Fusion protein construct containing interleukin-15 as an active ingredient and its use

Abstract

The present invention relates to a fusion protein construct containing interleukin 15 as an active ingredient and its use. Specifically, the fusion protein construct comprises (1) a first structural unit which is the sushi domain of the interleukin 15 (IL-15) receptor subunit α, (2) a second structural unit which is IL-15, (3) a third structural unit which is an antibody Fc or a mutant Fc fragment, and an important linking fragment 1, the amino acid sequence of which is an integer multiple repeat of GGGGS.

Description

[Technical Field]

[0001] The present invention belongs to the fields of medicine and biology, and more specifically, relates to a fusion protein construct containing interleukin 15 as an active ingredient and its use. [Background technology]

[0002] With the rapid development of immune checkpoint therapy, PD-1 / PD-L1 blocking antibodies have become one of the important methods in clinical treatment. PD-1 / PD-L1 immunoblocking antibodies reduce tumor inhibition on tumor-infiltrating lymphocytes (TILs), reactivating lymphocyte function to recognize and kill tumors. However, in the later stages of treatment, excessive activation causes T cells to gradually become depleted and lose their function. In clinical trials, patients with tumors generally have a low response rate to PD-1 / PD-L1 therapy and are at risk of resistance or relapse. Therefore, overcoming the bottlenecks in the clinical application of PD-1 / PD-L1 is the most important challenge in current immunotherapy.

[0003] T cell growth factors are important targets in immunotherapy due to their ability to proliferate T lymphocytes. Among them, IL-2 is a cytokine necessary for T cell proliferation and is one of the oldest immunotherapeutic drugs approved by the U.S. Food and Drug Administration (FDA) for malignant melanoma and renal cancer. However, IL-2 is not widely used in clinical practice due to (1) its short half-life, (2) activation of immunosuppressive regulatory T cells (Tregs), (3) elimination of activated effector T cells by activation-induced cell death (AICD), and (4) severe toxic side effects caused by activation of vascular endothelial cells. IL-15 is another important T cell growth factor and pleiotropic cytokine that can maintain and activate innate and adaptive immune responses. IL-15 can promote the activation, proliferation, and survival of CD8+ T cells and can activate and maintain memory T cells. IL-15 can also promote the activation and proliferation of NK and NKT cells. Furthermore, IL-15 can also promote the activation, proliferation, and differentiation of dendritic cells through autocrine mechanisms, enhance the expression of MHC-II and CD80 / CD86, and improve antigen cross-presentation by dendritic cells. Compared to IL-2, IL-15 has the following advantages in tumor therapy: (1) IL-15 can avoid activation-induced cell death and induce the generation of long-lived memory T cells and steady-state proliferation; (2) IL-15 does not proliferate Treg cells; and (3) IL-2 causes systemic toxicity by acting on vascular endothelial cells, but IL-15 does not cause this side effect. Therefore, IL-15 is currently a potential cytokine for tumor immunotherapy.

[0004] The clinical use of IL-15 is limited primarily by its short half-life and low therapeutic efficacy. Some researchers have linked IL-15 mutations to the IL-15Rα and IgG1 Fc domain (ALT-803) to extend the half-life. However, the combination with ALT-803 does not offer significant advantages compared to immune checkpoint inhibitor therapy. Simultaneously, increased systemic circulating NK cell counts and CD8+ T cell counts were detected in all patients, indicating significant toxic side effects of IL-15 at therapeutic doses. Currently, many preclinical studies use IL-15 mutations to reduce IL-15's binding to its receptor. This can reduce IL-15 binding to peripheral NK cells, but it also affects IL-15's antitumor activity. Furthermore, artificially modified IL-15 is more likely to induce anti-IL-15 antibodies in the human body, thereby further impairing the therapeutic effect of IL-15.

