Compositions and methods for cancer treatment

Abstract

The present invention relates to a fusion polypeptide comprising the extracellular domain of human CTLA-4 and the extracellular domain of human PD-L1, and an immunocomposition comprising the same, for use in the treatment of cancer in animals such as dogs and cats.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 510,286, filed on 26 June 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, the contents of which are incorporated herein by reference in their entirety. The XML copy was created on 18 June 2024, is named "063700-501001WO_SeqList_ST26.xml", and has a size of 5,997 bytes. [Background technology]

[0003] Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) belongs to the immunoglobulin superfamily. CTLA-4 has been reported to play an important role in the regulation of the immune system (Keilholz, U., J. Immunother 31, 431-439).

[0004] Programmed cell death protein 1 (PD-1) is a member of the CD28 superfamily. When PD-1 binds to its ligands, programmed cell death ligands 1 (PD-L1) and 2 (PD-L2), it induces a negative signaling pathway (Riley, JL, Immunol Rev 229, 114-125). This PD-1 / PD-ligand pathway plays a crucial role in immune tolerance and immunity. [Overview of the project]

[0005] This disclosure is based, at least in part, on the discovery that exemplary immunocompositions comprising CTLA-4-linker-PD-L1 polypeptide and an adjuvant unexpectedly enhanced the immune response and antitumor activity in a canine lung tumor model. Therefore, such immunocompositions are expected to be effective in inducing antitumor immune responses and treating tumors in pet animals such as cats and dogs.

[0006] Accordingly, the present invention relates to compositions and methods for treating cancer in dogs or cats, comprising using a fusion polypeptide comprising a human CTLA-4 fragment and a human PD-L1 fragment to enhance the immune response in cancer patients in dogs or cats.

[0007] In some embodiments, the disclosure provides a fusion polypeptide comprising (a) the extracellular domain of human CTLA-4 and (b) the extracellular domain of human PD-L1. In some embodiments, the extracellular domain of human CTLA-4 comprises the 123rd amino acid substitution of SEQ ID NO: 1 compared to the wild-type counterpart. In some examples, the 123rd amino acid substitution is C123S. 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 of (b). In some examples, the extracellular domain of human PD-L1 may comprise the amino acid sequence of SEQ ID NO: 3.

[0008] In some embodiments, the fusion polypeptides provided herein may further include a flexible peptide linker located between (a) and (b). In some embodiments, the flexible peptide linker may be a (G4S)n linker, where n is 1, 2, 3, 4, 5, or 6. In some examples, the flexible peptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 5).

[0009] In a specific example, the fusion polypeptide may contain the amino acid sequence of SEQ ID NO: 4.

[0010] In some embodiments, the Disclosure provides nucleic acids comprising a nucleotide sequence encoding any of the fusion polypeptides disclosed above and herein. In some embodiments, the nucleic acid may be a vector, such as an expression vector. The Disclosure also provides a host cell containing the nucleic acid, such as an expression vector. The fusion polypeptide can be produced within the host cell. Furthermore, the Disclosure also provides a method for producing the fusion polypeptide by culturing such a host cell and harvesting the resulting fusion polypeptide.

[0011] In some embodiments, the Disclosure provides an immunocomposition comprising a fusion polypeptide or nucleic acid as described above and herein, and one or more pharmaceutically acceptable carriers which may include an adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some examples, the immunocomposition disclosed herein comprises a fusion polypeptide and a saponin adjuvant as disclosed herein. In some embodiments, the immunocomposition may be a dosage form comprising about 20 to 200 μg of fusion polypeptide and about 10 to 50 μg of adjuvant. In some examples, the dosage form may comprise about 50 μg of fusion polypeptide and about 20 μg of adjuvant. Alternatively, the composition is a dosage form comprising about 50 to 500 μg of fusion polypeptide. In some examples, the composition does not contain an adjuvant.

[0012] In a further embodiment, the Disclosure provides a method for treating cancer in a subject. This method may include administering an effective amount of any of the immunocompositions disclosed above and herein to a patient. The subject may be a canine or feline patient with cancer.

[0013] In some cases, canine or feline cancer patients may have lung cancer, oral fibrosarcoma, oral squamous cell carcinoma, mast cell tumor, leukemia, malignant tumors of breast cancer, hepatocellular carcinoma, melanoma, head and neck cancer, or lymphoma.

[0014] In some embodiments, the immune composition may be administered subcutaneously. In some embodiments, the immune composition may be administered to a patient every two weeks or every three weeks in doses of about 20-200 μg of fusion polypeptide and about 10-50 μg of adjuvant. In some examples, the immune composition is administered to a patient every two weeks or every three weeks in doses of about 50 μg of fusion polypeptide and about 20 μg of adjuvant. In other examples, the composition is administered to a subject every two weeks or every three weeks in doses of about 50-500 μg of fusion polypeptide. Such compositions may not contain an adjuvant.

[0015] In some embodiments, the subject may be receiving one or more anti-cancer therapies, including but not limited to chemotherapy, targeted therapy, radiotherapy, immunotherapy, or a combination thereof.

[0016] In some cases, chemotherapy may include, but is not limited to, vincristine, chlorambucil, cyclophosphamide, cytarabine, epirubicin, doxorubicin, sorafenib, L-asparaginase, rabacfosazine, or combinations thereof.

[0017] In some cases, targeted therapy includes, but is not limited to, EGFR inhibitors, ALK inhibitors, or combinations thereof. Also, in some cases, targeted therapy includes, but is not limited to, gefitinib, imatinib, toceranib, masitinib, dasatinib, toceranib, lomustine, or combinations thereof.

[0018] In some examples, immunotherapy includes, but is not limited to, immune checkpoint inhibitors (e.g., antibodies specific to immune checkpoints), cytokines, oncolytic virus therapy, or cell transfer therapy.

[0019] In yet another embodiment, the present invention relates to a vaccine comprising any of the immunological compositions described above and disclosed herein.

[0020] Details of one or more embodiments of the present invention are described in the following description. Other features or advantages of the present invention will become apparent from the following drawings, detailed description of the embodiments, and appended claims.

Brief Description of Drawings

[0021] [Figure 1] Figures 1A - 1C show seroconversion analysis for adjuvant selection. Figure 1A: hPD-L1 as the target antigen. Figure 1B: cPD-L1 as the target antigen. Figure 1C: cCTLA-4 as the target antigen. [Figure 2] Figures 2A - 2C show vaccination at two-week intervals in rats. Figure 2A: Low-dose group (20 μg of API and 20 μg of Quil-A). Figure 2B: Medium-dose group (50 μg of API and 20 μg of Quil-A). Figure 2C: High-dose group (200 μg of API and 20 μg of Quil-A). [Figure 3] Figures 3A - 3C show vaccination at three-week intervals in rats. Figure 3A: Low-dose group (20 μg of API and 20 μg of Quil-A). Figure 3B: Medium-dose group (50 μg of API and 20 μg of Quil-A). Figure 3C: High-dose group (200 μg of API and 20 μg of Quil-A). [Figure 4] Figures 4A - 4C show the change in antibody titer in dogs administered three times at two-week intervals. Figure 4A: 50 μg of API and 20 μg of Quil-A. Figure 4B: 50 μg of API and 50 μg of Quil-A. Figure 4C: 250 μg of API and 250 μg of Quil-A. [Figure 5] Figure 5 shows seroconversion analysis of the immune memory response and the change in antibody titer in dog PBN1-EG-001. This dog received three vaccinations as the primary immunization, additional vaccinations at V7 (D99) and V13 (D238), and it has been shown that the antibody titer increased at V8 (D120) and V14 (D273). [Figure 6]Figures 6A-6B show the analysis of the immune cell profile of PBN1-EG-001 dogs, including the percentage of CD8 cells (percentage relative to the cell count at the D0 time point, Figure 6A) and the percentage of NKT cells (percentage relative to the cell count at the D0 time point, Figure 6B) at D0, D14, D28, D49, D63, and D77. [Figure 7] Figures 7A-7B are CT images of the lungs of dogs using a contrast agent. Figure 7A: Dorsal section. Figure 7B: Sagittal section (left lobe). "m" indicates the average tumor mass. The arrow indicates the possibility of infiltration into the hilar vein. [Figure 8] Figures 8A-8B are CT images of the lungs of dogs without contrast agent administration. Figure 8A: Dorsal section, Figure 8B: Sagittal section (left lobe). When comparing Figure 8A and Figure 7A, plaque-like calcification is observed within the tumor. [Figure 9] Figures 9A-9B are X-ray images of the lungs of dogs. Figure 9A is a supine chest X-ray image. Figure 9B is a chest X-ray image of the left lung.

