammatory cytokines and lysed PDL-expressing tumor cells and also reduced tumor burden and increased tumor-free survival in lymphoma-bearing mice. In addition, the chPDI receptor containing a Dap10 co-stimulator), domain was functionally superior compared with the chPDI receptor-containing a CD28 co-stimulatory domain,
Many new mechanisms to shield T cells from PDI inhibition are being developed. In addition to PDI blockade, expression of a PDI-CD28 switch receptor, which replaces the cytoplasmic domain of PDI with the cytoplasmic domain of CD28, has been shown to prevent T-cell inhibition:37-40 When co-expressed with a tumor-specific T-cell receptor or CAR, the PDI-CD28 switch receptor induces T-cell activation as shown by extracellular signal regulated kinase phosphorylation, cytokine secretion, proliferation, granzyme B expression and enhanced anti-tumor function.37-40 A primary goal of our study was to test the efficacy of T cells expressing a CAR that directly connects the PDI-extracellular domain to the intracellular domains of Dap10 of CD28 and CD3ζ, so providing both the activation and co-stimulatory signal all within the same receptor and eliminating the need for co-expressing two receptors in the T cells. Furthermore, many of the previous PDI switch receptor studies tested the anti-tumor efficacy of human T cells in immunodeficient mouse models.37,38 However, CAR T cells often require the induction of host immune responses for full anti-tumor efficacy.15,30-32 In addition, testing human CAR T-cell efficacy in immunodeficient mice does not investigate the role of other immune cells, including myeloid-derived suppressor cells and regulatory T cells, in the anti-tumor immune response. Therefore, the creation of a murine chPDI receptor allows the study of chPDI T-cell efficacy in an immunocompetent host and represents the tumor microenvironment the T cells would be likely to encounter in patients.
The inclusion of a co-stimulatory domain in CARs increases antitumor efficacy and the majority of CAR T-cell clinical trials are using second-generation CARs consisting of CD3ζ and CD28 or 4-IBB co-stimulatory domains,1,3,4,15,16,33,41 One difference that is observed between CAR T cells with CD28 or 4-IBB signalling domains is that inclusion of 4-IBB induces a central memory phenotype, and these T cells persist longer in vivo, and have stronger anti-tumor efficacy whereas T cells with a CD28-CAR induce an effector memory or effector cell phenotype and do not live as long in vivo 3,42 The induction of a central memory phenotype in T cells expressing a 4-IBB-containing CAR was partially caused by a distinct metabolic signature that included enhanced respiratory capacity, increased fatty acid oxidation and enhanced mitochondrial biogenesis, whereas CAR T cells with CD28 domains induced effector memory cells and had an enhanced glycolysis signature.42 In the present study, the inclusion of the Dap10 co-stimulatory domain induced superior anti-tumor immunity in vivo. Without being bound by theory, this could potentially be caused by the induction of a central memory phenotype and enhanced in vivo survival of the chPDI-Dap10 T cells. Stimulation of NKG2D/Dap10 has recently been shown to induce a central memory phenotype in murine effector CD8 cells in part due to differential activation of mTOR.29 Interestingly, mTOR activates specific metabolic pathways in T cells such as aerobic glycolysis and compared with CD28 co-stimulation, activation through NKG2D/Dap10 shows weaker activation of mTOR.29,43 Hence, the induction of mTOR activation, metabolism and cell differentiation are likely key characteristics in CAR T-cell success.