[0005] As a common target, PD-1 itself offers significant advantages. PD-1 is expressed more highly in tumors compared to other tissues throughout the body. More importantly, the high levels of PD-1 expression in CD8+ T cells within tumors allow αPD-1 antibodies to directly act on the killer T cell population. Simultaneously, αPD-1 blocking antibodies can also block the binding of PD-1 and PD-L1, mitigating T cell inhibition and further enhancing the antitumor capacity of T cells. [Overview of the project] [Problems that the invention aims to solve]

[0006] As mentioned above, the effect of PD-1 / PD-L1 antibody blockade therapy is not significant, likely due to gradual T cell depletion. As a T cell agonist, IL-15 can promote the proliferation of CD8+ T cells, thus potentially enhancing the T cell death capacity during PD-1 / PD-L1 antibody blockade therapy. While the concept of PD-1 / PD-L1 antibody-cytokine fusion proteins is very common, most forms of fusion proteins still produce significant toxic side effects. This is because cytokine receptors (such as the IL-15 receptor expressed on T cells and NK cells) are widely distributed, and cytokines have high affinity for their receptors (Kd~10 -9 This is found in M). As a result, in antibody-cytokine fusion proteins, the affinity between the cytokine and the receptor is likely to be greater than the affinity between the antibody and the antigen, thus failing to produce an effective tumor targeting effect and resulting in significant toxic side effects during systemic injection.

[0007] The advantage of the fusion protein constructed in this disclosure is that the length of the first linker between hIgG globulin and IL-15 is regulated to prevent IL-15 from binding to its receptor IL-2 / 15Rβ (CD122) due to steric hindrance. In peripheral circulation, the fusion protein targeted drug reduces peripheral toxic side effects by reducing binding to IL-15 receptor-expressing NK cells. In the tumor microenvironment, the fusion protein specifically binds to CD8+ T cells that highly express PD-1 via an αPD-1 antibody, and simultaneously assists in the binding of IL-15 to its receptor on CD8+ T cells by utilizing the high affinity of the αPD-1 antibody, thereby specifically activating CD8+ T cells. By utilizing the αPD-1 antibody to block PD-1 / PD-L1 inhibition on T cells, while the fusion protein further promotes T cell proliferation and enhances the T cell death ability via IL-15 signaling, the antitumor effect of the fusion protein targeted drug can be enhanced while eliminating peripheral toxic side effects. [Means for solving the problem]

[0008] This disclosure relates to a fusion protein, wherein the fusion protein is (1) The first structural unit is the sushi domain of the interleukin-15 (IL-15) receptor subunit α, (2) The second structural unit is IL-15, (3) A third structural unit located at the N-terminus of a fusion protein which is an antibody Fc fragment or a mutant Fc fragment, (4) A block comprising a second linker connecting the first structural unit and the second structural unit, The first linker, when the C-terminus of the fusion protein is the first structural unit, links the second structural unit and the third structural unit. The first linker links the first structural unit and the third structural unit when the C-terminus of the fusion protein is the second structural unit.

[0009] The aforementioned first linker is (G4S) n The amino acid sequence is an integer multiple repeat of GGGGS represented by , preferably n is any integer from 1 to 7, more preferably n is any integer from 1 to 5, and most preferably n is 3.

[0010] The aforementioned fusion protein further (5) comprising a block of a fourth structural unit linked to the N-terminus of the third structural unit which is the Fab of the therapeutic antibody, The aforementioned therapeutic antibodies are not particularly limited, but include anti-PD1 / PD-L1 antibodies, Her2 antibodies, anti-CD20 antibodies, anti-CD19 antibodies, anti-RANKL antibodies, anti-VEGFR antibodies, and anti-EGFR antibodies. Preferably, the Fab of the therapeutic antibody is the anti-PD-1 Fab (the Fab of the PD1 antibody).