Mode for Carrying Out the Invention

[0022] The present invention relates to a fusion polypeptide comprising the extracellular domain of human CTLA-4 and the extracellular domain of human PD-L1, which can be used, for example, as an antigen in an immunological composition (such as a vaccine) for cancer treatment in cancer patients of dogs or cats. As described herein, the CTLA-4 fragment and PD-L1 fragment derived from humans have successfully induced antibodies specific to canine CTLA-4 and PD-L1. These antibodies function as immune checkpoint inhibitors that enhance the cytotoxic activity of T cells in dogs and have shown significant antitumor activity. [[ID=I9]]

[0023] In some embodiments, the extracellular domain of human CTLA-4 contains an amino acid substitution at position C123 of SEQ ID NO: 1 compared to the wild-type counterpart. This amino acid substitution eliminates the formation of a disulfide bond at this position by replacing the original cysteine ​​residue with another amino acid residue (e.g., S or its conserved substitution). The resulting variants are expected to be less likely to aggregate and / or precipitate during manufacturing and / or formulation.

[0024] Alternatively, or in addition, the fusion polypeptides disclosed herein may include a flexible peptide linker (e.g., the G / S linker described herein) linking the CTLA-4 and PD-L1 moieties. Surprisingly, the inclusion of this additional peptide linker did not affect the desired immunogenicity of the fusion polypeptide, which is the production of anti-CTLA-4 and anti-PD-L1 antibodies in dogs administered the fusion polypeptide as an antigen.

[0025] This disclosure also relates to an immunocomposition comprising one of the fusion polypeptides disclosed herein and a suitable adjuvant, such as a saponin adjuvant. Furthermore, this disclosure relates to a method for treating cancer using one of the immunocompositions disclosed herein.

[0026] A. Fusion Polypeptides The fusion polypeptides provided herein include the extracellular domain of human cytotoxic T lymphocyte-associated protein 4 (CTLA-4, e.g., hCTLA-4) and the extracellular domain of human PD-L1 (hPD-L1). In some cases, the CTLA-4 extracellular domain is located N-terminally to the PD-L1 extracellular domain. Alternatively, the CTLA-4 extracellular domain is located C-terminally to the PD-L1 extracellular domain. In some cases, the CTLA-4 extracellular domain and the PD-L1 extracellular domain may be linked via a peptide linker (e.g., one provided herein). In some examples, the fusion polypeptides disclosed herein may be CTLA-4-linker-PD-L1, comprising the hCTLA-4 extracellular domain (e.g., a modified fragment compared to its wild-type counterpart), a peptide linker, and the hPD-L1 extracellular domain from N-terminus to C-terminus. In other examples, the fusion polypeptides disclosed herein may also include a PD-L1 linker CTLA-4 comprising an hPD-L1 extracellular domain, a peptide linker, and an hCTLA-4 extracellular domain (e.g., a modified fragment compared to its wild-type counterpart) from the N-terminus to the C-terminus.

[0027] (i) CTLA-4 extracellular domain Cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152, is an immune checkpoint receptor that suppresses the immune response. CTLA-4 is constitutively expressed in regulatory T cells but is upregulated only in conventional T cells after activation. When CTLA-4 binds to CD80 or CD86 on the surface of antigen-presenting cells, it functions as an "off" switch. The CTLA-4 protein contains an extracellular V domain, a transmembrane domain, and a cytoplasmic terminal. The human CTLA-4 gene is registered under GeneID:1493, and its relevant information is incorporated herein by reference for the purposes and purposes of this text. Furthermore, multiple CTLA-4 isoforms exist within cells due to alternative splicing.

[0028] In some cases, the CTLA-4 extracellular domain used in the fusion polypeptide may be the entire extracellular V domain of human CTLA-4. Alternatively, the CTLA-4 extracellular domain used in the fusion polypeptide disclosed herein may be an antigenic fragment of the extracellular V domain of the human CTLA-4 protein.

[0029] In some cases, the extracellular domain of CTLA-4 used in fusion polypeptides may be a fragment of the wild-type (natural) hCTLA-4 protein. The amino acid sequence of the wild-type hCTLA-4 extracellular domain is shown below (position C123 is bold and underlined).

[0030] Amino acid sequence of the extracellular domain of wild-type hCTLA-4 MKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEP C PDSD (Sequence ID: 1)

[0031] Alternatively, the CTLA-4 extracellular domain may be a variant of the wild-type counterpart, for example, containing one or more amino acid substitutions. In some embodiments, the CTLA-4 extracellular domain used in the fusion polypeptide disclosed herein may contain an amino acid substitution (e.g., C123S) at position C123 of SEQ ID NO: 1. Substituting the C123 residue with another amino acid residue (e.g., S or a conservative substitution) can avoid the formation of undesirable homodimers via disulfide bonds. Cysteine ​​residues are known to contribute to an irreversible unfolding pathway that promotes protein aggregation, and mutating cysteine ​​to serine can avoid protein aggregation and improve expression levels and purification processes in E. coli. Therefore, mutations in free cysteine ​​residues can significantly improve the conservation, reconstitution, and pharmacokinetic properties of protein-based therapeutics.

[0032] For example, the extracellular domain of hCTLA-4 used in the fusion polypeptide disclosed herein is modified from the wild-type hCTLA-4 counterpart (SEQ ID NO: 1) by substituting cysteine ​​(C) with serine (S) at position 123 (C123S).

[0033] The amino acid sequence of an example of a modified hCTLA4 extracellular domain is shown below. Substitutions from the wild-type sequence are shown in bold.

[0034] Amino acid sequence of the modified hCTLA-4 extracellular domain MKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPSPDSD(Sequence ID 2)

[0035] (ii) PD-L1 extracellular domain The human PD-L1 gene is GeneID:29,126 and is located on chromosome 9p24.2 (NCBI gene resource CD274 molecule [Homo sapiens (human)]-gene-NCBI. 2017 ncbi.nlm.nih.gov / gene). The full-length PD-L1 is a 40 kDa protein consisting of 290 amino acids. PD-L1 is a type I transmembrane protein composed of IgV-like and IgC-like extracellular domains, a hydrophobic transmembrane domain, and a short cytoplasmic tail of 30 amino acids. Its signaling properties are unknown (Dong, et al., Nat Med 1999; 5: 1365-9; Chen, et al., Ann Oncol 2016;27:409-16). Relevant information is incorporated herein by reference with respect to the matters and purposes cited herein.

[0036] In some cases, the PD-L1 extracellular domain used in the fusion polypeptide may be the entire IgV-like and IgC-like extracellular domains of human PD-L1. Alternatively, the PD-L1 extracellular domain used in the fusion polypeptide disclosed herein may be antigenic fragments of the IgV-like and IgC-like extracellular domains of human PD-L1. An example of the amino acid sequence of an hPD-L1 extracellular domain is shown below.

