Compositions and methods for treating cancer

Abstract

A fusion polypeptide comprising an extracellular domain of human CTLA-4 and an extracellular domain of human PD-L1 and an immune composition comprising such for use in treating cancer in animals such as dogs and cats.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING CANCER

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 510,286, filed June 26, 2023, the contents of which is incorporated by reference herein in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 18, 2024, is named 063700-501001WO_SeqList_ST26.xml and is 5,997 bytes in size.

[0006] BACKGROUND OF THE INVENTION

[0007] Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a member of the immunoglobulin superfamily. It has been reported that CTLA-4 plays a critical role in regulating immune system (Keilholz, U., J. Immunother 31, 431-439).

[0008] Programmed cell death proten- 1 (PD- 1 ) is a member of the CD28 superfamily. It triggers negative signaling pathway upon binding to its ligands, programmed cell death ligand 1 and 2 (PD-L1 and PD-L2, respectively) (Riley, J. L., Immunol Rev 229, 114-125). The PD-l / PD-ligand pathway plays an important role in tolerance and immunity.

[0009] SUMMARY OF THE INVENTION

[0010] The present disclosure is based, at least in part, on the discovery that an exemplary immune composition as claimed, comprising a CTLA-4-linker-PD-Ll polypeptide and an adjuvant, unexpectedly exhibited enhanced immune responses and anti-tumor activities in a dog lung tumor model. Such immune compositions are thus expected to be effective in eliciting anti-tumor immune responses and treating tumor in pet animals such as cats and dogs.

[0011] Accordingly, the present invention relates to compostions and methods for treating canine or feline cancer, which involves the use of a fusuion polypeptide comprising a human CTLA-4 fragment and a human PD-L1 fragment for enhancing immune responses in the canine or feline cancer patients.

[0012] In some aspects, the present disclosure provides a fusion polypeptide comprising (a) an extracellular domain of human CTLA-4, and (b) an extracellular domain of human PD-L1. In some embodiments, the extracellular domain of human CTLA-4, as compared with a wildtype counterpart, comprises amino acid substitution at position 123 in SEQ ID NO: 1. In some instances, the amino acid substitution at position 123 is Cl 23 S. In some examples, the extracellular domain of human CTLA-4 may comprise the amino acid sequence of SEQ ID NO: 2. In some embodiments, the fusion polypeptide may comprise the extracellular domain of human PD-L1 in (b). In some instances, the extracellular domain of human PD-L1 may comprise the amino acid sequence of SEQ ID NO: 3.

[0013] In some embodiments, the fusion polypeptide provided herein may further comprise a flexible peptide linker, which is located between (a) and (b). In some embodiments, the flexible peptide linker may be a (G4S)nlinker, in which n is 1, 2, 3, 4, 5, or 6. In some instances, the flexible peptide linker linker is GGGGSGGGGSGGGGS (SEQ ID NO: 5).

[0014] In specific examples, the fusion polypeptide may comprise the amino acid sequence of SEQ ID NO: 4.

[0015] In some aspects, the present disclosure provides a nucleic acid, comprising a nucleotide sequence encoding any of the fusion polypeptides disclosed above and herein. In some embodiments, the nucleic acid can be a vector such as an expression vector. Also provided herein are host cells comprising the nucleic acid such as the expression vector. The fusion polypeptide can be produced in the host cells. Also provided herein is a method for producing the fusion polypeptide by culturing such host cells and harvesting the fusion polypeptide thus produced.

[0016] In some aspects, the present disclosure provides an immune composition comprising any of the fusion polypeptides or any of the nucleic acids disclosed above and herein, and one or more pharmaceutically acceptable carriers, which may comprise an adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some examples, the immune composition as disclosed herein comprises a fusion polypeptide as disclosed herein and a saponin adjuvant. In some embodiments, the immune composition may be a dosage form comprising about 20-200 pg of the fusion polypeptide and about 10-50 pg of the adjuvant. In some example, a dosage form may comprise about 50 pg of the fusion polypeptide and about 20 pg of the adjuvant. Alternatively, the composition is a dosage form comprising about 50- 500 pg of the fusion polypeptide. In some instances, the composition is free of adjuvant.

[0017] In further aspects, the present disclosure provides a method for treating a cancer in a subject. This method may comprise administering to the patient an effective amount of any of the immune compositions disclosed above and herein. The subject may be a canine or feline cancer patient.

[0018] In some examples, the canne or feline cancer patient may have lung carcinoma, oral fibrosarcoma, oral squamous cell carcinoma, mast cell tumor, leukemia, malignant neoplasm of breast, hepatocellular carcinoma, melanoma, head and neck cancer, or lymphoma.

[0019] In some embodiments, the immune composition may be administered subcutaneously. In some embodiments, the immune composition may be administered to the patient at a dose of about 20-200 pg of the fusion polypeptide and about 10-50 pg of the adjuvant once every 2 weeks or once every 3 weeks. In some examples, the immune composition is administered to the patient at a dose of about 50 pg of the fusion polypeptide and about 20 pg of the adjuvant once every 2 weeks or once every 3 weeks. In other examples, the composition is administered to the subject at a dose of about 50-500 pg of the fusion polypeptide once every 2 weeks or once every 3 weeks. Such a composition may be free of adjuvant.

[0020] In some embodiments, the subject may be undergoing one or more anti-cancer therapies, including, but not limited to, chemotherapy, targeted therapy, radiation therapy, immunotherapy, or a combination thereof.

[0021] In some examples, the chemotherapy includes, but is not limited to, Vincristine, Chlorambucil, Cyclophosphamide, Cytarabine, Epirubicin, Doxorubicin, Sorafenib, L- asparaginase, Rabacfosadine, or a combination thereof.

[0022] In some examples, the targeted therapy includes, but is not limited to, an EGFR inhibitor, an ALK inhibitor, or a combination thereof. In some instances, the targeted therapy includes, but is not limited to, Gefitinib, Imatinib, Toceranib, Masitinib, Dasatinib, Toceranib, Lomustine, or a combination thereof.

[0023] In some examples, the immunotherapy includes, but is not limited to, immune checkpoint inhibitors (e.g., antibodies specific to an immune checkpoint), cytokines, oncolytic virus therapy, or cell transfer therapy.

[0024] In additional aspects, the invention relates to vaccines comprising any of the immune compositions disclosed above and herein.

[0025] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGS. 1A-1C illustrate seroconversion analyses for adjuvant selection. FIG. 1A: hPD-Ll as target antigen. FIG. IB: cPD-Ll as target antigen. FIG. 1C: cCTLA-4 as target antigen.

