Patent Application
20230331795
A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Apr. 18, 2023 having the file name “23-0366-US-CIP.xml” and is 57,441 bytes in size.
Activation of the stimulator of interferon genes (STING) pathway through cyclic dinucleotides (CDNs) could be used as a potent vaccine adjuvant against infectious diseases as well as to increase tumor immunogenicity towards cancer immunotherapy in solid tumors. Despite the promise of CDNs, such as cGAMP, as immune adjuvants, they suffer from several limitations: (1) CDNs exhibit fast clearance from the injection site, which may induce systemic toxicity; (2) naturally derived CDNs are susceptible to enzymatic degradation, which can lower the efficacy of adjuvanticity potential; and (3) CDNs have inefficient intracellular transport properties due to limited endosomal escape or reliance on the expression of a specific transporter protein. Hence, there is an urgent need to find new strategies for delivering CDNs.
In one aspect, the present disclosure provides a composition comprising a fusion protein comprising a STINGΔTM protein fused to a nanobody. Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the STINGΔTM comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequence selected from SEQ ID NOs: 3-6. In some embodiments, the nanobody is fused to the N-terminus of the STINGΔTM. In some embodiments, the nanobody is capable of binding to a cancer cell or or tumor extracellular matrices. In some embodiments, the nanobody is capable of binding to Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4), Programmed death protein 1 (PD-1), Programmed death-ligand 1 (PD-L1), Programmed death-ligand 2 (PD-L2), adenosine A2a receptor (A2AR), B7 homolog 3 protein (B7-H3), B7 homolog 4 protein (B7-H4), B- and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), Lymphocyte-activation gene 3 (LAG3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), Fibronectin extra type III B module (EIIIB), T cell immunoreceptor with Ig and ITIM domains (TIGIT), Natural killer group 2, member A (NGK2A), P-selectin glycoprotein ligand-1 (PSGL-1), or V-domain immunoglobulin suppressor of T-cell activation (VISTA). In one embodiment, the nanobody comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 7-10. In another embodiment, the fusion protein comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from SEQ ID NO: 11-42, wherein residues in parentheses are optional and may be present or may be deleted in whole or in part.
In further embodiments, the disclosure provides nucleic acids encoding the fusion protein of any embodiment or combination of embodiments herein, expression vectors comprising the nucleic acid operatively linked to a suitable control sequence, and host cells comprising the fusion protein, nucleic acid, and/or expression vector of any embodiment or combination of embodiments disclosed herein.
In another aspect, the disclosure provides compositions, comprising the fusion protein of any embodiment or combination of embodiments herein and a STING agonist. In one embodiment, the STING agonist is a cytosolic cyclic dinucleotide (CDN). In another embodiment, the CDN is c-di-GMP, 3′,3′cGAMP, 2′,3′cGAMP, c-di-AMP, cAIMP, cAIMP Difluor, cAIM(PS)2 Difluor (Rp,Sp), 2′2′-cGAMP, 2′3′-cGAM(PS)2 (Rp,Sp), 3′3′-cGAMP Fluorinated, c-di-AMP Fluorinated, 2′3′-c-di-AMP, 2′3′-c-di-AM(PS)2 (Rp,RP), 2′3′-c-di-AM(PS)2, c-di-GMP Fluorinated, 2′3′-c-di-GMP, or c-di-IMP. In a further embodiment, the STING agonist is a non-nucleotidyl small molecule. In various embodiments, the non-nucleotidyl small molecule is 5,6-dimethylxanthenone-4-acetic acid 7 (DMXAA), flavone-8-acetic acid, 2,7-bis(2-diethylamino ethoxy)fluoren-9-one, 10-carboxymethyl-9-acridanone, 2,7,2″,2″-dispiro[indene-1″,3″-dione]-tetrahydro dithiazolo[3,2-a:3′,2′-d]pyrazine-5,10(5aH,10aH)-dione, 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-yl methyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 6-Bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide, 3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]thiazine-7-carboxamide, 2-oxo-1,2,3,4-tetrahydroquinoline-7-carboxamide, or 2-Oxo-1,2,3,4-tetrahydroquinazoline-7-carboxamides.
In another aspect, the disclosure provides methods of treating cancer or an infectious disease, comprising administering to a patient in need thereof an effective amount of the composition of any embodiment or combination of embodiments herein. In one embodiment, the method is for treating cancer, incuding but not limited to a colon carcinoma, a melanoma, or breast cancer.
In one aspect, the present disclosure provides fusion proteins comprising STINGΔTM protein fused to a nanobody. The fusion proteins of the disclosure can be used, for example in methods to treat cancer or infectious disease, as disclosed herein. The cytosolic domain of STING protein (STINGΔTM) is fused to a nanobody, which can for example be complexed with a STING agonist for use in the therapeutic methods disclosed herein.
The term “STING”, also known as stimulator of interferon genes (STING). STING is a protein that in humans is encoded by the STING1 gene. STING plays an important role in innate immunity. STING induces type I interferon production when cells are infected with intracellular pathogens, such as viruses, mycobacteria and intracellular parasites. Type I interferon, mediated by STING, protects infected cells and nearby cells from local infection by binding to the same cell that secretes it (autocrine signaling) and nearby cells (paracrine signaling.)
Below are non-limiting examples of STING proteins.
MPYSNLHPAI PRPRGHRSKY VALIFLVASL MILWVAKDPP
NHTLKYLALH LASHELGLLL KNLCCLAEEL CHVQSRYQGS
YWKAVRACLG CPIHCMAMIL LSSYFYFLQN TADIYLSWMF
GLLVLYKSLS
MLLGLQSLTP AEVSAVCEEK KLNVAHGLAW
MPHSSLHPSI PCPRGHGAQK AALVLLSACL VTLWGLGEPP
EHTLRYLVLH LASLQLGLLL NGVCSLAEEL RHIHSRYRGS
YWRTVRACLG CPLRRGALLL LSTYFYYSLP NAVGPPFTWM
LALLGLSQAL
NILLGLKGLA PAEISAVCEK GNFNVAHGLA
In one embodiment, the STINGΔTM protein comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from SEQ ID NO: 3-6.
The STINGΔTM protein and nanobody may be directly linked in the fusion protein, or may be separated by an amino acid linker. Any suitable amino acid linker may be used. The nanobody may be fusion N-terminal to the STINGΔTM, or may be fused C-terminal to the STINGΔTM.