[0037] Amino acid sequence of the extracellular domain of hPD-L1 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER (Sequence ID 3)

[0038] (iii) Peptide linker In some cases, the hCTLA-4 and hPD-L1 fragments are linked via a peptide linker. In some examples, the peptide linker may be a polyglycin-serine (G4S) linker, a type of flexible, unstructured synthetic peptide linker sequence (underlined in SEQ ID NO: 4 above). Such protease-resistant linkers are widely used in protein engineering to link various proteins. Linkers are short amino acid sequences that occur in nature to separate multiple proteins in a single fusion protein. Most linkers are fixed and serve to prevent undesirable interactions between individual domains. For example, the (G4S)3 linker (i.e., GGGGSGGGGSGGGGS, SEQ ID NO: 5) links the modified hCTLA-4 extracellular domain to the hPD-L1 extracellular domain in the fusion polypeptide described above.

[0039] (iv) Exemplary fusion polypeptide The fusion polypeptides disclosed herein may comprise any of the hCTLA-4 extracellular domains and any of the hPD-L1 extracellular domains described herein, and optionally a peptide linker linking the hCTLA-4 extracellular domain and the hPD-L1 extracellular domain. In some examples, the human CTLA-4 extracellular domain contains the 123rd amino acid substitution of SEQ ID NO: 1 compared to its wild-type counterpart. In some examples, the amino acid substitution comprises C123S. In a specific example, the hCTLA-4 fragment contains (e.g., consists of) the amino acid sequence of SEQ ID NO: 2. In some examples, the fusion polypeptide comprises an hPD-L1 fragment (e.g., SEQ ID NO: 3). In some examples, the peptide linker is a (G4S)n linker, where n is an integer from 1 to 6. For example, the flexible peptide linker is a (G4S)3 linker, i.e., GGGGSGGGGSGGGGS (SEQ ID NO: 5).

[0040] In some cases, the fusion polypeptide may include an hCTLA-4 extracellular domain, a peptide linker, and an hCTLA-4 extracellular domain from the N-terminus to the C-terminus. Alternatively, the fusion polypeptide may include an hPD-L1 fragment, a peptide linker, and an hCTLA-4 extracellular domain from the N-terminus to the C-terminus. As an example, the fusion polypeptide disclosed herein includes the amino acid sequence of SEQ ID NO: 4 shown below.

[0041] Amino acid sequence of an exemplary fusion protein MKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPSPDSD GGGGSGGGGSGGGGSFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER(Sequence ID 4) *The underlined section is the linker arrangement.

[0042] All fusion polypeptides disclosed herein may further contain a signal peptide at their N-terminus.

[0043] (v) Preparation of fusion polypeptides The disclosed fusion polypeptides can be prepared by conventional methods, such as recombinant techniques as illustrated below.

[0044] Generally, nucleic acid sequences encoding any of the fusion polypeptides disclosed herein can be cloned into a suitable expression vector, operably linked to a suitable promoter, using methods known in the art. For example, under appropriate conditions, the nucleotide sequence and vector can be contacted with restriction enzymes to form complementary ends for each molecule, which can then be paired and ligated. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector / promoter depends on the type of host cell used for antibody production.

[0045] The antibody expression described herein can utilize a variety of promoters, including but not limited to the cytomegalovirus (CMV) intermediate early promoter, viral latent promoters such as Rous sarcoma virus LTR, HIV-LTR, and HTLV-1 LTR, simian virus 40 (SV40) early promoter, Escherichia coli lac UV5 promoter, and herpes simplex virus tk promoter.

[0046] Adjustable promoters are also available. Such adjustable promoters include those that use an E. coli-derived lac repressor as a transcription modulator to control transcription from mammalian cell promoters containing a lac operator [Brown, M. et al., Cell, 49:603-612 (1987)], and those that use a tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include those utilizing the FK506 dimer, VP16, or p65, which are derivable with estradiol, RU486, diphenol murislerone, or rapamycin. Derivable systems are available from Invitrogen, Clontech, and Ariad.

[0047] As a controllable promoter, one containing a repressor along with an operon can be used. In one embodiment, a lac repressor derived from E. coli functions as a transcription modulator and can control transcription from mammalian cell promoters containing a lac operator [M. Brown et al., Cell, 49:603-612 (1987). Also, Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] created a tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP 16) by fusing a tetracycline repressor (tetR) with a transcription activator (VP16). This fusion protein binds to a minimal promoter containing a tetO sequence derived from the human cytomegalovirus (hCMV) major immediate early promoter, constructing a tetR-tet operator system for controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operator is appropriately positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone can function as a potent transmodulator that controls gene expression in mammalian cells, rather than as a tetR-mammalian cell transcription factor fusion derivative (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)). One of the special advantages of this tetracycline-inducible switch is that it does not require a tetracycline repressor-mammalian cell transactivator or repressor fusion protein to achieve a tunable effect, as these fusion proteins can sometimes be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0048] Furthermore, the vector may include, for example, some or all of the following: a selection marker gene such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; an enhancer / promoter sequence derived from the human CMV immediate early gene for high levels of transcription; a transcription termination and RNA processing signal derived from SV40 for mRNA stability; an SV40 polyomatous replication origin and ColE1 for proper episomal replication; an internal ribosome binding site (IRES); a versatile multi-cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Methods for constructing vectors containing suitable vectors and transgenes are well known and available in the art.

[0049] Examples of polyadenylation signals useful for carrying out the methods described herein include, but are not limited to, the polyadenylation signals of human collagen I, human collagen II, and SV40.

[0050] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into a suitable host cell for producing a fusion polypeptide. The host cell can be cultured under conditions suitable for the expression of the antibody or any of its polypeptide chains. Such an antibody or its polypeptide chain can be recovered from the cultured cell (e.g., from the cell or culture supernatant) by conventional methods, such as affinity purification. If necessary, the antibody polypeptide chain can be incubated for a suitable time under suitable conditions that enable antibody production.

[0051] In some embodiments, the antibody preparation methods described herein utilize recombinant expression vectors encoding fusion polypeptides also described herein. Recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positively transformed host cells are selected and cultured under appropriate conditions to enable the expression of the two polypeptide chains constituting the antibody, and the resulting antibody can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under appropriate conditions to enable antibody formation.

[0052] Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a matrix conjugated with protein A or protein G.

[0053] The nucleic acids encoding the fusion polypeptides described herein, vectors containing such nucleic acids (e.g., expression vectors), host cells containing such vectors, and methods for producing the fusion polypeptides are all within the scope of this disclosure.

[0054] B. Immune composition Any of the fusion polypeptides disclosed herein can be incorporated into an immunocomposition for use in modulating (e.g., enhancing) the immune response in a recipient (e.g., a canine or feline cancer patient disclosed herein).

[0055] The immunocompositions used in accordance with this disclosure can be formulated by conventional methods using one or more pharmaceutically acceptable carriers or excipients. Both fusion polypeptides and coding nucleic acids can be mixed with pharmaceutically acceptable carriers (excipients) to form immunocompositions for use in the treatment of target diseases. The terms “excipient” and “carrier” are used interchangeably herein. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (preferably able to stabilize the active ingredient) and is not harmful to the target of treatment. Pharmaceutically acceptable excipients (carriers) include buffers well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover. In some embodiments, the pharmaceutically acceptable excipients (carriers) include adjuvants such as saponins, inorganic salts, gels, particulate adjuvants or pathogen-related molecules, or inert media. See, for example, “Overview of Vaccine Adjuvants: Introduction, History, and Current Status” (Methods Mol Biol. 2017; 1494:1-13). The immunocompositions used in this method may be in the form of lyophilized preparations or aqueous solutions and may contain pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover).Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used, and include, for example, buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; and preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexa This may include: 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, and lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextran; chelating agents such as EDTA, and sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0056] In some examples, the immunocompositions described herein may include liposomes containing fusion polypeptides (or nucleic acids encoding them). These liposomes can be prepared by methods known in the art, for example, 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. Patents 4,485,045 and 4,544,545. Liposomes with extended circulation times are disclosed in U.S. Patent 5,013,556. Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter having a specified pore size to obtain liposomes with a desired diameter.