[0027] FIGS. 2A-2C illustrate two weeks interval vaccination in rat. FIG. 2A: low dose groups (20 pg API and 20 pg Quil-A). FIG. 2B: middle dose groups (50 pg API and 20 pg Quil-A). FIG. 2C: high dose column (200 pg API and 20 pg Quil-A).

[0028] FIGS. 3A-3C illustrate three weeks interval vaccination in rat. FIG. 3A: low dose groups (20 pg API and 20 pg Quil-A). FIG. 3B: middle dose groups (50 pg API and 20 pg Quil-A). FIG. 3C: high dose column (200 pg API and 20 pg Quil-A).

[0029] FIGS. 4A-4C illurstarte the the antibody titer performance in dogs with 3 does per 2 weeks interval. FIG. 4A: 50 pg API and 20 pg Quil-A. FIG. 4B: 50 pg API and 50 pg Quil- A. FIG. 4C: 250 pg API and 250 pg Quil-A.

[0030] FIG. 5 illustrates seroconversion analysis of immune memory response and performance of antibody titer in PBN1-EG-001 dog. The dog received three fundamental vaccination and injected boosts on V7 (D99) and V13 (D238). It is shown that the antibody titers were increased on V8 (D120) and V14 (D273).

[0031] FIGS. 6A-6B illustrate analysis of the immune cell profile for PBN1-EG-001 dog, including on DO, D14, D28, D49, D63, D77, percentage of CD8 cells relative to the count on DO (FIG. 6A), and percentage of NKT cells relative to the count on DO (FIG. 6B).

[0032] FIGS. 7A-7B are CT images of dog lungs with contrast media. FIG. 7A: is the dorsal section. FIGS. 7B: is the sagittal section (left lobe), “m” means mean tumor mass. An arrow means the pulmonary portal vein may be infiltrated.

[0033] FIGS. 8A-8B are CT images of dog lungs without contrast media. FIG. 8A: is the dorsal section. FIG. 8B: is the sagittal section (left lobe). Comparison of FIG 8A with FIG. 7A, there are some intratumoral plaque-like mineralization.

[0034] FIGS. 9A-9B are X-ray images of dog lungs. FIG. 9A: is a laydown chest x-ray image. FIG. 9B: is the left-side lung chest x-ray image.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] The present disclosure relates to a fusion polypeptide comprising an extracellular domain of human CTLA-4, and an extracellular domain of human PD-L1, which can be used as an antigen in an immune composition (e.g., a vaccine) for treating cancer in, e.g., canine or feline cancer patients. As reported herein, the human versions of the CTLA-4 fragment and the PD-L1 fragment successfully induced antibodies specific to dog CTLA-4 and PD-L1, these antibodies successfully act as immune checkpoint inhibitors that increase T cell cytotoxicity in dog, leading to significant anti-tumor activity.

[0037] In some embodiments, the extracellular domain of human CTLA-4, as compared with a wild-type counterpart, comprises an amino acid substitutions at position C123 in SEQ ID NO: 1. This amino acid substitution replaces the original cysteine residue with another amino acid residue (e.g., S or a conservative substitution thereof) so as to eliminate formation of a disulfide bond at this position. It is expected that the resultant variant would less likely to aggregate and / or precipitate in manufacturing and / or formulation.

[0038] Alternatively or in addition, the fusion polypeptide disclosed herein may contain a flexible peptide linker (e.g., the G / S linker provided herein) connecting the CTLA-4 portion and the PD-L1 portion. Surprisingly, inclusion of the extra peptide linker did not affect the desired immunogenicity of the fusion polypeptide to produce anti-CTLA-4 and anti-PD-Ll antibodies in dogs receiving the fusion polypeptide as an antigen.

[0039] The present disclosure also relates to an immune composition comprising any of the fusion polypeptides disclosed herein and a suitable adjuvant, such as a saponin adjuvant. The present disclosure further relates to a method for treating a cancer using any of the immune compositions disclosed herein.

[0040] A. Fusion Polypeptide

[0041] The fusion polypeptide provided herein comprises an extracellular domain of human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4 such as hCTLA-4) and an extracellular domain of human PD-L1 (hPD-Ll) . In some instances, the CTLA-4 extracellular domain may be N-terminal to the PD-L1 extracellular domain. Alternatively, the CTLA-4 extracellular domain may be C-terminal to the PD-L1 extracellular domain. In some instances, the CTLA-4 cellular domain and the PD-L1 extracellular domain may be connected via a peptide linker (e.g., those provided herein). In some examples, the fusion polypeptide disclosed herein may be, from N-terminus to C-terminus, CTLA-4-linker-PD- Ll, which comprises, a hCTLA-4 extracellular domain (e.g., a modified fragment relative to the wild-type counterpart), a peptide linker, and a hPD-Ll extracellular domain. In other examples, the fusion polypeptide disclosed herein may be, from N-terminus to C-terminus, PD-Ll-linker-CTLA-4, which comprises, a hPD-Ll extracellular domain, a peptide linker, and a hCTLA-4 extracellular domain (e.g., a modified fragment relative to the wild-type counterpart). (i) CTLA-4 Extracellular Domain

[0042] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152, is an immune checkpoint receptor that downregulates immune responses. CTLA-4 is constitutively expressed in regulatory T cells but only upregulated in conventional T cells after activation. It acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. A CTLA-4 protein contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail. The human CTLA-4 gene is under the Gene ID: 1493, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. Multiple isoforms of CTLA-4 present in cells due to alternative splicing.

[0043] In some instances, the CTLA-4 extracellular domain for use in the fusion polypeptide may be the whole extracellular V domain of a human CTLA-4. Alternatively, the CTLA-4 extracellular domain for use in the fusion polypeptide disclosed herein can be an antigenic fragment of the extracellular V domain of a human CTLA-4 protein.

[0044] In some instances, the CTLA-4 extracellular domain for use in the fusion polypeptide may be a fragment of a wild-type (naturally -occurring) hCTLA-4 protein. The amino acid sequence of a wild-type hCTLA-4 extracellular domain is provided below (the C 123 position is in boldface and underlined).

[0045] Amino acid sequence of the wild-type hCTLA-4 extracellular domain MKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQ IYVIDPEPCPDSD (SEQ ID NO: 1)

[0046] Alternatively, the CTLA-4 extracellular domain may be a variant of a wild-type counterpart, for example, comprising one or more amino acid substitutions. In some embodiments, the CTLA-4 extracellular domain for use in the fusion polypeptide disclosed herein may contain amino acid substitution at position Cl 23 (e.g., C123S) in SEQ ID NO: 1 . The replacement of Cl 23 residue with another amino acid residue (e.g., S or a conservative) could avoid formation of undesired homodimers via disulfide bonds. It is known that cysteine residues contribute to an irreversible unfolding pathway that promotes protein aggregation, mutation of cysteine to serine may avoid protein aggregation and improve the expression level and purification process in E. coli. Consequently, mutation of free cystein residues can result in significant improvement in the storage, reconstitution, and pharmacokinetic properties of protein-based therapeutics.