In the clinic, one of the adverse effects following infusion of CAR T cells is the onset of immune activation, known as cytokine release syndrome.42,45 This may include elevation of cytokines including IFN-γ, GM-CSF, IL-10 and IL-6 following CAR T cell infusion and the dramatic increase in cytokines generally correlates with expansion and activation of adoptively transferred cells.45 In this study, one difference observed between chPDI-Dap10 and chPDI-CD28 T cells was the differential expression of cytokines, with chPDI-Dap10 T cells secreting, higner amounts of pro-inflammatory cytokines TNF-60 , GM-CSF, TL-17 and IL-21 and chPDl-CD28 T cells secreting more IL-2 and T helper type 2/anti-inflammatory cytokines IL-5 and IL-10 (
In this study, inclusion of the Dap10 co-stimulatory domain in the chPDI receptor did not induce secretion of IL-10 whereas inclusion of the CD28 domain did. CD28-induced secretion of IL-10 has been shown to alter T-cell, anti-tumor responses through down-regulation caf MHC niolecules, CD28 ligands and intercellular adhesion molecule-1 on antigen-presenting cells.48 As a consequence, host T-cell responses are inhibited and the secretion of pro-inflammatory eytokines is repressed. In addition, secretion of anti nflammatory cytokines has been shown to not only inhibit CAR T-cell efficacy but also to induce chronic toxicity in some studies,49 Therefore, without being bound by theory, the decrease in IL-10 secretionfrom chPDI-Dap10 T cells may contribute to their enhanced in vivo anti-tumor efficacy.
In sununary, a new chPDI receptor s developed that induces strong antitumor T-cell responses and induction of long-term, tumor-free survival in an immunocompetent mouse model of lymphoma. The strong induction of pro-inflammatory cytokines induced by inclusion of a Dap10 co-stimulatory receptor may be beneficial for anti-tumor therapy.
EXAMPLE 2. Human T cells expressing a chimeric-PDI-Dap10 receptor as an immunotherapy
Adoptive transfer of tumor-reactive T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. As presented in Example 1, one receptor that is a target for a new chimeric antigen receptor is PDI because the ligands for the PDI receptor are expressed on ninny cancer types. In the present study, a human chimeric PDI receptor (chPDI) consisting of the PDI receptor extracellular domain and the activation domain of CD3 zeta was developed, The Dap10 costimulatory domain was also included in the chPDI receptor as discussed in Example 1. The nucleic acid sequence of the CAR is presented in SEQ ID NO: 2 and the amino acid sequence is presented in SEQ ID NO: 3. To determine if this novel CAR could target a wide variety of tumors, the anti-tumor efficacy of chPDI T cells against human lymphoma, melanoma, myeloma, pancreatic, breast, and ovarian cancer cell lines was measured. Of the eight cell lines tested, all expressed PDI ligands on their cell surface, making them potential targets for chPDI T cells. The chPDI receptor was successfully expressed on the surface of human T cells and expression of chPD T cells induced significant tumor cell lysis of all tumor cell lines and secreted pro-inflammatory TNFα, IL-2, GM-CSF, IL-17, and IL-21) cytokines, Therefore, adoptive transfer of chPDI-Dap10 T cells represents a novel therapeutic strategy to treat multiple types of cancer.
References1 Johnson L A. June C H. Driving gene-engineered T cell immunotherapy of cancer, Cell Res 2017; 27:38-58.
2 Chmielewski M, Hombach A A, Abken H. Antigen-specific T-cell activation independently of the MHC: chimeric antigen receptor-redirected T-cells. Front Immunol 2013; 4:371.
3 Holzinger A, Barden M, Abken H. The growing world of CAR -F cell trials: a systematic review. Cancer Immunother 2016 65:1433-50.
4 Yong C S, Dardalhon V. Devaud C, Taylor N, Darcy P K, Kershaw M H, CAR T-cell therapy of solid tumors. Immunol Cell Biol 2017; 95:356-63.
5 Wherry E J. T cell exhaustion.Nat Immunol 2011: 12.492-9.
6 Speiser D E, Ho P C, Verdeil G, Regulatory circuits of T cell function in cancer. Nat Rev Inammol 2016: 16:599-611.
7 Chemnitz J M, Parry R V, Nichols K E, June C H, Riley J L. SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation, J Immunol 2004; 173:945-54.