[0057] Fusion polypeptides, or nucleic acids encoding them, can be encapsulated in microcapsules prepared, for example, by coacervation or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, respectively, or in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are well known in the art; see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0058] Immunotherapy compositions used for in vivo administration must be sterile. This can be easily achieved, for example, by passing them through a sterile filtration membrane. Therapeutic antibody compositions are typically filled into containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be punctured with a subcutaneous needle.

[0059] In some embodiments, the immunocomposition is an injectable formulation for subcutaneous or intramuscular administration. Injectable formulations may include various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous administration, water-soluble antibodies can be administered by drip infusion, in which the immunocomposition containing the antibody and physiologically acceptable excipients is injected. Physiologically acceptable excipients include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Formulations for intramuscular administration, such as sterile formulations of a suitable soluble salt form of the antibody, can be administered dissolved in immunoexcipients such as sterile water for injection, 0.9% saline, or 5% glucose solution.

[0060] The immunocompositions used in accordance with this disclosure can be formulated as vaccines to immunize canine or feline patients for the treatment of cancer. In some embodiments, the immunocomposition may comprise any of the fusion polypeptides disclosed herein (e.g., a fusion polypeptide comprising SEQ ID NO: 4) and a saponin adjuvant.

[0061] In some embodiments, the immunocomposition is a dosage form. A dosage form may refer to a pharmaceutical preparation in a form indicated depending on its intended use. The dosage form comprises one or more active ingredients (here, any of the fusion polypeptides disclosed herein, e.g., SEQ ID NO: 4) and excipients (e.g., adjuvants such as saponin adjuvants disclosed herein) in a specific form for the intended therapeutic use. In some cases, the dosage form may contain a specific amount of the active ingredient and a specific amount of the excipient in an amount equivalent to a single dose of the medicine administered to a subject in need of treatment. Furthermore, in some cases, the dosage form may be a unit dosage form containing a specific amount of the active ingredient and a specific amount of the excipient in a commercially available immunocomposition.

[0062] In some examples, the immunocompositions provided herein are dosage forms comprising about 20–200 μg of fusion polypeptide and about 10–50 μg of adjuvant (e.g., saponin adjuvant). In some specific examples, the immunocomposition may comprise about 50 μg of fusion polypeptide and about 20 μg of adjuvant.

[0063] In some examples, the immunocompositions provided herein are in dosage forms containing approximately 50–500 μg of fusion polypeptide and without adjuvants.

[0064] In some embodiments, the CTLA-4-linker-PD-L1 fusion polypeptide is mixed with a buffer to form a formulation. In some examples, the buffer contains 10 mM sodium citrate and has a pH of 6.5. In other examples, the buffer contains 10 mM sodium citrate, 50 mM NaCl, and 50 mM trehalose and has a pH of 6.5.

[0065] The safety of this vaccine is supported by the following two studies. (1) Rat toxicity test (a) No significant difference in body weight was observed between the vaccinated group and the control group. (b) In both the vaccinated group and the control group, the results of blood tests were within the normal range, and no significant difference was observed between the two groups. (c) No remarkable findings were observed in any of the animals during ophthalmic examination. (d) Nonspecific lesions when interpreted histologically. (2) Clinical research in dogs (a) Aside from swelling at the injection site and a mild fever on the first day, no other side effects were reported. (b) Vital signs (body temperature, blood pressure, heart rate, etc.) were stable before and after vaccination. (c) Serum biochemical tests showed no significant changes in CREA, AST, and ALT levels during the study period. (d) There were no reports of serious adverse events (SAEs) or adverse events (AEs), and the quality of life was generally good.

[0066] C. How to use The immunocompositions can be used to treat cancer in a subject. In certain embodiments, a method for treating cancer in a subject is provided. This method involves administering an effective amount of any of the pharmaceutical compositions or immunocompositions disclosed above and herein to a subject in need of treatment. The subject may be a canine or feline patient. In some cases, the subject is a dog or a cat. In some cases, the subject may have lung cancer, oral fibrosarcoma, oral squamous cell carcinoma, mast cell tumor, leukemia, malignant neoplasm of the mammary gland, hepatocellular carcinoma, melanoma, head and neck cancer, or lymphoma.

[0067] In some embodiments, the immune composition may be administered subcutaneously or intramuscularly to a subject at doses of approximately 20–200 μg of fusion polypeptide and approximately 10–50 μg of adjuvant every two or three weeks. In some cases, the immune composition may be administered to a subject at doses of approximately 50 μg of fusion polypeptide and approximately 20 μg of adjuvant every two or three weeks.

[0068] Effective amounts of the immunocompositions described above and herein can be administered to subjects in need of treatment (e.g., dogs or cats) via appropriate systemic or topical routes. In some embodiments, the fusion polypeptide can be administered intramuscularly or subcutaneously.

[0069] As used herein, “effective dose” refers to the amount of each active ingredient required to impart a therapeutic effect to a subject, either alone or in combination with one or more other active ingredients. In some embodiments, the therapeutic effect is an enhancement of the anti-tumor immune response in the tumor microenvironment. As is recognized by those skilled in the art, the effective dose varies depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, health status, physique, sex, and weight, the duration of treatment, the nature of any combination therapy, the specific route of administration, and similar factors within the scope of the knowledge and expertise of the healthcare professional. These factors are well known to those skilled in the art and can be addressed through normal experimentation. Generally, it is preferable to use the maximum dose of each ingredient or combination thereof, i.e., the highest safe dose based on appropriate medical judgment.

[0070] Empirical considerations, such as half-life, generally contribute to dosage determination. Dosage frequency can be determined and adjusted throughout the course of treatment, usually based on the treatment and / or suppression and / or improvement and / or delay of the target disease or disorder, but not limited to this. Alternatively, a sustained and continuous release formulation of the fusion polypeptide may be appropriate. Various formulations and devices for achieving sustained release are well known to those skilled in the art.

[0071] For the purposes of this disclosure, the appropriate dosage of the fusion polypeptide described herein will vary depending on the type and severity of the disease or disorder, whether the purpose of administration of the fusion polypeptide is preventive or therapeutic, the patient's medical history and response to treatment, and the discretion of the veterinarian. Typically, the veterinarian administers the fusion polypeptide until the desired result is achieved. In some embodiments, the desired result is enhancement of the antitumor immune response in the tumor microenvironment. A method for determining whether the dosage has produced the desired result is obvious to those skilled in the art. The administration of one or more polypeptides may be continuous or intermittent, depending, for example, the physiological state of the recipient, whether the purpose of administration is therapeutic or preventive, and other factors known to those skilled in the art. The administration of the fusion polypeptide may be substantially continuous over a pre-selected period, or it may be administered as multiple doses at intervals, for example, before, during, or after the onset of the disease or disorder in question.

[0072] As used herein, the term "treat" means applying or administering a composition containing one or more active ingredients to a target disease or disorder, symptoms of such disease or disorder, or a subject predisposed to such disease or disorder, with the aim of curing, restoring, reducing, alleviating, modifying, treating, improving, enhancing, or influencing the disorder, symptoms of such disease or disorder, or predisposition to such disease or disorder.