[0047] In one example, the hCTLA-4 extracellular domain for use in the fusion polypeptide disclosed herein was modified from the wild type hCTLA-4 counterpart (SEQ ID NO:1) from cysteine (C) to serine (S) at position 123 (C123S).

[0048] The amino acid sequence of one exemplary modified hCTLA4 extracellular domain is provided below. The substitutions from the wild-type sequence are in bold.

[0049] Amino acid sequence of the modified hCTLA-4 extracellular domain

[0050] MKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQ IYVIDPEPSPDSD (SEQ ID NO: 2)

[0051] (ii) PD-L1 Extracellular Domains

[0052] The human PD-L1 gene is under the Gene ID: 29,126 and is located at the chromosome 9p24.2 (NCBI gene resource CD274 molecule [Homo sapiens (human)] -gene- NCBI. 2017 ncbi.nlm.nih.gov / gene). The full length of PD-L1 is a 40 kDa protein of 290 amino acids. PD-L1 is a type 1 transmembrane protein and consists of IgV-like and IgC-like extracellular domains, a hydrophobic transmembrane domain and a short cytoplasmic tail made from 30 amino acids, with unclear signal transduction properties (Dong, et al., Nat Med 1999; 5: 1365-9; Chen, et al., Ann Oncol 2016;27:409-16). The relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.

[0053] In some instances, the PD-L1 extracellular domain for use in the fusion polypeptide may be the whole IgV-like and IgC-like extracellular domain of a human PD-L1. Alternatively, the PD-L1 extracellular domain for use in the fusion polypeptide disclosed herein can be an antigenic fragment of the IgV-like and IgC-like extracellular domain of a human PD-L1. The amino acid sequence of an exemplary hPD-Ll extracellular domain is provided below.

[0054] Amino acid sequence of the hPD-Ll extracellular domain

[0055] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLK VQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVN APYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEK LFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER (SEQ ID NO: 3)

[0056] ( Hi ) Peptide Linkers

[0057] In some instances, the hCTLA-4 fragment and the hPD-Ll fragment may be connected via a peotide linker. In some examples, the peptide linker may be a poly-Glycine- Serine (G4S) linker, which is a type of flexible, unstructured synthetic peptide linker sequence (underlined in SEQ ID NO: 4 above). Such protease resistant linkers are used extensively in protein engineering to connect various protein. Linkers are short amino acid sequences created in nature to separate multiple proteins in a single fused-protein. Most of them are rigid and function to prohibit unwanted interactions between the discrete domains. For example, (G4S)3 linker (i.e., GGGGSGGGGSGGGGS, SEQ ID NO: 5) links the modified hCTLA-4 extracellular domain and the hPD-Ll extracellular domain in the aboveillustrated fusion polypeptide.

[0058] (iv) Exemplary Fusion Polypeptides

[0059] The fusion polypeptide as disclosed herein may comprise any of the hCTLA-4 extracellular domain and any of the hPD-Ll extracellular domain provided herein, and optionally a peptide linker connecting the hCTLA-4 and hPD-Ll extracellular domains. In some examples, the the extracellular domain of human CTLA-4, as compared with a wildtype counterpart, comprises amino acid substitution at position 123 in SEQ ID NO: 1. In some instances, the amino acid substitution comprises C123S. In specific examples, the hCTLA-4 fragment comprises (e.g., consisting of) the amino acid sequence of SEQ ID NO: 2. In some instances, the fusion polypeptide comprises a hPD-Ll fragment (e.g., SEQ ID NO: 3). In some instances, the peptide linker is a (G4S)nlinker, in which n is an integer of any one of 1-6. For example, the flexible peptide linker is a (GrSh linker, i.e.. GGGGSGGGGSGGGGS (SEQ ID NO: 5).

[0060] In some instances, the fusion polypeptide may comprise, from N-terminus to C- terminus, the hCTLA-4 extracellular domain, the peptide linker, and the hPD-Ll fragment. Alternatively, the fusion polypeptide may comprise, from N-terminus to C-terminus, the hPD-Ll fragment, the peptide linker, and the hCTLA-4 extracellular domain. In one example, the fusion polypeptide disclosed herein comprises the amino acid sequence of SEQ ID NO: 4 shown below.

[0061] Amino acid sequence of the exemplary fusion protein MKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQ IYVIDPEPSPDSDGGGGSGGGGSGGGGSFTVTVPKDLYVVEYGSNMTIECKFPVEKQL DLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDV KLQDAGVYRCMISYGGADYKRITVKVNAPYNKTNQRILVVDPVTSEHELTCQAEGYP KAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENH TAELVIPELPLAHPPNER (SEQ ID NO: 4) *The underlined is the linker sequence. Any of the fusion polypeptides disclosed herein may further comprise a signal peptide at the N-terminus.

[0062] (v) Preparation of the Fusion Polypeptide

[0063] The fusion polypeptide as disclosed may be prepared via a conventiona method, for example, by the recombinant technology as exemplified below.

[0064] Generally, a nucleic acid sequence encoding any of the fusion polypeptides as disclosed herein can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors / promoter would depend on the type of host cells for use in producing the antibodies.

[0065] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (S V40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.

[0066] Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Fluman Gene Therapy, 9: 1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

[0067] Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992) | combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR- VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. The tetracycline repressor (tetR) alone, rather than the tetR- mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16): 1392-1399 (2003)). One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Set. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.

[0068] Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0069] Examples of polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.

[0070] One or more vectors (e.g. , expression vectors) comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the fusion polypeptides. The host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g. , from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary, polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.

[0071] In some embodiments, methods for preparing an antibody described herein involve a recombinant expression vector that encodes a fusion polypeptide as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g. , a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium. When necessary, the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.

[0072] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.

[0073] Any of the nucleic acids encoding the fusion polypeptide as described herein, vectors (e.g., expression vectors) containing such, host cells comprising the vectors, and methods for producing the fusion polypeptide are within the scope of the present disclosure.

[0074] B. Immune Compositions

[0075] Any of the fusion polypeptides disclosed herein may be formulated into an immune composition for use in modulating (e.g., enhancing) immune responses in a recipient (e.g., a dog or cat cancer patient as disclosed herein).