Any nanobody as suitable for an intended use may be present in the fusion proteins. A nanobody is a single-domain antibody (sdAb); an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, nanobodies are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (˜50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (˜25 kDa, two variable domains, one from a light and one from a heavy chain). Examples of nanobodies that may be included in the fusion protein include, but are not limited to, anti-Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) nanobodies, anti-Programmed death protein 1 (PD-1) nanobodies, anti-Programmed death-ligand 1 (PD-L1) nanobodies, anti-Programmed death-ligand 2 (PD-L2) nanobodies, anti-adenosine A2a receptor (A2AR) nanobodies, anti-B7 homolog 3 protein (B7-H3) nanobodies, anti-B7 homolog 4 protein (B7-H4) nanobodies, anti-B- and T-lymphocyte attenuator (BTLA) nanobodies, anti-killer cell immunoglobulin-like receptor (KIR) nanobodies, anti-Lymphocyte-activation gene 3 (LAG3) nanobodies, anti-T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) nanobodies, anti-Fibronectin extra type III B module (EIIIB) nanobodies, anti-T cell immunoreceptor with Ig and ITIM domains (TIGIT) nanobodies, anti-Natural killer group 2, member A (NGK2A) nanobodies, anti-P-selectin glycoprotein ligand-1 (PSGL-1) nanobodies, or anti-V-domain immunoglobulin suppressor of T-cell activation (VISTA) nanobodies.
In certain embodiments, the the nanobody comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 7-10.
ISSDGNINYAD
SVKDRFTISRDNASNTMYLQMNNLKPEDTAVYVCNIRGS
YGNTYYSRW
GQGTQVTVSSGGLPETGG (EIIIB binder (SEQ ID
In further specific embodiments, the fusion proteins comprise an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from SEQ ID NO: 11-42, wherein residues in parentheses are optional and may be present or may be deleted in whole or in part. The residues in parentheses are signal sequences that may be present or absent, and when present may be substituted with any other signal sequence as deemed appropriate for an intended use.
FSHNAGG
WYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNNLKPED
FSHNAGG
WYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNNLKPED
FSHNAGG
WYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNNLKPED
FSHNAGG
WYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNNLKPED
In further embodiments, the fusion proteins of the disclosure may further comprise a cell penetrating peptide, as disclosed herein.
In another aspect, the disclosure provides nucleic acids encoding a fusion protein of the disclosure. The nucleic acid sequence may comprise RNA (such as mRNA) or DNA. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the proteins of the invention.
In another aspect, disclosure provides expression vectors comprising the nucleic acid of any embodiment or combination of embodiments of the disclosure operatively linked to a suitable control sequence. “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence is still considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited to plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive).
In one aspect, the present disclosure provides cells comprising the fusion protein, the nucleic acid, and/or the expression vector of any embodiment or combination of embodiments of the disclosure, wherein the cells can be either prokaryotic or eukaryotic, such as mammalian cells. In one embodiment, the cells may be transiently or stably transfected with the nucleic acids or expression vectors of the disclosure. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art. A method of producing a fusion protein according to the invention is an additional part of the invention. The method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the fusion protein, and (b) optionally, recovering the expressed fusion protein.
The host cells can be fungal cells including yeast such as Saccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomyces pombe. The host cells also be any of various animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS-7, NIH-3T3, NS0, PER.C6®, and hybridoma). In further embodiments, the host cells is a CHO cell selected from CHO-K, CHO-0, CHO-Lec10, CHO-Lec13, CHO-Lec1, CHO Pro-5, and CHO dhfr−. In particular embodiments, the host cell is a hybridoma.
In one aspect, the disclosure provides compositions, comprising the fusion protein of any embodment or combination of embodiments herein and a STING agonist. The compositins may be used, for example, in the therapeutic methods of the disclosure.
Examples of STING agonists include, but are not limited to:
In one embodiment, the STING agonist is a cytosolic cyclic dinucleotide (CDN). In another embodiment, the CDN is c-di-GMP, 3′,3′cGAMP, 2′,3′cGAMP, c-di-AMP, cAIMP, cAIMP Difluor, cAIM(PS)2 Difluor (Rp,Sp), 2′2′-cGAMP, 2′3′-cGAM(PS)2 (Rp,Sp), 3′3′-cGAMP Fluorinated, c-di-AMP Fluorinated, 2′3′-c-di-AMP, 2′3′-c-di-AM(PS)2 (Rp,RP), 2′3′-c-di-AM(PS)2, c-di-GMP Fluorinated, 2′3′-c-di-GMP, or c-di-IMP.
In another embodiment, the STING agonist is a non-nucleotidyl small molecule. In exemplary embodiments, the non-nucleotidyl small molecule is 5,6-dimethylxanthenone-4-acetic acid 7 (DMXAA), flavone-8-acetic acid, 2,7-bis(2-diethylamino ethoxy)fluoren-9-one, 10-carboxymethyl-9-acridanone, 2,7,2″,2″-dispiro[indene-1″,3″-dione]-tetrahydro dithiazolo[3,2-a: 3′,2′-d]pyrazine-5,10(5aH,10aH)-dione, 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 6-Bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide, 3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]thiazine-7-carboxamide, 2-oxo-1,2,3,4-tetrahydroquinoline-7-carboxamide, or 2-Oxo-1,2,3,4-tetrahydroquinazoline-7-carboxamides.
In another aspect, the present disclosure provides methods of treating cancer or an infectious disease, comprising administering the composition of any embodiment or combination of embodiments of the disclosure.
The cytosolic DNA sensing pathway involving cyclic GMP-AMP synthase (cGAS) and the stimulator of interferon genes (STING) represents an essential innate immune mechanism in response to foreign pathogens. Upon detection of cytosolic DNA, the intracellular nucleic acid sensor cGAS catalyzes the productions of cyclic dinucleotides (CDNs) such as 2′3′-cyclic GMP-AMP (cGAMP), which functions as a second messenger to bind the adaptor protein
STING to initiate type I interferon (IFN) production and boost dendritic cell (DC) maturation and T cell infiltration. Meanwhile, the cGAS-STING signaling pathway is profound at sensing neoplastic progression by promoting type I IFN production and initiating cytotoxic T cell-mediated anti-tumor immune response. Synthetic STING agonists can be utilized to activate the innate and adaptive immune responses as a monotherapy or in combination with immune checkpoint blockade (ICB) for cancer immunotherapy.