[0073] Alleviation of the disease or disability in question includes delaying the onset or progression of the disease, reducing the severity of the disease, or extending survival time. Alleviation of the disease or extension of survival time does not necessarily require a cure. As used herein, “delaying the progression of the disease or disability in question” means slowing, hindering, slowing, inhibiting, stabilizing, and / or postponing the progression of the disease. This delay can vary in length depending on the disease history and / or the individual being treated. A method for “delaying” or alleviating the progression of the disease, or delaying the onset of the disease, is a method that reduces the probability of developing one or more symptoms of the disease within a given period, and / or reduces the severity of symptoms within a given period, compared to not using the method. Such comparisons are usually based on clinical trials with a sufficient number of subjects to yield statistically significant results.

[0074] The “onset” or “progression” of a disease means the onset of initial symptoms and / or subsequent progression of the disease. The onset of a disease can be detected and evaluated using standard clinical techniques well known in the art. However, “onset” also refers to progression that may not be detectable. For the purposes of this disclosure, “onset” or “progression” means the biological course of symptoms. “Onset” includes manifestation, recurrence, and disease onset. As used herein, “disease onset” or “manifestation” includes the initial onset and / or recurrence of the disease or disorder in question.

[0075] Conventional methods well known to those skilled in the medical field can be used to administer an immunocomposition to a subject, depending on the type or site of the disease being treated. The composition may also be administered via other conventional routes, such as parenteral administration. As used herein, “parenteral” includes injection or infusion techniques into the subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional, and intracranial regions. Furthermore, the composition may also be administered to the subject via routes of administration of injectable depot formulations, such as using 1-month, 3-month, or 6-month depot formulations or biodegradable materials and methods. In some examples, the immunocomposition is administered intraocularly or intravitreously.

[0076] Injectable compositions may include, for example, vegetable oil, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and various carriers of polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by infusion of a pharmaceutical preparation containing the antibody and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% dextrose solution, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile preparation in a suitable soluble salt form of the antibody can be administered dissolved in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0077] A specific administration regimen, i.e., dosage, timing, and frequency of administration, is determined according to the specific patient and their medical history. The therapeutic effect on the targeted disease or disorder can be evaluated by methods well known in the art.

[0078] In some embodiments, the fusion polypeptides disclosed above and herein may be administered simultaneously with any of the second therapeutic agents disclosed herein. In some embodiments, the fusion polypeptide may be administered before the second therapeutic agent, or after the second therapeutic agent. In some examples, the second therapeutic agent is administered systemically. In other examples, the second therapeutic agent is administered topically. In some examples, the second therapeutic agent is administered intravenously, e.g., by bolus administration or continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intra-articular, intravesical, synovial, intrathecal, intratumoral, or suburothelial pathways. In one embodiment, the second therapeutic agent is administered to the subject by intravenous infusion.

[0079] Combination therapy of the immunocompositions disclosed above and herein with other therapeutic agents may enhance therapeutic effects. In some embodiments, the subject may receive one or more other therapeutic agents selected from the group consisting of chemotherapy, targeted therapy, radiotherapy, and immunotherapy.

[0080] In some cases, immunological compositions can be used in combination with chemotherapeutic agents, and are particularly useful in non-small cell lung cancer (NSCLC) and melanoma. Chemotherapy agents can act synergistically with the immunological compositions disclosed herein by reducing tumor burden and enhancing immunogenicity. Common chemotherapeutic agents include, but are not limited to, vincristine, chlorambucil, cyclophosphamide, cytarabine, epirubicin, doxorubicin, sorafenib, L-asparaginase, and labacfosazine. Table 1 lists representative chemotherapeutic agents and their indications, classified based on their common pharmacological mechanisms of action in chemotherapy.

[0081] Table 1: List of common chemicals TIFF2026523254000002.tif65166

[0082] In other cases, the immunocompositions disclosed above and herein can be used in combination with targeted therapy agents such as EGFR inhibitors and ALK inhibitors to treat lung cancer. This combination therapy can simultaneously inhibit tumor cell proliferation signaling pathways and enhance the immune response. Table 2 below shows representative drugs used in targeted therapy for lung cancer, classified by their common pharmacological mechanisms of action.

[0083] Table 2: List of Common Targeted Therapies TIFF2026523254000003.tif54136

[0084] In further examples, the immunocompositions disclosed above and herein can be used in combination with radiotherapy to enhance local tumor control and promote the immune response. This combination therapy is commonly used in various cancers, including head and neck cancer and lymphoma.

[0085] In further examples, the immune compositions disclosed above and herein can 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 to tumors at various levels.

[0086] The anticancer activity of the therapies disclosed herein can be monitored by conventional methods. For example, target cancer cells and / or biomarkers can be measured, for example, in blood, serum, plasma, urine, ascites, and / or biopsy samples from tissue or organs before, during, and after administration of the fusion polypeptide and optionally the second therapeutic agent. The therapeutic effect can be evaluated by analyzing the results obtained in this manner.

[0087] D. Kit In certain embodiments, kits comprising a fusion polypeptide or any of the immunocompositions disclosed above and herein, and optionally instructions on a method of administration, are described herein. Such a kit may include one or more containers containing a fusion polypeptide (e.g., any of those described herein) and optionally a second therapeutic agent (also described herein) to be used in combination with the fusion polypeptide.

[0088] In some embodiments, the kit may include instructions for use in accordance with any of the methods described herein. The included instructions may include instructions for administering the fusion polypeptide and optionally a second therapeutic agent to treat, delay the onset of, or alleviate the target diseases described herein. In other embodiments, the instructions may include instructions for administering the fusion polypeptide to an individual at risk of the target disease.

[0089] Instructions for the use of a fusion polypeptide typically include information regarding the dosage, administration schedule, and route of administration in the intended treatment. Containers may be unit doses, bulk packaging (e.g., multi-dose packaging), or sub-unit doses. Instructions accompanying a kit of the present invention are typically printed on a label or accompanying document (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions recorded on a magnetic or optical disc) are also acceptable.

[0090] The label or accompanying leaflet indicates that the composition is used to treat, delay the onset of, and / or alleviate, a targeted malignant blood disorder. In some embodiments, instructions are provided for carrying out any of the methods described herein.

[0091] The kit of the present invention is packaged in appropriate packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar bags or plastic bags). Packaging for use in combination with specific devices such as inhalers, nasal administration devices (e.g., atomizers), or infusion devices such as minipumps is also envisioned. In some embodiments, the kit has a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured with a subcutaneous needle). In some embodiments, the container also has a sterile access port (e.g., the container is an intravenous solution bag or a vial with a stopper that can be punctured with a subcutaneous needle). At least one active ingredient in the composition is a fusion polypeptide as described herein.

[0092] The kit may include additional components, such as buffers and interpretation information, as needed. Typically, the kit includes a container and a label or accompanying documentation associated with the container. In some embodiments, the present invention provides a product comprising the contents of the above-mentioned kit.

[0093] General technology In implementing this disclosure, unless otherwise specified, prior art in molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, and immunology is used, and these fall within the scope of the relevant technical fields. These techniques are described, for example, in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1989) Academic Press; Animal Cell Culture (RI Freshney, ed. 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM 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 (lRL Press, (1986)); and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al.(eds.).This is described in detail in the literature. Without requiring further detailed explanation, those skilled in the art will likely be able to utilize the present invention to the fullest extent based on the above description. Accordingly, the following specific embodiments should be construed as merely illustrative and not limit the remainder of this disclosure in any way. All publications referenced herein are incorporated herein by reference for the purposes or subjects referenced herein. [Examples]

[0094] Adjuvant Selection Assay Adjuvants act by stimulating the immune system, often by activating antigen-presenting cells such as dendritic cells, which then present antigens to T cells, resulting in a stronger and more effective immune response. Adjuvants can also be used in research to enhance immune responses to experimental antigens or to stimulate immune cells for therapeutic purposes. Adjuvants can be prepared from a variety of substances, including inorganic salts, lipids, proteins, and carbohydrates. Quil-A®, CpG1018®, aluminum hydroxide, or nanoparticles were used as adjuvants in combination with the above-mentioned fusion polypeptide (SEQ ID NO: 4) to immunize mice.