[0076] Immune compositions for use in accordance with the present disclosure may be formulated in the conventional manner using one or more pharmaceutically acceptable carriers or excipients. Any of the fusion polypeptides, as well as the encoding nucleic acids, can be mixed with a pharmaceutically acceptable carrier (excipient) to form a immune composition for use in treating a target disease. The terms “excipients” and “carriers” are used herein interchangeably. “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. In some embodiments, pharmaceutically acceptable excipients (carriers) comprise adjuvant such as saponin, mineral salts, gels, particulate adjuvant or pathogen- associated molecular, or inert vehicles. See, e.g., Overview of Vaccine Adjuvants: Introduction, History, and Current Status (Methods Mol Biol . 2017: 1494:1- 13. )The immune compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0077] In some examples, the immune composition described herein comprises liposomes containing the fusion polypeptides (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0078] The fusion polypeptides, or the encoding nucleic acid(s), may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0079] The immune compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0080] In some examples, the immune composition is an injectable formulation for subcutaneous or intramuscular administration. Injectable formulations may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous infusion, water soluble antibodies can be administered by the drip method, whereby a immune formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients. Intramuscular preparations, e.g. , a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a immune excipient such as Water-for- Injection, 0.9% saline, or 5% glucose solution.

[0081] Immune compositions for use in accordance with the present disclosure may be formulated as a vaccine to immunize a canine or feline patient to treat cancer. In some embodiments, an immune composition may comprise any of the fusion polypeptides disclosed herein (e.g. the fusion polypeptide comprising SEQ ID NO: 4) and a saponin adjuvant.

[0082] In some embodiments, the immune composition is in a dosage form. A dosage form may refer to a pharmaceutical formulation for a drug product in the form as its is marked for use. The dosage form comprises one or more active ingredient (here any of the fusion polypeptides disclosed herein such as SEQ ID NO: 4) and excipients (e.g. , comprising adjuvants such as the saponin adjuvant disclosed herein) in a specific format for the intended therapeutic uses. In some instances, the dosage form may comprise a specific amount of the active ingredient and a specific amount of the excipient(s) that amount to one dose of the drug product to be given to a subject who needs the treatment. In some instances, the dosage form may be a unit dosage form, which may comprise a specific amount of the active ingredient and a specific amount of the excipient(s) in the immune composition as sold on market.

[0083] In some examples, the immune composition provided herein is a dosage form comprising about 20-200 pg of the fusion polypeptide and about 10-50 pg of the adjuvant (e.g., the saponin adjuvant). In some specific examples, the immune composition may comprise about 50 pg of the fusion polypeptide and about 20 pg of the adjuvant. In some examples, the immune composition provided herein is a dosage form comprising about 50-500 pg of the fusion polypeptide and no adjuvant.

[0084] In some embodiments, the fusion polypeptide of CTLA-4-linker-PD-Ll is mixed with a buffer to form a formulation. In some examples, the buffer comprises 10 mM sodium citrate, and has a pH of 6.5. In other examples, the buffer comprises 10 mM sodium citrate, 50 mM NaCl, and 50 mM treaholse, and has a pH value of 6.5.

[0085] The safety of the vaccine was supported by the following two studies:

[0086] (1) Rat toxicity study

[0087] (a) no significant difference in body weight was observed in both vaccinated group and the control group;

[0088] (b) both vaccinated and control groups had nomal range of blood analysis and no significant difference was observed in the two groups;

[0089] (c) all animals were unremarkable in ophthalmic examination; and

[0090] (d) non-specific lesions when interpreted histologically.

[0091] (2) Canine clinical study

[0092] (a) except for swelling at the injection site and mild fever on first day, no other adverse reactions were reported;

[0093] (b) vital signs were stable before and after vaccination (including temperature, blood pressure, and heart rate);

[0094] (c) the values of CREA, AST, and ALT from serum biochemical tests had no significant changes during the period of entry; and

[0095] (d) no SAE or AE report and life quality was good as usual.

[0096] C. Methods of Use

[0097] The immune composition may be used for treating a cancer in a subject. In certain aspects, a method for treating a cancer in a subject is provided. The method comprises administering to the subject in need thereof an effective amount of any of the pharmaceutical compositions or any of the immune compositions disclosed above and herein. The subject may be a canine or feline patient. In some instances, the subject is a dog or a cat. In some instances, the subject may be suffering from lung carcinoma, oral fibrosarcoma, oral squamous cell carcinoma, mast cell tumor, leukemia, malignant neoplasm of breast, hepatocellular carcinoma, melanoma, head and neck cancer, or lymphoma.

[0098] In some embodiments, the immune composition may be administered to the subject subcutaneously or intramuscularly at a dose of about 20-200 pg of the fusion polypeptide and about 10-50 pg of the adjuvant once every 2 weeks or once every 3 weeks. In some instances, the immune composition is administered to the patient at a dose of about 50 pg of the fusion polypeptide and about 20 pg of the adjuvant once every 2 weeks or once every 3 weeks.

[0099] An effective amount of the immune composition described above and herein can be administered to a subject (e.g. a dog or a cat) in need of the treatment via a suitable route, systemically or locally. In some embodiments, the fusion polypeptide may be administered intramuscularly or subcutaneously.

[0100] As used herein, “an effective amount” refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is increased antitumor immune responses in the tumor microenvironment. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

[0101] Empirical considerations, such as the half-life, generally contribute to the determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and / or suppression and / or amelioration and / or delay of a target disease / disorder. Alternatively, sustained continuous release formulations of a fusion polypeptide may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0102] For the purpose of the present disclosure, the appropriate dosage of a fusion polypeptide as described herein will depend on the type and severity of the disease / disorder, whether the fusion polypeptide is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the treatment, and the discretion of the attending veterinarian. Typically the veterinarian will administer a fusion polypeptide, until a dosage is reached that achieves the desired result. In some embodiments, the desired result is an increase in anti-tumor immune response in the tumor microenvironment. Methods of determining whether a dosage resulted in the desired result would be evident to one of skill in the art. Administration of one or more polypeptides can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of a fusion polypeptide may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a target disease or disorder.

[0103] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease / disorder, or a predisposition toward the disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, a symptom of the disease or disorder, or the predisposition toward the disease or disorder.

[0104] Alleviating a target disease / disorder includes delaying the development or progression of the disease or reducing disease severity or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require curative results. As used therein, "delaying" the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0105] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and / or recurrence.

[0106] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the immune composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered parenterally. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. In some examples, the immune composition is administered intraocularly or intravitreally.

[0107] Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for- Injection, 0.9% saline, or 5% glucose solution.

[0108] The particular dosage regimen, i.e.., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history. Treatment efficacy for a target disease / disorder can be assessed by methods well-known in the art.