8 Ahmadzadeh M, Johnson L A, Heemskerk Wunderlich J R, Dudley M E, White D E, Rosenberg S A. Tumor antigen-specific CD8 T cells infiltrating, the tumor express high levels of PD-1 and are functionally impaired, Blood 2009; 114:1537 1
9 Pardon D M. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012; 12:252-64.
10 Kamphorst A O, Ahmed R. Manipulating the PD-1 pathway to improve immunity. Curr Opin Immunol 2013; 25:381-8.
11 Postow M A, Callahan M K, Wolchok J D. Immune checkpoint blockade in cancer therapy. J Clin Oncol 2015; 33:1974-82.,
12 Bardhan K. Anagnostou T, Boussiotis V A. The PDI1:PD-L1/2 pathway from discovery to clinical implementation. Front Immunol 2016; 7:550.
13 Xia Y, Jeffrey Medeiros L, Young K H. Signaling pathway and dysregulation of PDI and its ligands in lymphoid maligancies. Biochim Biophys Acta 2016; 1865:58-71.
14 Ansell S M. Where do programmed death-1 inhibitors fit in the management of malignant lymphoma? J Oncol Pract 2016; 12:101-6.
15 Redeker A, Arens R. Improving adoptive T cell therapy: the particular role of T cell costimulation, cytokines, and post-transfer vaccination, Front Immunol 2016; 7:345.
16 Hornbuch A A, Holzinger A. Ahken H. The weal and woe of costimulation in the adoptive therapy of cancer with chimeric antigen receptor tCAR y-redirected T cells, Curr Mol Med 2013; 13:1079-88.
17 Savoldo B, Ramos C A, Liu. E, Mims M P, Keating M I, Carrum G et al. CD28 costimulation improves expansion and persistence of chimeric antigen recptor-modified T cells in lymphoma patients. J Clin Invest 2011; 121:1822-6.
18 Kowolik C M, Topp M S, Gonzalez S, Pfeiffer T, Olivares S. Gonzalez N et al. CD28 costimulation provided through a CD19 specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res 2006; 66:10995-1004.
19 Sadelain M, Brent R. Riviere I. The basic principles of chimeric antigen receptor design, Cancer Diseov 2013;3:388-98.
20 Benitez A C, Dai Z, Mann H H, Reeves R S. Margineantu D R, Gooley T A et al. Expression, signaling proficiency, and stimulatory function of the NKG2D lymphocyte receptor in human cancer cells. Proc Natl Acad Sci USA 2011; 108:4081-6.
21 Kim E H, Sullivan J A, Plisch E H, Tejera M M, Jatzek A, Choi K Y et at, Signal integration by Akt regulates CD8 T cell effector and memory differentiation, J Immunol 2012; 188:4305-14.
22 Colombetti S, Basso V, Mueller D L, Mondino A. Prolonged TCR/CD28 engagement drives IL-2-independent T cell clonal expansion through signaling, mediated by the mammalian target of rapamycin. J Immunol 2006; 176:2730-8.
23 Upshaw J L, Leibson P I, NKG2D-mediated activation of cytotoxic lymphocytes: unique signaling pathways and distinct functional outcomes, Semin Immunol 2006; 18:167-75.
24 Maasho K, Opoku-Ariane J, Marusina A I, Coligan J E, Borrego F. NKG2D is a costimulatory receptor for human naive CD8+ T cells, J Immunol 2005; 174:4480-4.
25 Markiewicz M A, Carayannopoulos L N, Naidenko O V, Matsui K, Burack W R, Wise E L et Costimulation through NKG2D enhances marine CD28+CTL, function: similarities and differences between NKG2D and CD 28 costimulation. J Immunol 2005; 175:2825-33.
26 Rajasekaran K, Xiong V, Fong L, Gorski J, Malarkannan S. Functional dichotomy between NKG2D and CD28-mediated co-stimulation in human CD8+ T cells, PLoS ONE 2010; 5:e12635.
27 Barber A. Sentman C L. NKG2D receptor regulates human effector T-cell cytokine production. Blood 2011; 117:6571-81.
28 Whitman E, Barber A. NKG2D receptor activation of enhances inflammatory cytokine production in murine effector CD8+ T cells. Mol Imnumol 2015; 63:268-78.