[0011] The anti-PD-1 Fab comprises a heavy chain (variable region + constant region) and a light chain (variable region + constant region), wherein the heavy chain is located at the N-terminus of the fusion protein. Preferably, the anti-PD-1 Fab is mouse-derived or human-derived anti-PD-1 Fab. More preferably, the mouse-derived or human-derived anti-PD-1 Fab has a light chain having the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 18, or SEQ ID NO: 19, and the anti-PD-1 Fab has a heavy chain having the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 20, or SEQ ID NO: 21. Sequence ID 4: YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTV LGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 18: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 19: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 5: EVRLLESGGGLVKPEGSLKLSCVASGFTFSDYFMSWVRQAPGKGLEWVAHIYTKSYNYATYYSGSVKGRFTISRDDSRSMVYLQMNNLRTEDTATYYCTRDGSGYPSLDFWGQGTQVTVSSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC Sequence number 20: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRV Sequence number 21: QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK

[0012] The IL-15 is mouse-derived or human-derived IL-15 having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 15. SEQ ID NO: 1: NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS SEQ ID NO: 15: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0013] The sushi domain of the mouse-derived or human-derived IL-15 receptor subunit α has the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 17. Sequence ID 3: GTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT Sequence ID 17: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP

[0014] The Fc fragment or mutant Fc fragment has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 16. Sequence ID 2: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK Sequence ID 16: EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

[0015] The second linker has the amino acid sequence shown in SEQ ID NO: 6. Sequence ID 6: SGGGSGGGGSGGGGSGGGGSGGGSLQ

[0016] Furthermore, this disclosure relates to homodimers composed of fusion proteins, preferably in which the monomers of the homodimer are linked to each other via dimerization of the third structural unit.