[0109] In some embodiments, the fusion polypeptide disclosed above and herein may be administered concurrently with any of the second therapeutic agent as disclosed herein. In some embodiments, the fusion polypeptide can be administered before the second therapeutic agent. Alternatively, the fusion polypeptide can be administered after the second therapeutic agent. In some instances, the second therapeutic agent is administered systemically. In other instances, the second therapeutic agent is administered locally. In some examples, the second therapeutic agent is administered by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intra-articular, intravesical, intrasynovial, intrathecal, intratumoral, or sub-urothelial route. In one embodiment, the second therapeutic agent is administered to the subject by intravenous infusion.

[0110] Combination therapy of the immune composition disclosed above and herein and other treatment modalities may enhance therapeutic efficacy. In some embodiments, the subject may be under one or more other treatment modalities selected from the group consisting of chemotherapy, targeted therapy, radiation therapy, and immunotherapy. In some instances, the immune composition may be combined with chemotherapy drugs, particularly in non-small cell lung cancer (NSCLC) and melanoma. Chemotherapy drugs can synergistically work with the immune composition disclosed herein by reducing tumor burden and increasing immunogenicity. Some of the common chemotherapy drugs include, but are not limited to, Vincristine, Chlorambucil, Cyclophosphamide, Cytarabine, Epirubicin, Doxorubicin, Sorafenib, L-asparaginase, Rabacfosadine. Table 1 lists a selection of representative chemotherapy drugs, categorized by their common pharmacological mechanisms in chemotherapy, along with their indications. Tablel: Common Chemical Drug List

[0111] In other instances, the immune composition disclosed above and herein may be combined with targeted therapy drugs such as EGFR inhibitors or ALK inhibitors to treat lung cancer. This combination therapy can simultaneously inhibit tumor cell growth signaling pathways and enhance immune response. Table 2 below lists a selection of representative drugs for targeted therapy for lung cancer, categorized by their common pharmacological mechanism. Table 2: Common Target Therapy Drug List

[0112] In still other instances, the immune composition disclosed above and herein may be combined with radiation therapy to increase local tumor control and promote immune response. This combination therapy is commonly used in various cancer types, including head and neck cancers and lymphomas.

[0113] In still other instances, the immune composition disclosed above and herein may be combined with other immunotherapy approaches such as immune checkpoint inhibitors (e.g., antibodies specific to immune checkpoints), cytokines, oncolytic virus therapy, or cell transfer therapy. This combination therapy can enhance the immune response against tumors at different levels.

[0114] Anti-cancer activity of the treatment methods disclosed herein may be monitored by conventional approaches. For example, before, during, and after the administration of the fusion polypeptide and optionally the second therapeutic agent, cancerous cells and / or biomarkers in a subject can be measured in a biological sample, such as blood, serum, plasma, urine, peritoneal fluid, and / or a biopsy from a tissue or organ. Results thus obtained can be analyzed to assess treatment efficacy.

[0115] D. Kits

[0116] In certain aspects, kits comprising any of the fusion polypeptides or any of the immune compositions disclosed above and herein, optionally together with instructions for administration are described herein. Such kits can include one or more containers comprising a fusion polypeptide, e.g., any of those described herein, and optionally a second therapeutic agent to be co-used with the fusion polypeptide, which is also described herein.

[0117] In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of the fusion polypeptide, and optionally the second therapeutic agent, to treat, delay the onset, or alleviate a target disease as those described herein. In other embodiments, the instructions comprise a description of administering a fusion polypeptide to an individual at risk of the target disease.

[0118] The instructions relating to the use of an fusion polypeptide generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine- readable instructions (e.g. , instructions carried on a magnetic or optical storage disk) are also acceptable.

[0119] The label or package insert indicates that the composition is used for treating, delaying the onset and / or alleviating the target hematologic malignancy. In some embodiments, instructions are provided for practicing any of the methods described herein.

[0120] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. In some embodiments, a kit has a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container also has a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an fusion polypeptide as those described herein.

[0121] The kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above. General Techniques

[0122] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. ( 1986» ; Immobilized Cells and Enzymes (IRL Press, (1986)); and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0123] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. Example 1: Adjuvant Selection Assay

[0124] Adjuvants work by stimulating the immune system, often by activating antigen- presenting cells such as dendritic cells, which then present the antigen to T cells, resulting in a stronger and more effective immune response. Adjuvants may also be used in research to enhance the immune response to experimental antigens or to stimulate immune cells for use in therapies. Adjuvants can be made from a variety of substances, including mineral salts, lipids, proteins, and carbohydrates. Quil-A®, CpG1018®, Aluminum hydroxice, or nanoparticles, was / were used as adjuvants in combination with the fusion polypeptide described above (SEQ ID NO: 4) to immunize mice.

[0125] Quil-A® adjuvant is a saponin adjuvant produced under GMP by Croda used in a wide variety of veterinary vaccines, as well as in immunological research into human and veterinary applications. Saponins can induce strong cytotoxic CD8+ lymphocyte responses and potentiate the response to mucosal antigens.

[0126] Aluminum hydroxide (Alum) (Imject®, Thermo Scieneific) is the most commonly used vaccine adjuvant. Induction of inflammatory cell death at the injection site, activation of innate immune-sensing pathways and production of chemokines and cytokines at the injection site and / or draining lymph nodes (LNs).

[0127] CpG 1018 (CpG 1018®, DYNAVAX) adjuvant increases antibody concentrations, stimulates helper (CD4+) and cytotoxic (CD8+) T cell populations and generates robust T and B cell memory responses. CpG 1018 targets a single, well defined receptor (TLR9) expressed on only a few key cell types and the mechanisms of action as an adjuvant are quite well understood.

[0128] Nanoparticles (NanoCherub®) produce balanced Thl / Th2 (cell-mediated) immunity across a range of antigens and uses quantum mechanics parameters to encapsulate antigens and form densely positive-surface-charged nanoparticles.

[0129] Balb / c mice (n = 3 / group) aged 6-7 weeks were vaccinated by subcutaneous injection into 100 pl mixture (containing 20 pg API with or without different kinds of adjuvants). Quil- A: 15 pg, CpG1018 / Alu: 50 pg / 23.3 pl, and Nanopaticle: 50 pl. Vaccinations were conducted three injections every week. Vaccinations were conducted three injections every week. At Day-2, 7, 14, 21, 28, 35, 42, 49, 56 and 63, serum samples from mice were withdrawn and analyzed by ELISA (FIGS. 1A-1C). Serum samples containing specific antibodies against human PD-L1 (hPD-Ll) (FIG. 1A), canine PD-L1 (cPD-Ll) (FIG. IB) and canine CTLA-4 (cCTLA-4) (FIG. 1C) were observed at Day 14, reached the peak at Day 21 and maintained up to Day 63. Comparing three different adjuvant combination groups, Quil-A combining active pharmaceutical ingredient (API) groups showed the better antibody production performance (FIGS. 1A-1C). Finally, Quil-A adjuvant combined with API could assist to produce more specific antibodies and then Quil-A was selected.