[0095] Quil-A® adjuvant is a saponin adjuvant manufactured by Croda under GMP standards and is used in various animal vaccines, as well as in immunological studies on its application in humans and animals. Saponins can induce a potent cytotoxic CD8+ lymphocyte response and enhance the response to mucosal antigens.

[0096] Aluminum hydroxide (Aram) (Imject®, Thermo Science) is the most commonly used vaccine adjuvant. It induces inflammatory cell death at the injection site, activates innate immune sensing pathways, and stimulates the production of chemokines and cytokines at the injection site and / or lymph nodes (LNs).

[0097] CpG1018 (CpG1018®, DYNAVAX) adjuvant enhances antibody concentrations, stimulates helper T cells (CD4+) and cytotoxic T cells (CD8+), and generates potent T-cell and B-cell memory responses. CpG1018 targets a single, clearly defined receptor (TLR9) expressed only in a few major cell types, and its mechanism of action as an adjuvant is well understood.

[0098] Nanoparticles (NanoCherub®) encapsulate antigens using quantum mechanical parameters, forming nanoparticles with a high density of positively charged surfaces, thereby inducing balanced Th1 / Th2 (cell-mediated) immunity across various antigens.

[0099] Balb / c mice (n=3 / group) aged 6-7 weeks were vaccinated by subcutaneous injection of 100 μl of a mixture (containing 20 μg of API, with or without various adjuvants). The dosages of each adjuvant were Quil-A: 15 μg, CpG1018 / Alu: 50 μg / 23.3 μl, and nanoparticles: 50 μl. Vaccination was performed by injection three times a week. Serum samples were collected from the mice on days 2, 7, 14, 21, 28, 35, 42, 49, 56, and 63 after administration and analyzed by ELISA (Figures 1A-1C). Serum samples containing specific antibodies against human PD-L1 (hPD-L1) (Figure 1A), canine PD-L1 (cPD-L1) (Figure 1B), and canine CTLA-4 (cCTLA-4) (Figure 1C) were observed on day 14, peaked on day 21, and were maintained until day 63. Comparing three different adjuvant combination groups, the group using Quil-A in combination with the active pharmaceutical ingredient (API) showed superior antibody production performance (Figures 1A-1C). Ultimately, Quil-A was selected because the Quil-A adjuvant combined with API may promote more specific antibody production. [Examples]

[0100] Rat vaccination with CTLA4-PD-L1 fusion polypeptide In this example, we investigate the effect of an immunotherapy composition containing a CTLA-4-linker-PD-L1 fusion polypeptide (SEQ ID NO: 4) and the saponin-based adjuvant Quil-A® in physiological saline on inducing an antibody response in a rat model.

[0101] Six-to-seven-week-old Sprague Dawley (SD) rats were administered an immunization composition subcutaneously a total of three times. The administrations were performed at 14-day (2-week) intervals (vaccination days 0, 14, and 28) or 21-day (3-week) intervals (vaccination days 0, 21, and 42). Each group (n=3) was divided into a low-dose group (LD, API 20 μg and Quil-A 20 μg), a medium-dose group (MD, immunization composition 50 μg and Quil-A 20 μg), and a high-dose group (HD, API 200 μg and Quil-A 20 μg).

[0102] In the 2-week interval group, antibody titers peaked at D42 after three vaccinations and were able to maintain the peak until at least D56 (Figures 2A-2C). Furthermore, similar antibody titer performance was observed with different doses. In the 3-week interval group, antibody titers peaked at D42 and were able to maintain the peak until at least D56 (Figures 3A-3C). Comparing the medium-dose group with the low-dose and high-dose groups, the medium-dose vaccination group showed stronger antibody titers than the other two groups. In the 3-week interval group, antibody titers in most rats peaked at D63. The high-dose group appeared to have superior antibody performance.

[0103] To efficiently reach the peak antibody titer for cancer treatment, a two-week dosing interval was chosen, and a moderate dose was used as the treatment regimen. [Examples]

[0104] Canine vaccination with CTLA4-PD-L1 fusion polypeptide This example investigates the effect of an immunocomposition containing a human-derived CTLA-4-linker-PD-L1 fusion polypeptide (SEQ ID NO: 4) and the saponin-based adjuvant Quil-A in physiological saline on inducing an antibody response in a dog model.

[0105] Dogs were administered an immunotherapy vaccine by subcutaneous injection a total of three times at 14-day intervals (D0, D14, D28). Three different dose groups were used, each receiving different amounts of API and adjuvant (Figure 4A: 50 μg API and 20 μg Quil-A; Figure 4B: 50 μg API and 20 μg Quil-A; Figure 4C: 250 μg API and 250 μg Quil-A). To investigate specific antibody production, blood samples were collected on the dates shown in the figures, and antibody titers were analyzed and measured by ELISA according to the following protocol.

[0106] Coat the plate: 1. Dilute the antigens (canine PD-L1 and canine CTLA-4) with coating buffer (0.1 M NaHCO3) to a final concentration of 0.5 μg / ml, and transfer 50 μl to each well of a high-affinity protein-binding ELISA plate. 2. Seal the plate and incubate overnight at 4°C.

[0107] Block the plate: 1. Return the plate to room temperature (RT), remove the capture antibody solution, wash three times with PBST0.1 (0.1% Tween20 in PBS), and add 200 μl of blocking solution (1% BSA in PBS) to each well to block nonspecific binding sites. 2. Seal the plate and incubate at room temperature for 2 hours.

[0108] Add the standard and sample: 1. Dilute immunized dog serum in blocking solutions (400x, 800x, 1600x, 3200x, 6400x, 12800x, 25600x, 512000x, 1024000x). 2. Add 50 μl to each well of the ELISA plate. 3. Seal the plate and incubate at room temperature for 1 hour. 4. Wash three times with PBST0.1.

[0109] Add the detection antibody: 1. Dilute goat anti-canine IgG-HRP in blocking solution at a ratio of 1:5,000. 2. Add 50 μl of diluted antibody to each well. 3. Seal the plate and incubate at room temperature for 1 hour. 4. Wash three times with PBST0.1.

[0110] Addition of substrate (color development): 1. Thaw the TMB substrate solution within 10 minutes of use. 2. Add 50 μl of TMB to each well and incubate at room temperature for 10 minutes to allow color development. 3. Add 50 μl of stop solution to stop the color reaction. 4. Set the microplate reader to 450 nm and measure the optical density (OD) of each well.