29 McQueen B, Trace K, Whitman P. Bedsworth T, Barber A. NKG2D and CD28 receptors differentially activate mTOR to alter murine effector CD8+ T cell differentiation. Immunology 2016; 147:305-20.
30 Barber A, Zhang T, Sentman Immunotherapy with shimchimeric NKG2D receptors leads to long-term tumor-free survival and development of host antitumor immunity in murine ovarian cancer. J Immunol 2008; 180:72-8.
31 Zhang T. Barber A, Sentmart C L. Chimeric NKG2D modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways. Cancer Res2007; 67:11029-36.
32 Barber A, Sentman C L. Chimeric NKG2D T cells require both T cell- and host-derived cytokine secretion and perform expression to increase to increase tumor antigen presentation and systemic immunity. J Immunol 2009; 183:2365-72.
33 Lim W A, June C H. The principles of engineering immune cells to treat cancer. Cell 2017; 168:724-40.
34 Ito T, Ueno T, Clarkson M R, Yuan X, Jurewicz M M, Yagita H et al. Analysis of the role of negative T cell costimulatory pathways in CD4 and CD8 T cell-mediated alloimmune responses in vivo, J Immunol 2005; 174:6645-56.
35 Blank C. Mackensen A. Contribution of the PD-LI/PD1 pathway to T-cell exhaustion: an update on implications for chronic infections and tumor evasion. Cancer Immunol Immunother 2007; 56;739-45.
36 Morales-Kastresana A. Labiano S, Quetglas J I, Melero I. Better performance of CARs deprived of the PD-1 brake. Clin Cancer Res 2013; 19:5546-8.
37 Prosser M E, Brown C P, Shami A F, Forman S J, Jensen MC. Tumor PD-LI co-stimulates primary human CD8+ cytotoxic T cells modified to express PDI:CD28 chimeric receptor. Mol Immunol. 2012; 51;263-72.
38 Ankri C, Shamalov K, Horovitz-Fried M, Mauer S, Cohen C J. Human V cells engineered to express a prorammed death 1/28 coxtimulatory retargeting molecule display enhanced antitumor activity. J Immunol 2013; 191:4121-9.
39 Lin X, Ranganathan R, Jiang S, Fan, C, Sun J, Kim S ask al. A chimeric switch-receptor targeting PDI augments the efficacy of second-generation CAR T cells in advanced solid tumors. Cancer Res 2016: 76;1578-90.
40 Kobold S, Grassmann S, Chaloupka M, Lampert C, Wenk S, Kraus F et al. Impact of a new fusion receptor on PD-1-mediated immunosuppression in adoptive T cell therapy. J Cancer Inst 2015; 107:djv146.
41 Hombach A A, Abken H. Of chimeric antigen receptors and antibodies: OX40 and 4IBB costimulation sharpen up T cell-based immunotherapy of cancer. Immunotherapy 2013; 5:677-81.
42 Kawalekar O U, O'Connor R S, Fraietta J A, Guo L, McGettigan S E, Posey A D et al. Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells. Immunity 2016; 44:380-90.
43 Yang K. Chi H. mTOR and metabolic pathways in cell quiescence and functional activation. Semin Immunol 2012; 24:421-8.
44 Lee D W, Gardner K, Porter Louis C U,pt, Ahmed N, Jensen M et al Current concepts in the diagnosis and management of cytokine release syndrome. Blood 2014; 124:188-95.
45 Bonifant C L, Jackson H U, Brentjens R, Curran K J. Toxicity and management in CAR T-cell therapy. Mol. Ther Oncolytics 2016; 3:16011.
46 Davila M L, Riviere I, Wang X, Bartido S, Park I, Curran K et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 2014; 6:224ra.25.