[0130] Example 2: Rat Vaccination with CTLA4-PD-L1 Fusion Polypeptide

[0131] This example explores the effect of an immune composition comprising a fusion polypeptide of CTLA-4-linker-PD-Ll (SEQ ID NO: 4) and a saponin-based adjuvant Quil- A® in normal saline solution, for eliciting antibody responses in a rat model.

[0132] Sprague Dawley (SD) rats aged 6-7 weeks were vaccinated by the immune composition via subcutaneous injection, a total of three doses, 14 days (2 weeks) intervals (vaccination on DO, D14 and D28) or 21 days (3 weeks) intervals (vaccination on DO, D21 and D42). In each group (n = 3), there were low doses (LD, 20 pg API and 20 pg Quil-A), middle doses (MD, 50 pg immune composition and 20 pg Quil-A), and high doses (HD, 200 pg API and 20 pg Quil-A) groups.

[0133] In the two weeks interval group, after three vaccinations, the antibody titer reached the peak at D42 and the peak could be maintained to D56 at least (FIGS. 2A-2C). Additionally, it was also observed that antibody titer performance induced by different doses was similar. In the three weeks interval group, the antibody titer reached the peak at D42 and the peak could be maintained to D56 at least (FIGS. 3A-3C). Compared the middle dose group with low dose and high dose groups, it was revealed that the middle dose vaccination group showed stronger antibody titer than other two groups. In the three weeks interval groups, most mouse antibody titers were up to the peak at D63. The high dose group seemed to have better antibody performance.

[0134] In order to reach the peak of antibody titer efficiently for cancer therapy, two weeks administration interval was selected, and middle dose was used as treatment regimen.

[0135] Example 3: Dog Vaccination with CTLA4-PD-L1 Fusion Polypeptide

[0136] This example explores the effect of an immune composition comprising fusion polypeptide of CTLA-4-linker-PD-Ll (SEQ ID NO: 4) with human source and a saponin- based adjuvant Quil-A in normal saline solution, for eliciting antibody responses in a dog model.

[0137] Dogs was vaccinated with the immune composition vaccine via subcutaneous injection with a total of three doses, 14 days apart (DO, D14 and D28). In the three dose groups, different amount of API and adjuvant were used (FIG. 4A: 50 pg API and 20 pg Quil-A. FIG. 4B: 50 pg API and 20 pg Quil-A. FIG. 4C: 250 pg API and 250 pg Quil-A). To investigate the specific antibody production, blood samples were drawn on the dates indicated in the figures, and antibody titer was analyzed / detremined by ELISA based on the following protocol:

[0138] Coat the Plate:

[0139] 1. Dilute antigens (Canine PD-L1 and canine CTLA-4) to a final concentration of 0.5 pg / ml in Coating Buffer (0. 1 M NaHCCL) and transfer 50 pl to each well of a high affinity, protein-binding ELISA plate.

[0140] 2. Seal the plate and incubate overnight at 4°C.

[0141] Block the Plate:

[0142] 1. Bring the plate to room temperature (RT), flick off the capture antibody solution, wash 3 times with PBSTo i (0.1% Tween 20 in PBS) and block non-specific binding sites by adding 200 pl of Blocking Solution (1% BSA in PBS) to each well.

[0143] 2. Seal plate and incubate at RT for 2 hours.

[0144] Add Standards and Samples:

[0145] 1. Dilute immunized canine sera (400x, 800x, 1600x, 3200x, 6400x, 12800x, 25600x, 512000x, 1024000x) to in Blocking Solution.

[0146] 2. Add 50 pl per well to the ELISA plate.

[0147] 3. Seal the plate and incubate for 1 hour at RT.

[0148] 4. Wash 3 times with PBSTo.i.

[0149] Add Detection Antibody:

[0150] 1. Dilute Goat anti-canine IgG-HRP (1:5,000) for samples in Blocking Solution.

[0151] 2. Add 50 pl of diluted antibody to each well.

[0152] 3. Seal the plate and incubate for 1 hour at RT.

[0153] 4. Wash 3 times with PBSTO.I.

[0154] Add Substrate (color development):

[0155] 1. Thaw TMB Substrate Solution within 10 min of use.

[0156] 2. Add 50 pl TMB into each well and incubate at RT for 10 min color development.

[0157] 3. To stop the color reaction, add 50 pl of Stop Solution.

[0158] 4. Read the optical density (OD) for each well with a microplate reader set to 450 nm.

[0159] After three vaccinations, the antibody titer reached the peak at D28. (FIGS. 4A-4C). Additionally, it was also observed that antibody titer performance induced by different doses was different. Among the three groups, antibodies against PD-L1 and CTLA-4 could be tiggered. It worthed mentioned that the sequence similarity between canine PD-L1 and haman PD-L1 or canine CTLA-4 or human CTLA-4 is 80.5% and 84.1%, respectively. It is unexpected that in all three groups, antibodies against canine PD-L1 and canine CTLA-4 can be deleted in dog serum after vaccinated with the immune composition, which is a fusion protein vaccine comprising human sequence.

[0160] Example 4: Antibody Production and Memory Response in Dog Vaccinated with CTLA4-PD-L1 Fusion Polypeptide

[0161] A dog (PBN1-EG-001) was vaccinated with an immune composition comprising fusion polypeptide of CTLA-4- linker- PD-L1 (SEQ ID NO: 4) via subcutaneous injection only, a total of three doses, 14 days apart (DO, D14 and D28). To investigate the specific antibody production, blood samples were drawn on DO, D14, D28, D49, D63, D77, and antibody titer was analyzed / detremined by ELISA based on the protocol described above in Example 3.

[0162] On D99, a boost was taken at the same dosage as vaccination when antibody titer was seen to go down comtinuously. Higher antibody titer production on DI 20 suggested that memory response was established in this dog.

[0163] When immune memory response was established in the dog, the antibody titer detection by ELISA was followed continuously. After performing ELISA experiments, the antibody titers against PD-L1 and CTLA-4 decreased on D141 comparing to DI 20 which was the peak of antibody titer by the boost shot (FIG. 5).

[0164] On D238, a second boost was taken at the same dosage. Higher antibody titer production on D273 was observed, suggesting that memory response was established in this dog (FIG. 5).