[0017] More preferably, the homodimer (1) Homodimer 1 (Fc-G4S-Rα-IL-15) The fusion protein as a monomer comprises, from the N-terminus to the C-terminus, human-derived IgG1-Fc, a first linker (G4S), the sushi domain of the IL-15 receptor subunit α, a second linker, and IL-15, and preferably a homodimer 1 having the amino acid sequence shown in SEQ ID NO: 7. Sequence ID 7: KLATMETDTLLLWVLLLWVPGSTGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKGGGGSGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTSGGGSGGGGSGGGGSGGGGSGG GSLQNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS (2) Homodimer 2(Fc-G4S-IL-15-Rα) The fusion protein as a monomer comprises, from the N-terminus to the C-terminus, human-derived IgG1-Fc, a first linker (G4S), IL-15, a second linker, and the sushi domain of the IL-15 receptor subunit α, preferably a homodimer 2 having the amino acid sequence shown in SEQ ID NO: 8. Sequence ID 8: KLATMETDTLLLWVLLLWVPGSTGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKGGGGSNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFL QSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT (3) Homodimer 3 (Anti-PD-1 Fab Fc-G4S-Rα-IL-15) The fusion protein as a monomer comprises, from the N-terminus to the C-terminus, anti-PD-1 Fab, human-derived IgG1-Fc, a first linker (G4S), the sushi domain of IL-15 receptor subunit α, a second linker, and IL-15, and preferably a homodimer 3 having the amino acid sequence shown in SEQ ID NO: 9. Sequence ID 9: YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECKLATMETDTLLLWVLLLWVPGSTGEVRLLESGGGLVKPEGSLKLSCVASGFTFSDYFMSWVRQAPGKGLEWVAHIYTKSYNYA300 TYYSGSVKGRFTISRDDSRSMVYLQMNNLRTEDTATYYCTRDGSGYPSLDFWGQGTQVTVSSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCRTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKGGGGSGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS (4) Homo-dimer 4 (anti-PD-1 Fab Fc-G4S-IL-15-Rα) The fusion protein as a monomer comprises, from the N-terminus to the C-terminus, anti-PD-1 Fab, human-derived IgG1-Fc, a first linker (G4S), IL-15, a second linker, and the sushi domain of the IL-15 receptor subunit α, preferably a homodimer 4 having the amino acid sequence shown in SEQ ID NO: 10. Sequence ID 10: YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECKLATMETDTLLLWVLLLWVPGSTGEVRLLESGGGLVKPEGSLKLSCVASGFTFSDYFMSWVRQAPGKGLEWVAHIYTKSYNYATYYSGSVKGRFTISRDDSRSMVYLQMNNLRTEDTATYYCTRDGSGYPSLDFWGQGTQVTVSSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCRTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKGGGGSNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT (5) Homo-dimer 5 (Fc-(G4S)3-IL-15-Rα) The monomer comprises, from the N-terminus to the C-terminus, human-derived IgG1-Fc, a first linker ((G4S)3), mouse-derived or human-derived IL-15, a second linker, and the sushi domain of the IL-15 receptor subunit α, preferably a homodimer 5 having the amino acid sequence shown in SEQ ID NO: 11. Sequence ID 11: KLATMETDTL LLWVLLLWVP GSTGEPKSSD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKGG GGSGGGGSGG GGSNWIDVRY DLEKIESLIQ SIHIDTTLYT DSDFHPSCKV TAMNCFLLEL QVILHEYSNM TLNETVRNVL YLANSTLSSN KNVAESGCKE CEELEEKTFT EFLQSFIRIV QMFINTSSGG GSGGGGSGGG GSGGGGSGGG SLQGTTCPPP VSIEHADIRV KNYSVNSRER YVCNSGFKRK AGTSTLIECV INKNTNVAHW TTPSLKCIRD PSLAHYSPVP T (6) Homodimer 6 (anti-PD-1 Fab Fc-(G4S)3-IL-15-Rα) The monomer comprises, from the N-terminus to the C-terminus, anti-PD-1 Fab, human-derived IgG1-Fc, a first linker ((G4S)3), IL-15, a second linker, and the sushi domain of the IL-15 receptor subunit α, preferably a homodimer 6 having the amino acid sequence shown in SEQ ID NO: 12. Sequence ID 12: YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECKLATMETDTLLLWVLLLWVPGSTGEVRLLESGGGLVKPEGSLKLSCVASGFTFSDYFMSWVRQAPGKGLEWVAHIYTKSYNYATYYSGSVKGRFTISRDDSRSMVYLQMNNLRTEDTATYYCTRDGSGYPSLDFWGQGTQVTVSSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCRTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKGGGGSGGGGSGGGGSNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT (7) Homo-dimer 7 (Fc-(G4S)5-IL-15-Rα) The monomer comprises, from the N-terminus to the C-terminus, human-derived IgG1-Fc, a first linker ((G4S)5), IL-15, a second linker, and the sushi domain of the IL-15 receptor subunit α, and preferably a homodimer 7 having the amino acid sequence shown in SEQ ID NO: 13, or Sequence ID 13: KLATMETDTLLLWVLLLWVPGSTGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKGGGGSGGGGSGGGGSGGGGSGGGGSNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKT FTEFLQSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT (8) Homodimer 8 (Anti-PD-1 Fab Fc-(G4S)5-IL-15-Rα) The monomer comprises, from the N-terminus to the C-terminus, anti-PD-1 Fab, human-derived IgG1-Fc, a first linker ((G4S)5), IL-15, a second linker, and the sushi domain of the IL-15 receptor subunit α, and preferably a homodimer 8 having the amino acid sequence shown in SEQ ID NO: 14. Sequence ID 14: YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLGRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECKLATMETDTLLLWVLL LWVPGSTGEVRLLESGGGLVKPEGSLKLSCVASGFTFSDYFMSWVRQAPGKGLEWVAHIYTKSYNYATYYSGSVKGRFTISRDDSRSMVYLQMNNLRTEDTATYYCTRDGSGYPSLD FWGQGTQVTVSSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCR TEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKGGGGSGGGG SGGGGSGGGGSGGGGSNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQ SFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT That is the case.

[0018] This disclosure also relates to nucleotide fragments encoding the fusion protein.