As disclosed in the examples that follow, compositions of the disclosure, exemplified using cGAMP as the STING agonist, eliminated subcutaneous MC38 and YUMM1.7 tumors in 70%-100% of mice and protected all cured mice against rechallenge. Mechanistic studies revealed a robust TH1 polarization and suppression of Treg of CD4+ T cells, followed by an effective collaboration of CD4+ T, CD8+ T and NK cells to eliminate tumors. The examples further demonstrate the potential to overcome host STING deficiency by significantly decreasing MC38 tumor burden in STING-KO mice, addressing the translational challenge for the 19% of human population with loss-of-function STING variants. The cytosolic DNA sensing pathway involving cyclic GMP-AMP synthase (cGAS) and the stimulator of interferon genes (STING) represents an essential innate immune mechanism in response to foreign pathogens. Upon detection of cytosolic DNA, the intracellular nucleic acid sensor cGAS catalyzes the productions of cyclic dinucleotides (CDNs) such as 2′3′-cyclic GMP-AMP (cGAMP), which functions as a second messenger to bind the adaptor protein STING to initiate type I interferon (IFN) production and boost dendritic cell (DC) maturation and T cell infiltration. Meanwhile, the cGAS-STING signaling pathway is profound at sensing neoplastic progression by promoting type I IFN production and initiating cytotoxic T cell-mediated anti-tumor immune response. Synthetic STING agonists can be utilized to activate the innate and adaptive immune responses as a monotherapy or in combination with immune checkpoint blockade (ICB) for cancer immunotherapy.
Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.
The terms “a,” “an” and “the” include plural referents unless the context in which the term is used clearly dictates otherwise. The terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein. Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two or more specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).
The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Wherever embodiments, are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of,” and/or “consisting essentially of” are also provided.
“Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
“Administering” or “administration of” a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In one embodiment, the administering is done intra-tumorally.
Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and/or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.
The term “a small molecule” is a compound having a molecular weight of less than 2000 Daltons, preferably less than 1000 Daltons. Typically, a small molecule therapeutic is an organic compound that may help regulate a biological process.
A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, and the nature and extent of the condition being treated, such as cancer. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.
The terms “cancer,” “tumor,” “cancerous,” and “malignant” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancers include but are not limited to, carcinoma including adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and leukemias. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer (including hormonally mediated breast cancer, see, e.g., Innes et al., Br. J. Cancer 94:1057-1065 (2006)), colon cancer, colorectal cancer, endometrial carcinoma, myeloma (such as multiple myeloma), salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, various types of head and neck cancer and cancers of mucinous origins, such as mucinous ovarian cancer, cholangiocarcinoma (liver) and renal papillary carcinoma. In particular embodiments, the cancer is breast, endometrial, or uterine cancer. In other embodiments, the cancer is a myeloma (e.g., multiple myeloma, plasmacytoma, localized myeloma, and extramedullary myeloma), or endometrial, gastric, liver, colon, renal or pancreatic cancer. In some embodiments, the cancer comprises a colon carcinoma, a melanoma, or breast cancer.
A “recombinant” polypeptide, protein or antibody refers to polypeptide, protein or antibody produced via recombinant DNA technology. Recombinantly produced polypeptides, proteins and antibodies expressed in host cells are considered isolated for the purpose of the present disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
The term “percent sequence identity” or “percent identity” between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence. The percentage of sequence identity is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using readily available software programs. Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site. Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI).
The term “cell-penetrating-peptide sequence” is used in the present specification interchangeably with “CPP”, “protein transducing domain” or “PTD”. It refers to a peptide chain of variable length that directs the transport of a protein inside a cell. The delivering process into cell commonly occurs by endocytosis but the peptide can also be internalized into cell by means of direct membrane translocation. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acid and non-polar, hydrophobic amino acids.
Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular intake and uptake of molecules ranging from nanosize particles to small chemical compounds to large fragments of DNA. The “cargo” is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions. CPPs deliver the cargo into cells, commonly through endocytosis.
CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar, charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPPs are the hydrophobic peptides, containing only apolar residues with low net charge or hydrophobic amino acid groups that are crucial for cellular uptake. Numerous CPPs are known in the art, any of which can be part of the heterologous fusion proteins of the present invention.
The fusion proteins comprising STINGΔTM protein fused to a cell-penetrating domain or a nanobody of the compositions may be produced by either synthetic chemical processes or by recombinant methods or a combination of both methods. The fusion proteins comprising STINGΔTM protein fused to a cell-penetrating domain or a nanobody may be prepared as full-length polymers or be synthesized as non-full length fragments and joined. Chemical synthesis of peptides is routinely performed by methods well known to those skilled in the art for either solid phase or solution phase peptide synthesis. For solid phase peptide synthesis, so called t-Boc (tert-Butyloxy carbonyl) and Fmoc (Fluorenyl-methoxy-carbonyl) chemistry, referring to the N-terminal protecting groups, on polyamide or polystyrene resin have become the conventional methods (Merrifield, R B. 1963 and Sheppard, R C. 1971, respectively). Unlike ribosomal protein synthesis, solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by these two groups and added onto a deprotected amino acid chain. Deprotection requires strong acid such as trifluoroacetic acid for t-Boc and bases such as piperidine for Fmoc. Stepwise elongation, in which the amino acids are connected step-by-step in turn, is ideal for small peptides containing between 2 and 100 amino acid residues.
Non-naturally occurring residues may be incorporated into fusion proteins comprising STINGΔTM protein fused to a cell-penetrating domain or a nanobody. Examples of non-ribosomally installed amino acids that may be used in accordance with a present invention and still form a peptide backbone include, but are not limited to: D-amino acids, β-amino acids, pseudo-glutamate, γ-aminobutyrate, ornithine, homocysteine, N-substituted amino acids (R. Simon et al., Proc. Natl. Acad. Sci. U.S.A. (1992) 89: 9367-71; WO 91/19735 (Bartlett et al.; incorporated by reference), U.S. Pat. No. 5,646,285 (Baindur; incorporated by reference), α-aminomethyleneoxy acetic acids (an amino acid-Gly dipeptide isostere), and α-aminooxy acids and other amino acid derivatives having non-genetically non-encoded side chain function groups etc. Peptide analogs containing thioamide, vinylogous amide, hydrazino, methyleneoxy, thiomethylene, phosphonamides, oxyamide, hydroxyethylene, reduced amide and substituted reduced amide isosteres and β-sulfonamide(s) may be employed.