47 Lee D W, Kochenderfer J N, Stetler-Stevenson M, Cui Y K, Delbrook C, Feldman S A et al. T cells expressing CD9 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 2015; 385:517-28.
48 Hombach A A, Heiders J, Foppe M. Chmielewski M, Ahkem H, Ox40 costimulation by a chimeric antigen receptor abrogates CD28 and IL-2 induced IL-10 secretion by redirected CD4 T cells. Oncoimmunology 2012; 1:458-66.
49 Cheadle E J, Sheard V, Rothwell D G, Bridgeman J S, Ashton G, Hanson V et al. Differential role of Th1 and Th2 cytokines in autotoxieity driven by CD19-specific second-generation chimeric antigen receptor T cells in a mouse model. J Immunol 2014; 192:3654-65.
While the invention has been described in its preferred embodiments, those of skill in the art will recognize the invention can be practiced with variations within the spirit and scope of the appended clams.
Claims
1. A chimeric antigen receptor (CAR) polypeptide comprising:
- a programmed death receptor 1 (PDI) extracellular domain;
- a transmembrane domain; a CD3ζ cytoplasmic domain; and a DNAX-activating protein 10 (Dap10) co-stimulatory domain.
2-5. (canceled)
6. A vector comprising a nucleic acid encoding the CAR polypeptide according to claim 1.
7. A T lymphocyte genetically modified to express a CAR according to claim 1.
8. A composition for adoptive cell transfer comprising T lymphocytes according to claim 7 and a pharmaceutically acceptable carrier.
9. The composition of claim 8, further comprising one or more chemotherapeutic or radiotherapeutic agents.
10. A method of treating cancer in a subject in need thereof, wherein cells of said cancer express at least one of PDL1 and PDL2, comprising: administering to the subject a therapeutically effective amount of a composition for adoptive cell transfer comprising T lymphocytes comprising a polynucleotide encoding the CAR polypeptide of claim 1.
11-14. (canceled)
15. The method of claim 10, wherein said cancer is selected from the group consisting of lymphoma, melanoma, myeloma, pancreatic cancer, breast cancer, and ovarian cancer, prostate cancer, brain cancer, liver cancer, kidney cancer, lung cancer, spleen cancer, gall bladder cancer, anal cancer, testicular cancer, ovarian cancer, cervical cancer, skin cancer, bone cancer, and colon cancer.
16. The method of claim 10, further comprising a step of administering one or more of a chemotherapeutic or radiotherapeutic agent.
17. The CAR polypeptide of claim 1, wherein said transmembrane domain is a T-cell receptor alpha chain, a T-cell receptor beta chain, a CD3 zeta chain, CD28, CD3s, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, a functional derivative thereof, or a combination thereof.
18. The CAR polypeptide of claim 1, wherein said transmembrane domain is an artificial transmembrane domain.
19. The method of claim 10, wherein said transmembrane domain is a T-cell receptor alpha chain, a T-cell receptor beta chain, a CD3 zeta chain, CD28, CD3s, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, a functional derivative thereof, or a combination thereof.
20. The method of claim 10, wherein said transmembrane domain is an artificial transmembrane domain.
21. The vector of claim 6, wherein said vector is a viral vector.
22. The vector of claim 21, wherein said viral vector is an adenoviral vector, an adeno-associated virus vector, a retroviral vectors, or a lentiviral vector.
23. The method of claim 16, wherein administration of said composition for adoptive cell transfer and said one or more of a chemotherapeutic or radiotherapeutic agent are administered sequentially.
24. The method of claim 10, further comprising administration of Cytokine Release Syndrome (CRS) therapy.
25. The method of claim 10, wherein said cancer is metastatic cancer.
26. A kit for cell therapy comprising a composition of T lymphocytes genetically modified to express a CAR according to claim 1 in a pharmaceutically acceptable form, said composition in a suitable container.
Type: Application
Filed: Dec 29, 2022
Publication Date: Sep 21, 2023
Inventor: Amorette BARBER (Farmville, VA)
Application Number: 18/147,879