[0111] After three vaccinations, antibody titers peaked on day 28 (Figures 4A-4C). Furthermore, it was observed that different doses resulted in different antibody titer performance. Antibodies against PD-L1 and CTLA-4 could be induced in all three groups. It is noteworthy that the sequence similarities between canine PD-L1 and human PD-L1, canine CTLA-4, or human CTLA-4 were 80.5% and 84.1%, respectively. Unexpectedly, antibodies against canine PD-L1 and canine CTLA-4 could be detected in canine serum in all three groups after administration of an immune composition that was a fusion protein vaccine containing human sequences. [Examples]

[0112] Antibody production and memory response in dogs vaccinated with CTLA4-PD-L1 fusion polypeptide. CTLA4-PD-L1 fusion polypeptide Dogs (PBN1-EG-001) were administered an immunotherapy composition containing a CTLA-4-linker-PD-L1 fusion polypeptide (SEQ ID NO: 4) by subcutaneous injection only, at 14-day intervals for a total of three doses (D0, D14, D28). To investigate specific antibody production, blood samples were collected on D0, D14, D28, D49, D63, and D77, and antibody titers were analyzed and measured by ELISA based on the protocol described in Example 3 above. Because a continuous decline in antibody titer was observed, a booster dose was administered at D99 using the same dosage as the initial vaccination. The increase in antibody titer at D120 suggested that a memory response had been established in this dog. After immunological memory was established in dogs, antibody titers were continuously measured by ELISA. Following the ELISA experiment, antibody titers against PD-L1 and CTLA-4 decreased at D141 compared to D120, which was the peak antibody titer after boost inoculation (Figure 5). D238 received a second boost injection at the same dose. D273 showed higher antibody titer production, suggesting that a memory response had been established in this dog (Figure 5). [Examples]

[0113] Flow cytometry analysis of immune cell markers An immunotherapy composition containing a CTLA-4-linker-PD-L1 fusion polypeptide (SEQ ID NO: 4) was administered subcutaneously to dogs (PBN1-EG-001) three times at 14-day intervals (D0, D14, D28). To investigate the immune cell profile, blood samples were collected on D0, D14, D28, D49, D63, and D77, and immune cells were analyzed by flow cytometry according to the following protocol.

[0114] Plasma separation: Peripheral blood was transferred to a 15 mL centrifuge tube, centrifuged at 1000 × g for 10 minutes, and the supernatant was transferred to a cryovial and stored in a -80°C ultra-low temperature freezer for subsequent testing.

[0115] Isolation of peripheral blood lymphocytes: 1. 1 mL of blood remaining after "plasma separation" was collected and red blood cell lysis was performed. Prepare a 2.1×RBC lysis buffer and heat it to 37°C. 3.1×RBC lysis buffer and the remaining blood were mixed in a 50 mL plastic centrifuge tube in a volume ratio of 20:1. 4. Place the mixture in a 3D shaker at room temperature (RT) and shake in the dark for 10 minutes. 5. An equal volume of 1×PBS was added to the above mixture to stop the dissolution reaction. 6. The mixture was centrifuged at 400 × g for 5 minutes at room temperature. 7. After removing the supernatant, 10 mL of 1×PBS was added and the mixture was centrifuged at 400×g for 5 minutes at room temperature. 8. After centrifugation, the supernatant was removed, and the cells were relysed in an appropriate amount of R-10 medium (10% FBS / 1% penicillin + streptomycin / 0.1% β-mercaptoethanol / RPMI medium 1640), and the number of PBMCs was counted.

[0116] PMA / ionomycin activation stimulation: 1. Collect an appropriate amount of cells, with a cell density of 0.2 × 10⁻⁶. 6 The samples were placed in a U-bottom 96-well plate at 200 μL per unit. 2. The cell stimulation cocktail (500-fold dilution) and GolgiStop® (1000-fold dilution) were added and thoroughly mixed. 3. The eggs were incubated in a carbon dioxide incubator at 37°C for 2 hours.

[0117] Immunological function tests: After 1.2 hours of stimulation, the cells were transferred to a V-bottom 96-well plate. 2. The mixture was centrifuged at 400 × g for 5 minutes at room temperature, and the supernatant was discarded. 3. Add 200 μL of staining buffer to each well and repeat step 2. 4. Add the surface antigen-antibody mixture to each well, adjust the volume to 50 μL using staining buffer, gently aspirate and mix using a multi-channel microdispenser, and then stand in a refrigerator at 4°C in the dark for 10 minutes. 5. Add 150 μL of staining buffer to each well to bring the total volume to 200 μL. 6. The mixture was centrifuged at 400 × g for 5 minutes at room temperature, and the supernatant was discarded. 7. Add 200 μL of staining buffer to each well and repeat step 6 twice. 8. Add 100 μL of fixation / permeabilization solution to each well and incubate in a refrigerator at 4°C for 20 minutes. 100 μL of 9.1× permeabilization buffer was added, and the mixture was centrifuged at 400×g for 5 minutes at room temperature. The supernatant was discarded. 200 μL of 10.1× permeabilization buffer was added, and the mixture was centrifuged at 400×g for 5 minutes at room temperature. The supernatant was discarded. 11. First, add 50 μL of 1× permeabilization wash buffer to each well, then add the necessary intracellular staining antibody or isotype antibody, mix well, and leave it to stand in the dark in a refrigerator at 4°C for 30 minutes (CD8 T cells: CD45 + , CD3 + , CD5 + and CD8 + , NKT cells: CD45 + , CD3 + and CD5dim). 12. Add 150 μL of 1× permeabilization wash buffer, centrifuge at 400 × g for 5 minutes at room temperature, and discard the supernatant. 13. Add 200 μL of 1× permeabilization wash buffer, centrifuge at 400 × g for 5 minutes at room temperature, and discard the supernatant.<000048​​​​​​​​​​​​​​​​​Two imaging techniques were used to evaluate the progression of lung tumors in PBN1-EG-001 dogs vaccinated with an immunotherapy composition containing the CTLA-4-linker-PD-L1 fusion polypeptide (SEQ ID NO: 4) (API 50 μg and Quil-A 20 μg). The first CT scan was taken before vaccination and revealed a solid tumor in the left lung with tumor cell infiltration into the pulmonary hilar vein (Figures 7A-7B). Subsequently, PBN1-EG-001 dogs were vaccinated with an immunotherapy composition containing the CTLA-4-linker-PD-L1 fusion polypeptide (SEQ ID NO: 4). Three doses were administered subcutaneously at two-week intervals. The size of the tumors induced in the left and right lungs differed. Compared to the previous scan (Figure 8A vs. Figure 7A), the lesion in the left posterior lobe occupied a larger portion of the left posterior lobe parenchyma. However, the second CT image showed several cavities (tumor necrosis) in the tumor and portal vein within the circled area, with no further invasion (Figures 8A-8B). Next, X-rays were taken of a PBN1-EG-001 dog approximately 6 months after vaccination, revealing a significant reduction in the volume of the tumor on the left side (Figure 9B). The tumor nodule on the right side was larger but contained cavities (Figure 9A). The final survival time was approximately 9 months after diagnosis, at least 4 months longer than the veterinarian's prediction. These results suggest that an immune composition as a fusion protein vaccine containing human sequences unexpectedly exhibits therapeutic efficacy in treating cancer in dogs. [Examples]

[0120] Efficacy evaluation in cats This example aims to investigate the effect of an immunocomposition containing a human-derived CTLA-4-linker-PD-L1 fusion polypeptide (SEQ ID NO: 4) on inducing an antibody response in cats.

[0121] To evaluate the efficacy of cats vaccinated with an immunotherapy composition containing a CTLA-4 linker-PD-L1 fusion polypeptide (SEQ ID NO: 4), the adjuvant-free immunotherapy composition is administered subcutaneously to healthy cats. The immunotherapy composition is administered in doses of 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, and 500 μg, for a total of three doses at two-week intervals. To investigate specific antibody production, blood samples are collected at several time points after vaccination, and antibody titers are analyzed / measured by ELISA based on a method similar to the protocol described in Example 3. In the ELISA for measuring antibody titers, the coating antigen is feline CTLA-4 or feline PD-L1, and the antibody to be detected is anti-feline IgG-HRP.

[0122] An adjuvant-free immune composition is administered subcutaneously to cats with tumors. The dosages of the immune composition are 50 μg, 100 μg, 150 μg, 250 μg, and 500 μg. The administration interval is 2 weeks apart for a total of 3 doses. To investigate specific antibody production, blood samples are collected at multiple time points after vaccination, and antibody titers are analyzed / measured using ELISA. The sequence similarity between feline PD-L1 and human PD-L1, or between feline CTLA-4 and human CTLA-4, is 76.9% and 85.71%, respectively. To evaluate therapeutic efficacy, tumor size, disease progression, or final survival rate in cats with tumors are assessed, and the vaccine containing the fusion protein vaccine CTLA-4-linker-PD-L1 fusion polypeptide (SEQ ID NO: 4), which includes a human sequence, is examined to determine whether it is effective in treating cancer in cats.