[0019] This disclosure also relates to the use of fusion proteins and homodimers composed of said fusion proteins in the manufacture of pharmaceuticals, preferably said pharmaceuticals being antitumor pharmaceuticals, and most preferably said pharmaceuticals being pharmaceuticals for B-cell lymphoma, colorectal cancer, melanoma, or lung cancer.

[0020] Furthermore, this disclosure relates to a method for preparing a fusion protein, and the method for preparing the fusion protein is: (1) A step of constructing an expression vector containing a gene encoding a fusion protein, preferably the expression vector being a pEE12.4 expression vector, (2) A step of constructing host cells containing the expression vector by transiently transfecting host cells, wherein the host cells are preferably 293F cells, (3) A step of culturing the host cells and collecting the cell supernatant, (4) A step of purifying the fusion protein using a protein A affinity chromatography column. [Effects of the Invention]

[0021] This disclosure has the following beneficial effects: 1. Two forms of IL-15 fusion proteins with similar structures were designed, in which the position of IL-15 and the sushi domain of IL-15 receptor subunit α were swapped at the C-terminus of Fc. The biological activity of the two IL-15 fusion proteins differs by approximately 10,000-fold. 2. Various linkers (first linkers) are designed to link the C-terminus of Fc to the IL-15 / sushi domain of the IL-15 receptor subunit α. The basic unit of a first linker consists of five amino acids GGGGS. The length of the first linker can directly affect the biological activity of IL-15. 3. MC38 small tumor model (7 days after inoculation of tumor cells, tumor size is approximately 30 mm) 3In the MC38 large tumor model (14 days after inoculation of tumor cells, tumor size was approximately 100 mm), the tumor could be completely eliminated after systemic injection of αPD-1 Fab Fc-G4S-IL-15-Rα fusion protein. 3 In this case, tumors can be substantially controlled after systemic injection of the αPD-1 Fab Fc-G4S-IL-15-Rα fusion protein. 4. Mice treated with fusion protein-targeting drugs did not lose body weight after systemic injection of αPD-1 Fab Fc-G4S-IL-15-Rα fusion protein at three times the therapeutic dose. 5. In a CD34+ humanized mouse A549 lung cancer tumor model, tumors can be substantially controlled after systemic injection of anti-human PD-1 Fab Fc-G4S-human IL-15-Rα or anti-human PD-1 Fab Fc-(G4S)3-human IL-15-Rα fusion protein. [Modes for carrying out the invention]

[0022] [Definition of Terms] The term "fusion protein" refers to a protein product obtained by linking the coding regions of two or more genes by genetic recombination, chemical methods, or other appropriate methods, and by recombinantly expressing these genes under the control of the same regulatory sequence. Unless otherwise specified, the C-terminus of the first block (polypeptide) at the N-terminus of a fusion protein is ligated to the N-terminus of the next block (or linker), and so on. Thus, the N-terminus of the N-terminal block of a fusion protein is the N-terminus of the fusion protein, and the C-terminus of the C-terminal block of a fusion protein is the C-terminus of the fusion protein.

[0023] The terms "IL-15 wild-type" or "wild-type IL-15" refer to naturally occurring human IL-15 or non-human mammalian IL-15 or non-mammalian IL-15. The terms "IL-15 wild-type" or "wild-type IL-15" may also refer to the general IL-15 polypeptide in the art.

[0024] The term "IL-15R subunit α" may refer to IL-15Rα of any species or a functional fragment thereof, such as human IL-15Rα, non-human mammalian IL-15Rα, or non-mammalian IL-15Rα. Examples of non-human mammals include pigs, rabbits, monkeys, chimpanzees, and mice. Examples of non-mammals include chickens. Preferably, IL-15R subunit α is human IL-15Rα, preferably an extracellular domain fragment of human IL-15Rα.