In another process, unnatural amino acids have been introduced into recombinantly produced proteins by a method of codon suppression. In one aspect, the use of codon suppression techniques could be adapted to introduce an aldehyde or ketone functional group or any other functional group in any suitable position within a polypeptide chain for conjugation (see e.g. WO 2006/132969; incorporated by reference).
Alternatively, recombinant expression methods are particularly useful. Recombinant protein expression using a host cell (a cell artificially engineered to comprise nucleic acids encoding the sequence of the peptide and which will transcribe and translate, and, optionally, secrete the peptide into the cell growth medium) is used routinely in the art. For recombinant production process, a nucleic acid coding for the amino acid sequence of the peptide would typically be synthesized by conventional methods and integrated into an expression vector. Such methods are particularly preferred for manufacture of the polypeptide compositions comprising the peptides fused to additional polypeptide sequences or other proteins or protein fragments or domains. The host cell can optionally be at least one selected from E. coli, COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Also provided is a method for producing at least one peptide, comprising translating the peptide encoding nucleic acid under conditions in vitro, in vivo or in situ, such that the peptide is expressed in detectable or recoverable amounts. The techniques well known in the art, see, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989).
Methods of fusing antibodies like nanobodies with proteins is known in the art, see, e.g., LaFleur, et al., MAbs. 2013 March-April;5(2):208-18. Small binding domains can be fused to multiple locations on antibodies and still retain binding affinity to ligand and antigen.
Methods of preparing and administering a composition comprising a fusion protein and a STING agonist, wherein the fusion protein comprises STINGΔTM protein fused to a cell-penetrating domain or a nanobody. The methods of administering the composition to a subject in need thereof are known to or are readily determined by those of ordinary skill in the art. The route of administration of the composition can be, for example, oral, parenteral, by inhalation or topical. The term parenteral includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, intraocular, subcutaneous, rectal, or vaginal administration. While all these forms of administration are clearly contemplated as being within the scope of the disclosure, another example of a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition can comprise a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer agent (e.g., human albumin), etc. In other methods compatible with the teachings herein, the composition as provided herein can be delivered directly to the organ and/or site of a fibrosis or tumor, thereby increasing the exposure of the diseased tissue to therapeutic agent.
As discussed herein, the composition can be administered in a pharmaceutically effective amount for the in vivo treatment of cancer. In this regard, it will be appreciated that the disclosed composition can be formulated so as to facilitate administration and promote stability of the active agent. Pharmaceutical compositions in accordance with the disclosure can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. Suitable formulations for use in therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).
Certain pharmaceutical compositions provided herein can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions or solutions. Such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.
The provided compositions comprising a fusion protein and a STING agonist, wherein the fusion protein comprises STINGΔTM protein fused to a cell-penetrating domain or a nanobody are useful in a variety of applications including, but not limited to, methods of treating and/or ameliorating various diseases and conditions. Methods are provided for the use of the disclosed compositions to treat subjects having a disease or condition associated with STING signaling, altered STING expression. The composition disclosed herein may be used to treat auto-inflammation, virus infection or cancers.
In certain embodiments, the disclosure provides a method of treating cancer that comprises contacting a cancer cell, tumor associated-stromal cell, tumor extracellular matrices, or endothelial cell with the disclosed composition. In additional embodiments, the cancer cell is a myeloma (e.g., multiple myeloma, plasmacytoma, localized myeloma, or extramedullary myeloma), ovarian, breast, colon, endometrial, liver, kidney, pancreatic, gastric, uterine and/or colon cancer cell. In some embodiments, the contacted cell is from a cancer line. In some embodiments, the cancer cell is contacted in vivo.
In some embodiments, the composition comprising a fusion protein and a STING agonist, wherein the fusion protein comprises STINGΔTM protein fused to a cell-penetrating domain or a nanobody is administered alone or as a combination therapy. In some embodiments, composition is administered in combination with one or more other therapies. Such therapies include additional therapeutic agents as well as other medical interventions. Exemplary therapeutic agents that can be administered in combination with the composition provided herein include, but are not limited to, chemotherapeutic agents, and/or immunomodulators. In various embodiments, the composition is administered to a subject before, during, and/or after a surgical excision/removal procedure.
Stimulator of interferon genes (STING) signaling is a promising target in cancer immunotherapy. Here, we report a distinct CD4-mediated, protein-based combination therapy of STING and ICB as an in situ vaccine. The treatment eliminated subcutaneous MC38 and YUMM1.7 tumors in 70%-100% of mice and protected all cured mice against rechallenge. Mechanistic studies revealed a robust TH1 polarization and suppression of Treg of CD4+ T cells, followed by an effective collaboration of CD4+ T, CD8+ T and NK cells to eliminate tumors. Finally, we demonstrated the potential to overcome host STING deficiency by significantly decreasing MC38 tumor burden in STING-KO mice, addressing the translational challenge for the 19% of human population with loss-of-function STING variants.
Immune checkpoint blockade (ICB) has revolutionized the landscape of cancer immunotherapy over the past decades as one of the most promising treatment options for diseases such as advanced melanoma, colorectal cancer, and non-small cell lung cancer. However, response rates towards ICB treatment varies widely across individuals and cancer types, leaving only a minority of patients benefiting from significant tumor regression and long-term protection from recurrence. While the exact mechanism of resistance is not fully understood, immunosuppression on multiple levels—genetic (e.g., impaired HLA class I and mutation of the PTEN gene), protein (e.g., VEGF, IL-10 and TGFβ), and cellular (e.g., inadequate antigen presentation and tumor infiltration of lymphocytes)—has been reported to be closely correlated to clinical outcome, suggesting the need for a more comprehensive approach in treatment.
To date, no FDA approval has been achieved for the widespread use of STING-agonist-based cancer treatments. This is primarily due to limitations in safety and efficacy, as prior studies have reported potential adverse effects such as dose-limited toxicity and a potent increase in serum interferon (IFN) levels. One challenge in terms of efficacy is the large human population with potentially defective STING due to genetic mutations. Studies have shown that the HAQ and H232 mutations of human STING, which account for ˜30% East Asian and ˜10% of European populations, are likely loss-of-function variants. As existing STING-targeting cancer immunotherapies rely on the intracellular delivery of STING agonists, the lack of fully functional endogenous STING could significantly abrogate signaling and treatment efficacy.