[0165] Example 5: Flow Cytometry Analysis of Immune Cell Markers

[0166] A dog (PBNl-EG-001) was vaccinated by an immune composition comprising fusion polypeptide of CTLA-4-linker-PD-Ll (SEQ ID NO: 4) via subcutaneous injection only, a total of three doses, 14 days interval (on DO, D14 and D28). To investigate the immune cell profiles, blood samples were drawn on DO, D14, D28, D49, D63, D77 and immune cells were analyzed by flow cytometry based on the following protocol: Plasma separation: transfer the peripheral blood to a 15 mL centrifuge tube, centrifuge at 1000 x g for 10 minutes, transfer the supernatant to a cryovial, and store it in a -80°C ultra-low temperature freezer for subsequent testing.

[0167] Separation of peripheral blood lymphocytes:

[0168] 1. Take 1 mL of the remaining blood from “Plasma separation” for erythrocyte lysis.

[0169] 2. Prepare lx RBC Lysis buffer and warm it up to 37°C.

[0170] 3. lx RBC Lysis buffer and remaining blood were mixed in a 50-mL plastic centrifuge tube at a volume ratio of 20: 1.

[0171] 4. Place the mixture on a 3D shaker at room temperature (RT) and shake for 10 minutes in the dark.

[0172] 5. Add an equal volume of IxPBS to the above mixture to stop the lysis reaction.

[0173] 6. Centrifuge at 400 x g for 5 minutes at RT.

[0174] 7. After removing the supernatant, add 10 mL IxPBS and centrifuge at 400 x g for 5 minutes at RT.

[0175] 8. Remove the supernatant after centrifugation, re-lyse the cells with an appropriate amount of R-10 medium (10% FBS / 1% Penicillin+Streptomycin / 0.1% P- mercaptoethanol / RPMI Medium 1640) and count PBMC numbers.

[0176] PMA / Ionomycin activation stimulation:

[0177] 1. Take an appropriate amount of cells, the cell density is 0.2xl06cells / 200 pL, in a U-bottom 96-well plate.

[0178] 2. Add cell stimulation cocktail (500 folds dilution) and GolgiStop™ (1000 folds dilution) and mix well.

[0179] 3. Incubate in a carbon dioxide incubator for 2 hours at 37°C.

[0180] Immune function tests:

[0181] 1. After the cells were stimulated for 2 hours, they were transferred to a V-bottom 96-well plate.

[0182] 2. Centrifuge at 400 x g for 5 minutes at RT and discard the supernatant.

[0183] 3. Add 200 pL staining buffer to each well and repeat step 2.

[0184] 4. Add the surface antigen antibody combination to each well and use staining buffer to adjust the volume to 50 pL, use a multi-channel micro-dispenser to slowly aspirate to mix the cells and antibodies, and place in a 4°C refrigerator for 10 minutes in the dark.

[0185] 5. Add 150 pL staining buffer to each well to make up the volume to 200 pL. 6. Centrifuge at 400 x g for 5 minutes at RT and discard the supernatant.

[0186] 7. Add 200 pL staining buffer to each well and repeat step 6 twice.

[0187] 8. Add 100 pL of Fixation / Permeabilization solution to each well and incubate in the refrigerator at 4°C for 20 minutes.

[0188] 9. Add 100 pL of Ixpermeabilization wash buffer, centrifuge at 400 x g for 5 minutes at RT and discard the supernatant.

[0189] 10. Add 200 pL of Ixpermeabilization wash buffer, centrifuge at 400 x g for 5 minutes at RT and discard the supernatant.

[0190] 11. First add 50 pL of Ixpermeabilization wash buffer to each well, then add the required internal staining antibody or isotype antibody to each well, mix well, and place in a 4°C refrigerator for 30 minutes in the dark (CD8 T cells: CD45+, CD3+, CD5+and CD8+; NK T cells: CD45+, CD3+and CD5dim).

[0191] 12. Add 150 pL of Ixpermeabilization wash buffer, centrifuge at 400 x g for 5 minutes at RT and discard the supernatant.

[0192] 13. Add 200 pL of Ixpermeabilization wash buffer, centrifuge at 400 x g for 5 minutes at RT and discard the supernatant.

[0193] 14. Add 200 pL staining buffer to mix the cells evenly, transfer the cell suspension into the flow tube, collect flow cytometry data, and analyze with Kaluza analysis software (version 2.1).

[0194] The numbers of CD8+ T cells and NKT cells were analyzed. After three vaccinations, it was found that the number of CD8+ and NKT cells tented to increase. The fold changes of CD8+ and NKT cells were 1.8 folds on D28 (FIGS. 6A-6B). CD8+ cytotoxic T lymphocytes (CTLs) are preferred immune cells for targeting cancer. NKT cells are a unique subset of T cells and have the capacity to mount strong anti-tumor responses. The results suggest that the immune composition is effective in cancer therapy via, e.g., enhanced immune responses.

[0195] Example 6: Alleviation of Lesions in PBN1-EG001 Dog

[0196] Two image tools were used to evaluate lung tumor progress in PBN1-EG-001 dog vaccinated with the immune composition comprising fusion polypeptide of CTLA-4-linker- PD-L1 (SEQ ID NO: 4) (50 pg API and 20 pg Quil-A). The first CT image was taken prior to vaccination and the results confirmed presence of a solid tumor at the left lung, and tumor cell infiltration in the pulmonary portal vein. (FIGS. 7A-7B). The PBN1-EG-001 dog was then vaccinated by the immune composition comprising fusion polypeptide of CTLA-4- linker-PD-Ll (SEQ ID NO: 4) via subcutaneous injection three times through 2 weeks intervals. Tumors induced at the left and right lungs had different sizes. Compared with the previous scan (FIG. 8A versus FIG. 7A), the lesion area of the left posterior lobe occupies more of the parenchyma of the left posterior lobe; however, the second CT image found some cavities (tumor necrosis) in the tumor and the pulmonary portal vein without further infiltration (FIGS. 8A-8B) in circled regions. Next, X-ray was performed on the PBN1-EG- 001 dog around 6-month post vaccination and it was found that the left tumor volume significantly decreased (FIG. 9B). The right tumor nodule was grosser but with cavities (FIG. 9A). The final survival period was about 9 months after diagnosis, which was prolonged for at least 4 months compared to veterinary surgeon’ s prediction. These results suggest that the immune composition, as a fusion protein vaccine comprising human sequence, unexpectedly exhibits therapeutical efficacy in treating cancer in dogs.

[0197] Example 7: Efficay Evalutaion in Cats

[0198] This example aims to explore the effect of an immune composition comprising fusion polypeptide of CTLA-4-linker-PD-Ll (SEQ ID NO: 4) with human source, for eliciting antibody responses in cat.