[0123] Other embodiments All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each disclosed feature is merely an example of a general group of equivalent or similar features.

[0124] From the above description, those skilled in the art will readily grasp the essential features of the present invention and can make various changes and modifications to the invention to adapt it to various uses and circumstances without departing from the spirit and scope of the invention. Accordingly, other embodiments are also included in the claims.

[0125] Equal parts While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures to perform the functions described herein and / or to obtain the results and / or one or more advantages, and each such variation and / or modification will be considered to fall within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations described herein are illustrative, and that actual parameters, dimensions, materials and / or configurations will depend on the particular application(s) to which the teachings of the present invention are applied. Those skilled in the art will be able to recognize or confirm many embodiments equivalent to the particular embodiments described herein, not beyond routine experimentation. Therefore, it should be understood that the embodiments described herein are presented for illustrative purposes only, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be carried out in ways different from those specifically described and claimed. Embodiments of the invention of this disclosure apply to each individual feature, system, article, material, kit and / or method described herein. Furthermore, any combination of these features, systems, articles, materials, kits, and / or methods is included in the scope of the invention of this disclosure, provided that these features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0126] Furthermore, unless explicitly stated otherwise, it should be understood that, where a claimed method in this specification includes multiple steps or actions, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

[0127] All definitions defined and used herein are understood to take precedence over dictionary definitions, definitions incorporated by reference in documents, and / or the usual meanings of the defined terms.

[0128] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter they cite, and in some cases may encompass the entirety of such references.

[0129] As used herein and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly indicated otherwise.

[0130] As used herein and in the claims, the phrase “and / or” should be understood to mean “either or both” of the elements thus combined, that is, elements that may exist as a combination and, in some cases, as separate elements. Multiple elements listed using “and / or” should be interpreted similarly, that is, they should be understood to mean “one or more” of the elements thus combined. Elements other than those specifically identified by the “and / or” phrase may exist as they may, whether or not they are related to those specifically identified elements. Thus, as an example without limitation, when used in conjunction with an open expression such as “including,” a reference to “A and / or B” may refer to A only (optionally including elements other than B) in one embodiment, B only (optionally including elements other than A) in another embodiment, and both A and B (optionally including other elements) in yet another embodiment.

[0131] As used herein 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” is interpreted as inclusive; that is, it includes at least one of the enumerated elements, may include more, and may optionally include additional elements not enumerated. Only terms that explicitly indicate the opposite, such as “one only” or “exactly one,” or “consisting of” as used in the claims, mean that it includes multiple elements or exactly one of the enumerated elements. In general, as used herein, the term “or” is interpreted as indicating an exclusive choice (i.e., “one or the other, but not both”) only when preceded by an exclusive expression such as “either one,” “one of,” “only one,” or “exactly one.” “Essentially consisting of” shall have the usual meaning as used in the field of patent law when used in the claims.

[0132] As used herein and in the claims, the terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which depends in part on the limits of the method or determination of the value, i.e., the measuring system. For example, “about” may mean within an acceptable standard deviation according to the practice in the art. Alternatively, “about” may mean within a range of up to ±20%, preferably ±10%, more preferably up to ±5%, and even more preferably ±1% of a given value. Where a particular value is described in this specification and in the claims, unless otherwise specified, the term “about” is implicitly included and in this context means within an acceptable margin of error for that particular value.

[0133] In this specification and in the claims, the expression “at least one” referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list, but not necessarily including at least one of all elements specifically enumerated in the element list, nor excluding any combination of elements in the element list. This definition also allows for the presence of elements other than those specifically identified in the element list referred to by the phrase “at least one,” whether or not they are related to those specifically identified elements. Therefore, as an example that is not limited, "at least one of A and B" (or, as a synonym, "at least one of A or B", or as a synonym, "at least one of A and / or B") may, in one embodiment, refer to at least one (including any multiple) A and no B (including any other element); in another embodiment, refer to at least one (including any multiple) B and no A (including any other element); and in yet another embodiment, refer to the presence of at least one (including one or more at will) A and at least one (including one or more at will) B (and other elements at will).

Claims

1. A fusion polypeptide comprising (a) the extracellular domain of human CTLA-4 and (b) the extracellular domain of human PD-L1, The extracellular domain of the human CTLA-4 contains the 123rd amino acid substitution of SEQ ID NO: 1, and / or Furthermore, a fusion polypeptide containing a flexible peptide linker (c) located between (a) and (b).

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

3. The aforementioned flexible peptide linker is (G ４ S)n linker, Here, n is one integer from 1 to 6. The fusion polypeptide according to claim 1 or claim 2, wherein optionally the flexible peptide linker linker is GGGGGSGGGGGS (SEQ ID NO: 5).

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

2.

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

3.

6. The fusion polypeptide according to claim 1, comprising the amino acid sequence of SEQ ID NO:

4.

7. A nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide according to any one of claims 1 to 6.

8. An immunocomposition comprising a fusion polypeptide according to any one of claims 1 to 6 or a nucleic acid according to claim 7, and one or more pharmaceutically acceptable carriers optionally comprising an adjuvant.

9. An immunoassay comprising a fusion polypeptide and a saponin adjuvant, wherein the fusion polypeptide comprises (a) the extracellular domain of human CTLA-4, (b) the extracellular domain of human PD-L1, and optionally (c) a flexible peptide linker linking (a) and (b), and optionally (G ４ S) an immunocomposition, which is an n linker, where n is an integer from 1 to 6.

10. The immunocomposition according to claim 9, wherein the fusion polypeptide is the fusion polypeptide according to any one of claims 1 to 6.

11. The immunocomposition according to any one of claims 8 to 10, comprising a dosage form containing about 20 to 200 μg of the fusion polypeptide and about 10 to 50 μg of the adjuvant, and optionally comprising a dosage form containing about 50 μg of the fusion polypeptide and about 20 μg of the adjuvant.

12. An immunoassay composition according to any one of claims 8 to 10, comprising a dosage form containing about 50 to 500 μg of the fusion polypeptide, wherein the dosage form optionally does not contain an adjuvant.

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

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

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

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

17. The method according to any one of claims 11 to 16, wherein the immune composition is administered to the subject once every two weeks or once every three weeks in a dose of about 20 to 200 μg of the fusion polypeptide and about 10 to 50 μg of the adjuvant.

18. The method according to claim 17, wherein the immune composition is administered to the subject once every two weeks or once every three weeks in a dose of about 50 μg of the fusion polypeptide and about 20 μg of the adjuvant.

19. The method according to any one of claims 11 to 16, wherein the immunotherapy composition is administered to the subject once every two weeks or once every three weeks in a dose of about 50 to 500 μg of the fusion polypeptide, and optionally the immunotherapy composition does not contain an adjuvant.

20. The method according to any one of claims 11 to 19, wherein the subject is receiving one or more anticancer therapies selected from the group consisting of chemotherapy, targeted therapy, radiotherapy, immunotherapy, and combinations thereof.

21. The method according to claim 20, wherein the chemotherapy comprises vincristine, chlorambucil, cyclophosphamide, cytarabine, epirubicin, doxorubicin, sorafenib, L-asparaginase, lavacosadin, or a combination thereof.

22. The method according to claim 20 or 21, wherein the targeted therapy comprises an EGFR inhibitor or an ALK inhibitor, optionally gefitinib, imatinib, toceranib, masitinib, dasatinib, toceranib, lomustine, or a combination thereof.

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

Images