To this end, we previously developed an approach for cyclic guanosine monophosphate—adenosine monophosphate (cGAMP) delivery by adapting the cytosolic domain of STING protein (STINGΔTM) as a biomimetic carrier. The resulting STINGΔTM-cGAMP complex was shown in vitro to restore STING signaling in cell lines without STING protein or expressing only HAQ mutant STING, demonstrating its use as a fully functional protein52, 53. In this work, we have engineered a novel fusion protein of ICB nanobody and STINGΔTM, which can then be complexed with cGAMP as an intratumorally-injected therapeutic. Through depletion studies, we observed that CD4+ T cells played a critical role in achieving antitumor immunity, to an extent that no significant differences were observed when compared to the untreated control. Mechanistic studies revealed a remodeling of the tumor microenvironment (TME) with proinflammatory cytokines, as well as the polarization of CD4+ T cells towards the TH1 phenotype, followed by NK and CD8+ T cells—mediated cytotoxicity towards tumor cells. The ICB-STINGΔTM-cGAMP complex was found to effectively eradicate tumors in the MC38 colon carcinoma and the YUMM1.7 melanoma model, inhibit metastasis in the 4T1 breast cancer model, and prevent cancer recurrence in cured mice. Finally, when treating tumors on STING-knockout mice, the complex was able to achieve significant tumor regression via restored STING signaling with STINGΔTM. These results underscore the use of this combination in cancer immunotherapy, while simultaneously providing new insights regarding the potential benefits of STING-activated CD4+ T cells for clinical translation.
In engineering the ICB-STINGΔTM-cGAMP complex, we fused two types of ICB nanobodies to the STINGΔTM protein (ΔTM)—an anti-cytotoxic T-lymphocyte associated protein 4 (CTLA4) nanobody (αCTLA4-ΔTM), and an anti-programmed death ligand 1 (PDL1) nanobody (ΔPDL1-ΔTM) (
ICB-ΔTM-cGAMP Eradicates Subcutaneous MC38 and YUMM1.7 Tumors with Immune Memory
Next, we evaluated the therapeutic efficacy with two subcutaneous mouse tumor models, MC38 and YUMM1.7. We observed significant antitumor effect and sustained antitumor immune memory in both models following treatment with ICB-ΔTM-cGAMP (
In both tumor models, all combinations of ICB-ΔTM-cGAMP were observed to significantly inhibit tumor growth and increase survival relative to STING-only (αGFP-ΔTM-cGAMP) and ICB-only controls (ΔPDL1&CTLA4-ΔTM). Tumors decreased in size and formed black scabs following treatment, which sloughed off and healed without further intervention (
To characterize the TME, we performed separate treatment experiments for both MC38 and YUMM1.7 models, resecting the tumors from mice after the 3rd dose for analysis (
ICB-ΔTM-cGAMP Engages CD4+ T, NK and CD8+ T Cells in a Multi-Pronged Immune Response
The therapeutic mechanism of ICB-ΔTM-cGAMP was observed to rely significantly on CD4+ T, NK, and CD8+ T cell-mediated pathways. Analysis of MC38 and YUMM1.7 tumors first revealed a significantly larger population of NK cells in the αPDL1&CTLA4-ΔTM-cGAMP treatment groups when compared to STING-only or ICB-only trials. These results were corroborated by an immune cell depletion study with YUMM1.7 model (
Despite the initial anti-tumoral effect of NK cells, the overall survival of the NK-depleted group indicated that NK cells were not essential for eradication of the tumor. Both NK and macrophage-depleted groups failed to exhibit any significant differences in overall survival compared to the no-depletion control, with all groups achieving a survival rate of 25%-30% (
Most notably, the depletion of CD4+ T cells proved to be critical to the therapeutic effect of ICB-ΔTM-cGAMP at all stages of the study, resulting in tumor progression similar to that of untreated controls. In comparison with NK- and CD8-depleted groups, CD4-depleted group exhibited no therapeutic benefit from either NK or CD8+ T cells throughout the study. To investigate this phenomenon, we sought to quantify the amount of tumor antigen-specific immune cells from peripheral blood mononuclear cells (PBMCs) via IFNγ ELISPOT and observed significantly compromised adaptive immunity in CD4-depleted mice, worse even than that of the untreated group (
The aforementioned results led us to speculate that the engagement of CD4+ T cells with ICB-ΔTM-cGAMP occurs at an early timepoint. To verify this hypothesis, we sought to characterize tumor immune cells 24 hours post-intratumoral injection of Cy5-labeled ICB-ΔTM-cGAMP. Flow cytometry analysis of the excised tumors demonstrated the cellular targeting of ICB nanobodies—αCTLA4 towards CD4+ T cells and ΔPDL1 towards macrophages. Overall, the highest percentage Cy5+ cells were the CD4+ T cells (
At this juncture, a therapeutic mechanism for the intratumoral delivery of ICB-ΔTM-cGAMP may be proposed. During the first 2 weeks, CD4+ T and NK cells are major contributors to tumor regression. The injected ICB-ΔTM-cGAMP primarily interacts with CD4+ T cells, giving rise to TH1 polarization, a subtype known for its supporting role in inducing cellular immunity, including activating and sustaining NK cells via IL-2 secretion55. On the other hand, while NK cells did not directly interact with ICB-ΔTM-cGAMP, our intracellular staining of granzyme B and perforin suggested a robust activation of NK cytotoxicity. We therefore hypothesize that CD4+ T cells play a role in stimulating NK-mediated anti-tumor immunity, though the specific mechanism remains to be uncovered. Concurrently, CD4+ T cells aid in antigen presentation and in priming CD8+ T cells, which leads to CD8+ T cell-mediated antitumor immunity at 2-3 weeks post-injection and CD8+ memory T cell-based protection against tumor recurrence (observed in YUMM1.7 and MC38 tumor models). The process has been illustrated in
To assess the systemic immune protection afforded by the intratumoral delivery of ICB-ΔTM-cGAMP, we next treated the 4T1 metastatic breast cancer model (
Finally, we explored the possibility of restoring STING signaling with the functional STINGΔTM carrier by treating MC38 tumors on STING KO mice with ΔPDL1&CTLA4-ΔTM-cGAMP. To decouple immune responses against this protein complex not induced via the STING pathway, we included ΔPDL1&CTLA4-S365AΔTM-cGAMP as a control (
Potent immunostimulation as well as effective approaches to reverse immunosuppression and capture tumor cells that evade immunosurveillance remains a challenge in clinical cancer immunotherapy
The cancer immunity cycle has traditionally been depicted as occurring between dendritic cells, which prime CD8+ T cells with tumor antigens, resulting in the elimination of tumor cells and the generation of antigens in self-sustaining loop. In recent years, NK cells have entered the picture, collaborating alongside CD8+ T cells. Here, we engineered a new strategy to effectively boost the cancer immunity cycle, elucidating the critical role of CD4+ T cells as an initiator for NK cell tumor elimination70 and an aide to CD8+ T cell priming and memory71 in STING-mediated antitumor immunity. This is a highly salient observation, as the role of CD4+ T cells in cancer immunotherapy has been reported to be bidirectional. Despite evidence that CD4+ T cells are fundamentally important in supporting anti-tumoral immunity2, many studies over the past two decades have shown that CD4+ T cell depletion results in improved therapeutic outcomes, including a wide range of therapies such as adoptive T cell transfer, inflammatory cytokines, and ICB antibody treatments7. There has even been a human clinical trial intravenously injecting anti-CD4 depletion antibodies to treat solid tumors, where tumor shrinkage was observed in 5/11 patients over three months of observation78. In these therapies, the presence of CD4+ T cells did not positively contribute to antitumor immunity, which was attributed to the immunosuppressive effects of Treg and TH2 outweighing that of TH1.