[0199] To evaluate the efficacy of cat vaccinated with the immune composition comprising fusion polypeptide of CTLA-4- linker-PD-Ll (SEQ ID NO: 4), non-adjuv anted immune composition is to be subcutaneously injected to healthy cats. The doses of the immune composition include 50 pg, 100 pg, 150 pg, 250 pg, and 500 pg. The dosing interval is three times through 2 weeks intervals. To investigate the specific antibody production, blood samples from several time points are obtained after vaccination, and antibody titer is to be analyzed / determined by ELISA based on a protocol similar to the one described above in Example 3. In the ELISA for determining antibody tier, the coating antigen is feline CTLA- 4 or feline PD-L1, and the detection antiboy is anti-feline IgG-HRP.

[0200] Non-adjuvanted immune composition is to be subcutaneously injected to tumorbearing cats. The doses of the immune composition include 50 pg, 100 pg, 150 pg 250 pg, and 500 pg. The dosing interval is three times through 2 weeks intervals. To investigate the specific antibody production, blood samples from several time points are obtained after vaccination, and antibody titer is to be analyzed / determined by ELISA. The sequence similarity between feline PD-L1 and haman PD-L1 or feline CTLA-4 and human CTLA-4 is 76.9% and 85.71%, respectively. To evaluate the therapeutical efficacy, tumor size, disease status, or final survival of the tumor-bearing cats are to be evaluated to understand the efficacy of the vaccine comprising fusion polypeptide of CTLA-4-linker-PD-Ll (SEQ ID NO: 4), as a fusion protein vaccine comprising human sequence, in treating cancer in cats.

[0201] OTHER EMBODIMENTS

[0202] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0203] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0204] EQUIVALENTS

[0205] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0206] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0207] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0208] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0209] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0210] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0211] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0212] The term “about” or “approximately” as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ± 20 %, preferably up to ± 10 %, more preferably up to ± 5 %, and more preferably still up to ± 1 % of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

[0213] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements), etc.

Claims

WHAT IS CLAIMED IS:

1. A fusion polypeptide comprising (a) an extracellular domain of human CTLA- 4, and (b) an extracellular domain of human PD-L1, wherein the extracellular domain of human CTLA-4, as compared with a wild-type counterpart, comprises amino acid substitution at position 123 in SEQ ID NO: 1, and / or wherein the fusion polypeptide further comprises (c) a flexible peptide linker, which is located between (a) and (b).

2. The fusion polypeptide of claim 1 , wherein the amino acid substitution is C123S.

3. The fusion polypeptide of claim 1 or claim 2, wherein the flexible peptide linker is a (G4S)nlinker, in which n is an integer of any one of 1-6; optionally wherein the flexible peptide linker linker is GGGGSGGGGSGGGGS (SEQ ID NO: 5).

4. The fusion polypeptide of any one of claims 1-3, wherein the extracellular domain of human CTLA-4 of (a) comprises the amino acid sequence of SEQ ID NO: 2.

5. The fusion polypeptide of any one of claims 1-4, wherein the extracellular domain of human PD-L1 of (b) comprises the amino acid sequence of SEQ ID NO: 3.

6. The fusion polypeptide of claim 1, which comprises the amino acid sequence of SEQ ID NO: 4.

7. A nucleic acid, comprising a nucleotide sequence encoding a fusion polypeptide set forth in any one of claims 1-6.

8. An immune composition, comprising the fusion polypeptide of any one of claims 1-6 or the nucleic acid of claim 7, and one or more pharmaceutically acceptable carriers, which optionally comprise an adjuvant.

9. An immune composition, comprising a fusion polypeptide and a saponin adjuvant, wherein the fusion polypeptide comprises (a) an extracellular domain of humanCTLA-4, (b) an extracellular domain of human PD-L1, and optionally (c) a flexible peptide linker connecting (a) and (b), which optionally is a (G4S)nlinker, wherein n is an integer of any one of 1-6.

10. The immune composition of claim 9, wherein the fusion polypeptide is set forth in any one of claims 1-6.

11. The immune composition of any one of claims 8-10, which is a dosage form comprising about 20-200 pg of the fusion polypeptide and about 10-50 pg of the adjuvant; optionally about 50 pg of the fusion polypeptide and about 20 pg of the adjuvant.

12. The immune composition of any one of claims 8-10, which is a dosage form comprising about 50-500 pg of the fusion polypeptide; optionally wherein the dosage form is free of adjuvant.

13. A method for treating a cancer in a subject, the method comprising administering to the subject in need thereof an effective amount of the immune composition of any one of claims 8-12, wherein the subject is a canine or feline cancer patient.

14. The method of claim 13, wherein the subject is a dog or a cat.

15. The method of claim 13 or claim 14, wherein cancer is lung carcinoma, oral fibrosarcoma, oral squamous cell carcinoma, mast cell tumor, leukemia, malignant neoplasm of breast, hepatocellular carcinoma, melanoma, head and neck cancer, or lymphoma.

16. The method of any one of claims 11-15, wherein the immune composition is administered to the subject subcutaneously or intramuscularly.

17. The method of any one of claims 11-16, wherein the immune composition is administered to the subject at a dose of about 20-200 pg of the fusion polypeptide and about 10-50 pg of the adjuvant once every 2 weeks or once every 3 weeks.

18. The method of claim 17, wherein the immune composition is administered to the patient at a dose of about 50 pg of the fusion polypeptide and about 20 pg of the adjuvant once every 2 weeks or once every 3 weeks.

19. The method of any one of claims 11-16, wherein the immune composition is administered to the subject at a dose of about 50-500 pg of the fusion polypeptide once every 2 weeks or once every 3 weeks; optionally wherein the immune composition is free of adjuvant.

20. The method of any one of claims 11-19, wherein the subject is under one or more anti-cancer therapy selected from the group consisting of chemotherapy, targeted therapy, radiation therapy, immunotherapy, and a combination thereof.

21. The method of claim 20, wherein the chemotherapy comprises Vincristine, Chlorambucil, Cyclophosphamide, Cytarabine, Epirubicin, Doxorubicin, Sorafenib, L- asparaginase, Rabacfosadine, or a combination thereof.

22. The method of claim 20 or 21, wherein the targeted therapy comprises an EGFR inhibitor or ALK inhibitor, which optionally is Gefitinib, Imatinib, Toceranib, Masitinib, Dasatinib, Toceranib, Lomustine, or a combination thereof.

23. The method of any one of claims 20-22, wherein the immunotherapy comprisesan immune checkpoint inhibitor, which optionally is an antibody specific to an immune checkpoint molecule, a cytokine, an oncolytic virus, or cell transfer therapy.

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