While the positive role of CD4+ T cells has not been extensively investigated in many immunotherapies, recent studies have suggested that significant potential benefit could be exploited from CD4+ T cells if properly activatedln our study, we observed depletion of CD4+ T cells. Additionally, we found that CD4+ T cells could directly interact with our STING signaling complex ex vivo (
Following initiation of the innate immune system via STING-mediated CD4 TH1-polarization and NK-mediated cytotoxicity, the ICB-ΔTM-cGAMP complex then acts upon the adaptive immune system through ICB. We sought to leverage the synergy of PDL1 and CTLA4 dual blockade and succeeded in eradicating both immunologically “cold” (YUMM 1.783) and “hot” (MC3884) tumors, gaining long-term protection against recurrence through memory T cells. When administrated as a neoadjuvant for the metastatic 4T1 model—a model recalcitrant to immunotherapy—our approach likewise resulted in prolonged post-op survival. These results demonstrate this system's utility as a modular platform for incorporation of other ICB or tumor-targeting nanobodies, such as anti-TIGIT and anti-NGK2A for NK cells, anti-PSGL-1 for CD4+ T cells, anti-EIIIB for tumor extracellular matrices85, allowing additional flexibility in treating a wide range of cancers.
One remaining challenge to clinical translation lies in host STING deficiency and the limited options to restore STING signaling in affected individuals. Our present system provides a tangible solution by eliciting significant antitumoral immunity via STING signaling in STING KO mice, both in terms of tumor burden and in the production of pro-inflammatory cytokines within the TME. The aforementioned findings emphasize this platform's immense utility in CD4+ T cell activation and overcoming host STING deficiency, offering new solutions to improving the clinical outcomes of ICB treatments.
The expression plasmid was cloned based on our previous pSH200_STINGΔTM plasmid, where STINGΔTM stands for the segment of amino acids 138-378 of mouse STING52. DNA sequences encoding for nanobodies of anti-PDL1 (PDB ID: 5DXW_A), anti-CTLA4 (PDB ID: 5E03_A), anti-GFP (PDB ID: 3OGO_E), anti-96G3m (PDB ID: 6X07_B) were synthesized by gBlock (IDT) and inserted at the N-terminus of the sequence encoding for STINGΔTM protein. Mutants such as S365A and R237A/Y239A of the STINGΔTM protein were created via site-specific mutagenesis. Histidine6-tagged nanobody-STINGΔTM proteins were expressed in Rosetta DE3 Escherichia coli (Rosetta E. coli, Millipore Sigma, Cat#: 70954). The purification method is slightly modified based on our previous published protocol86. Briefly, Rosetta E. coli transformed with the protein expression plasmid were cultured in 1 L volume of lysogeny broth (LB) at 37° C. until OD600 reaches 0.5-0.8. The culture was then induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG, Millipore Sigma, Cat#: I6758-10G) at 18° C. for 20 hours. Following induction, the cells were centrifuged, lysed, sonicated, and further centrifuged to obtain the cell lysate that contains the desired his-tagged protein. The lysate was then incubated with cobalt resin (Thermo Fisher Scientific, Cat#: 89964) followed by elution with 150 mM imidazole (Millipore Sigma, Cat#: I5513) and eventually buffer exchanged to 20 mM Hepes, 150 mM NaCl, 10% glycerol with 1 mM dithiothreitol (DTT, Millipore Sigma Cat#: 10197777001), aliquoted and stored in −80 ° C. freezer for future use.
In vitro STING Signaling Activation in 293T Reporter Cells
The STING deficient human embryonic kidney (HEK293T) cell line was used to study the capability of restoring STING signaling in vitro. The assay was based on our previous published protocol86. Briefly, a reporter cell line was generated from transfecting HEK293T cells with pGL4.45 [luc2p/ISRE/Hygro] plasmid (Promega), which was able to express luciferase driven by the interferon-sensitive response element (ISRE) stimulated by STING signaling. 100 μL of 3×105 cells/mL HEK293T-luc2p/ISRE/Hygro cells were seeded in 96 well plate in Dulbecco's modified Eagle's medium (DMEM, Corning, Cat#: 10-041-CV) supplemented with 10% fetal bovine serum (FBS, Gibco, Cat#: 10437-028) and 1% penicillin/streptomycin (Corning, Cat#: 30-002-CI). After an overnight incubation, 0.025 μg cGAMP (Invivogen, Cat#: tlrl-nacga23-02) mixed with 1.7 μg ICB-ΔTM protein and 1.7 μL TransIT-X2 commercial transfection reagent (Mirus, Cat#: MIR6004) were mixed in 20 μL Opti-MEM™ medium (Gibco, Cat#: 31985062), incubated for 15 minutes for protein complex formation before added to the wells. 24 hours post treatment, cells were lysed for firefly luciferase assay (Biotium, Cat#: 30075-2) to quantify the interferon-luciferase activity.
Ex vivo Cytotoxicity Assay with MC38-SIINFEKL Cells
MC38-SIINFEKL cells were created via lenti-viral vector transduction. pLenti-CMV-GFP-Puro™ plasmid was obtained from Addgene (Cat#: 17448). The DNA sequence encoding for SIINFEKL peptide was inserted at the N-terminus of GFP via mutagenesis. HEK293T cells were transfected with pLenti-CMV-SIINFEKL-GFP-Puro plasmid with the help of TransIT-X2 transfection reagent. The culture medium containing lentivirus were collected, filtered through 0.45 μm filter and added to plated MC38 cells with 5 μg/mL polybrene (Millipore Sigma, Cat#: H9268), followed by puromycin (Millipore Sigma, Cat#: P9620) selection in the concentration range of 5-20 μg/mL. The final transduction efficiency was confirmed with flow cytometry based on percentage GFP expression.
To perform the ex vivo cytotoxicity assay of CD8+ T cells against cancer cells, MC38-SIINFEKL cells were first treated with 50 ng/mL mouse IFNγ for 24 hours to induce PDL1 upregulation, which is confirmed by qPCR to be approximately 10-fold of untreated cells' PDL1 expression level. In parallel, splenocytes were harvested from C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT1) mice and activated with αCD3/CD28 Dynabeads™ (Gibco, Cat#: 11456D) at a density of 2×106 cells/mL Roswell Park Memorial Institute medium (RPMI, Corning, Cat#: 10-013-CV) according to the instruction manual. The IFNγ treated MC38-SIINFEKL cells were then washed and plated at a density of 4×105 cells/mL with stimulated OT1 splenocytes at a ratio of 1:2 in 1 mL RPMI medium in 12 well plates for 48 hours, with the addition of 17 μg ΔPDL1-ΔTM plus 0.25 μg cGAMP, 17 μg α96G3M-ΔTM plus 0.25 μg cGAMP, 16 μg ΔPDL1 mono antibody (clone 10F.9G2, BioXCell, Cat#: BE0101), along with an untreated control. After 48 hours of coculture, cells were stained with Zombie Aqua™ live-dead stain (Biolegend, Cat#: 423101) and analyzed with flow cytometry. MC38 tumor cells were gated with FITC channel due to their GFP expression, and their percentage viability were used to assess the ex vivo PDL1 checkpoint blockade efficiency.
MC38 and YUMM1.7 tumors were inoculated in C57BL/6 or B6(Cg)-Sting1tm1.2Camb/J (STING knockout) mice by subcutaneously (s.c.) injecting 106 cells suspended in 100 μL sterile phosphate buffered saline (PBS, Lonza, Cat#: 17-516F) into the shaved hind right flank. All C57BL/6 mice were female and purchased at the age of 8 weeks old, tumors were usually inoculated 1-2 weeks after mice arrival at the lab. STING knock-out mice were bred in house, for tumor treatment studies, each group contained the equal number of male and female mice of the same age (8-12 weeks old). For rechallenge, 105 cells in 100 μL sterile PBS were injected into the hind left flank. The tumor sizes were monitored every two days with caliper measurement, volumes calculated as length×width2 then divided by 2. Treatment by intratumoral (i.t.) injections started when the average tumor volume reached around 100 mm3. Each i.t. injection consisted of 170 μg of ICB-ΔTM protein with or without 2.5 μg cGAMP. For ICB antibody controls, each i.t. injection consists of 80 μg ΔPDL1 mAb (clone 10F.9G2, BioXCell, Cat#: BE0101) and 80 μg αCTLA4 mAb (clone 9D9, BioXCell, Cat#: BE0164). The humane endpoint tumor burden for euthanasia of all three mouse models was 1000 mm3.
4T1 cells were inoculated in Balb/c mice in the mammary fat pad. A small incision was made next to the 4th nipple, 106 4T1 cells suspended in 50 μL PBS were injected into the mammary fat pad through the incision, which is subsequently closed with suture (Ethicon, Cat#: R690G). Caliper measurement and intratumoral injections were performed similar to MC38 and YUMM1.7 models. The only caution was that intratumoral injections for 4T1 tumors were done very slowly to achieve a uniform distribution of drug inside the tumor. Fast injections would result in a pouch of liquid seeping outside the tumor.
Depletion of mouse immune cell populations were carried out according to the literature75,87 by intraperitoneally (i.p.) injecting mice with antibodies against mouse CD8α (clone 2.43, BioXCell, Cat#: BP0061, 400 μg twice every week), CD4 (clone GK1.5, BioXCell, Cat#: BP0003-1, 400 μg twice every week), NK1.1 (clone PK136, BioXCell, Cat#: BE0036, 400 μg twice every week), or CSF1R (clone AFS98, BioXCell, Cat#: BP0213, 300 μg every other day). 36 hours post i.p. injection, mice PBMCs were sampled via cheek bleeding and stained for flow cytometry analysis to confirm the depletion efficiency.
Tumor Digestion for Immune Cell Analysis with Flow Cytometry
Approximately 100 mg mouse tumor was resected, weighed, and then minced to small pieces with diameters <1 mm in 500 μL PBS with 1 mg/mL collagenase type IV (Gibco, Cat#: 17104019). The minced tumor in collagenase was then incubated at 37° C. under shaking for 20 mins (MC38 tumors) or 30 mins (YUMM1.7 and 4T1 tumors). After incubation, the mixture was diluted with 1 mL PBS, vortexed, and filtered through 70 μm nylon mesh cell strainer (Fisherbrand, Cat#: 22363548). In case there were a lot of red blood cells (RBC), the filtrate was resuspended in 5 mL of RBC lysis buffer (Millipore Sigma, Cat#: R7767-100 mL), incubated at room temperature for 5 mins, then pelleted with centrifugation. The filtrate was then washed twice with PBS, resuspended in 10 mL PBS, and sampled for cell count. For intracellular cytokine staining samples, an additional 4 hour 37° C. incubation was performed prior to staining in RPMI supplemented with non-essential amino acid (NEAA, Gibco, Cat#: 11140050) and Golgi inhibitor (GolgiStop™, BD Biosciences, Cat#: 554724).
Afterwards, the cells were collected and washed with PBS for FACS staining. 106 cells from each tumor samples were loaded into 96 V-bottom well plate in 50 μL PBS and first stained with Zombie Aqua™ live/dead dye, followed by 20 min incubation on ice avoiding light. The cells are then washed with FACS buffer (PBS with 1% BSA and 2 mM EDTA). After washing, each well of cells are stained with Fc-blocker anti-mouse CD16/32 (Thermo Fisher Scientific, Cat#: 14-9161-73) in 50 μL FACS buffer, followed by 15 min incubation on ice. When the blocking was complete, surface staining antibodies were added to the cells for 30 min incubation on ice, followed by two washes with FACS buffer. Intracellular cytokine staining was then performed with the BD Cytofix/Cytoperm™ kit (BD Biosciences, Cat #555028). 100 μL fixation/permeabilization solution was first added to each well followed by 20 min incubation on ice. The cells were then washed twice with Perm/wash buffer and stained with intracellular staining antibodies in 50 μL Perm/wash buffer for 30 min on ice. After staining, the cells were washed twice with Perm/wash buffer, resuspended in FACS buffer, and analyzed with LSR-II-Fortessa flow cytometer (BD Biosciences). Data were analyzed with FlowJo™ software.
Flow cytometry antibodies (all from Biolegend) used are anti-mouse CD4 (clone GK1.5, Cat#: 100434), CD3 (clone 17A2, Cat#: 100204 and 100232), CD8α (clone 53-6.7, Cat#: 100707), F4/80 (clone BM8, Cat#: 123116 and 123135), NK1.1 (clone S17016D, Cat#: 156514), NKp46 (clone 29A1.4, Cat#: 137618), Ly6C (clone HK1.4, Cat#: 128031), Ly6G (clone 1A8, Cat#: 127645), CD45 (clone 30-F11, Cat#: 103128), CD11b (clone M1/70, Cat#: 101241), IFNγ (clone XMG1.2, Cat#: 505813), IL4 (clone 11B11, Cat#: 504133), PU.1 (clone 7C2C34, Cat#: 681307), IL17 (clone TC11-18H10.1, Cat#: 506915), FoxP3 (clone MF-14, Cat#: 126419), granzyme B (clone GB11, Cat#: 515403), and Perforin (clone S16009B, Cat#: 154404).
Approximately 50 mg mouse tumor was resected and grinded with microcentrifuge tube pestles in T-PER buffer (Thermo Fisher Scientific, Cat#: 78510) with protease inhibitor (Thermo Fisher Scientific, Cat#: 78425). The protein extraction solutions were then centrifuged at 14,000×g for 15 min. Supernatant protein concentrations were quantified with Nanodrop based on absorption at 280 nm and were all diluted to 5 mg/mL. Samples were then frozen and shipped to Eve Technology for multiplex assay for the cytokine concentrations. Heat maps of the protein levels were plotted as log2(fold change of untreated groups), with hierarchical clustering based on one minus Pearson correlation with complete linkage method performed with Morpheus software.
Spleen from C57BL/6 mice were first grinded and filtered through 70 μm cell strainer and washed once by RBC lysis buffer. The cells were then washed and resuspended in FACS buffer. EasySep™ Mouse CD4+ T cell isolation kit (Stemcell Technology, Cat#: 19852) was used to isolate CD4+ T cells from splenocytes. Splenocytes were re-suspended in PBS containing 2% FBS and 1 mM EDTA at the concentration of 108 cells/mL. Per mL of the splenocytes, 50 μL rat serum and 50 μL isolation cocktail were added followed by a 10 mins incubation at room temperature. 75 μL of vortexed magnetic beads Rapidspheres™ was added per mL of sample, mixed and incubated for 2.5 mins. Then buffer was added to the sample to top it up to 2.5 mL, before placed in the magnet (EasySep™) for 2.5 mins. Afterwards, the separated CD4+ T cells were poured out in one continuous motion. The separation effect is verified with flow cytometry with anti-CD4 surface staining. For phenotyping, the isolated CD4+ T cells were then treated with ICB-ΔTM protein mixed with cGAMP in RPMI media with NEAA for 8 hours (GolgiStop™ added for the last 4 hours) before staining for flow cytometry analysis as described in the tumor cell FACS section.
CD4+ T cells treated with Cy5-labeled ICB-ΔTM protein mixed with cGAMP were collected in microcentrifugation tubes along with untreated controls. The cells were washed 3 times with PBS, fixed with PBS containing 4% formaldehyde (Millipore Sigma, Cat#: 47608-250ML-F) for 15 mins on ice, then permeabilized by PBS containing 0.1% Triton X-100 (Millipore Sigma, Cat#: T8787) on ice for 10 mins. Afterwards, cells were washed by PBS with 0.05% Tween20 (Millipore Sigma, Cat#: P9416) and 1% BSA and stained with Alexa™ Flour 488 phalloidin (Invitrogen, Cat#: A12379) and Hoechst dye (Thermo Fisher Scientific, Cat#: 62249) in the same buffer for 30 mins avoiding light. The cells were then washed 3 times with PBS containing 0.05% Tween20 and 1% BSA and loaded onto microscope slides with anti-fade mounting media and covered with cover slips. Images were acquired with Olympus FV1200 confocal microscope and analyzed with ImageJ software.
C57BL/6 mice inoculated with YUMM1.7 tumor were injected i.t. with 170 μg Cy5-labeled anti-GFP/PDL1/CTLA4-ΔTM protein mixed with 2.5 μg cGAMP in 25 μL PBS. 24 hours post treatment, mice were sacrificed and organs plus tumors and both inguinal lymph nodes were collected for fluorescent imaging with the In Vivo Imaging System (IVIS, Xenogen). Data analysis was performed with the Living Image software (Xenogen).
PBMCs were obtained from cheek bleeding of the immune cell depleted mice collected with EDTA-coated collection tubes (Greiner Bio-one™, K3EDTA Cat#: 450530). Whole blood was washed twice with RBC lysis buffer to obtain peripheral blood mononuclear cells (PBMCs). ELISPOT was performed according to the kit instruction manual (BD ELISPOT reagent kit). Briefly, ELISPOT plate was first coated with anti-IFNγ capture antibodies overnight. The following day, the plate was washed and blocked. Equal number of PBMCs from each mouse were co-cultured with X-ray irradiated YUMM1.7 cells in the plate overnight. On the third day, cell suspension was aspirated, wells were incubated with detection antibody, streptavidin-HRP and substrate solution with 5 washes between every incubation. Spots were enumerated with an ELISPOT plate reader.
Data were analyzed with Prism GraphPad™: tumor volume and body weight change were analyzed with two-way ANOVA, the survival curve was analyzed with Kaplan-Meier, the remaining plots were analyzed with one-way ANOVA. Outlier were analyzed and removed with the Grubb's test.
strategies to modulate the tumour immune microenvironment for systemic anti-tumour immunity. British Journal of Cancer (2022).
All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
This application is a continuation in part of U.S. Application Ser. No. 17/181,884 filed Feb. 22, 2021, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 62/979,733, filed Feb. 21, 2020, each of which is incorporated by reference herein in its entirety
| Number | Date | Country | |
|---|---|---|---|
| 62979733 | Feb 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17181884 | Feb 2021 | US |
| Child | 18312030 | US |