[0025] The term "sushi domain" is defined as the extracellular region of IL-15Rα or a fragment containing at least one sushi polypeptide containing an IL-15Rα sushi domain, and the extracellular region of IL-15Rα containing a domain called the sushi domain (Wei et al. 2001, J.Immunol. 167:277-282). The sushi domain of IL-15Rα has a β-sheet structure and is encoded by exon 2 of IL-15Rα, starting with a cysteine ​​residue (C1) encoded by the first exon 2 and ending with a cysteine ​​residue (C4) encoded by the fourth exon 2. When considering the standard N-terminal to C-terminal protein sequence of IL-15Rα, the "sushi domain of IL-15R subunit α" can be defined as beginning with the first cysteine ​​residue (C1) after the signal peptide and ending with the fourth cysteine ​​residue (C4) after the signal peptide, and both residues C1 and C4 can be included in the sequence of the sushi domain.

[0026] The term "Fc" is an abbreviation for the Fc region of immunoglobulins, referring to the constant region of immunoglobulins, particularly the carboxyl terminus or a portion thereof of the heavy chain constant region of immunoglobulins, which does not possess antigen-binding activity and is the site where antibodies interact with effector molecules and cells. In this disclosure, "Fc" may be any Fc or variant thereof derived from human or non-human mammals. For example, Fc may include a combination of two or more domains of CH1, CH2, CH3, and CH4 of the heavy chain and the immunoglobulin hinge region. Fc may be derived from different species, preferably human immunoglobulins. According to the amino acid sequence of the heavy chain constant region, immunoglobulins can be divided into five different classes, mainly IgA, IgD, IgE, IgG, and IgM. Some classes can be further divided into subclasses (isotypes) such as IgG-1, IgG-2, IgG-3, and IgG-4, or IgA-1 and IgA-2. The “Fc region” preferably comprises at least one immunoglobulin hinge region of IgG, as well as CH2 and CH3 domains. More preferably, the “Fc region” comprises the CH2 and CH3 domains of IgG1, as well as an immunoglobulin hinge region whose initial amino acid position can be altered. In some embodiments, Fc comprises Fc having increased antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cell-mediated cytotoxicity (CDC) activity due to enhanced or weakened binding affinity to Fc receptors such as CD16a, CD16b, CD32a, CD16b, CD64, and C1q proteins. In other embodiments, mutations are incorporated into the Fc domain to reduce ADCC or CDC activity. These mutations may include, but are not limited to, (1) mutations at N297, such as N297A and N297G, (2) mutations at L234, such as L234A and L234G, and / or mutations at L235, such as L235A or L235G, (3) mutations at P329, such as P329G, or (4) mutations at D265, such as D265A, or combinations of substitutions at any or all of these locations.In other embodiments, the Fc mutation (all numbering is the Eu index in the Kabat numbering system) includes mutations that increase the serum half-life. In one embodiment, Fc has a substitution of T250Q, or M428L, or a T250Q / M428L double mutation in CH3 (Hinton et al., J Biol Chem. 279(8):6213-6, 2004). In other embodiments, Fc has a triple mutation of M252Y / S254T / T256E (Dall'Acqua WF et al., J Immunol 169(9):5171-80, 2002). In other embodiments, Fc has the N434A mutation (Petkova SB et al., International Immunology 18(12):1759-1769, 2006), the M428L / N434S double mutation, or the M428 / N434A double mutation (Zalevsky J et al., Nat Biotechnol. 28(2):157-159, 2010). In other embodiments, the Fc region is modified to increase its serum half-life. In some embodiments, the modification that increases the serum half-life is M428L.

[0027] The term "antibody (Ab)" refers to an immunoglobulin molecule that specifically binds to a target antigen or exhibits immunoreactivity with a target antigen, and includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, recombinant antibodies and other modified antibodies (chimeric antibodies, humanized antibodies, fully human antibodies, heterospecific-conjugated antibodies (e.g., bispecific, triplicate, and quadruplicate antibodies), antibody conjugates and antigen-binding fragments of antibodies (e.g., Fab', F(Ab')2, Fab, Fv, rIgG, and scFv fragments)).

[0028] The term "Fab" fragment refers to one of two identical antigen-binding fragments produced by the digestion of an antibody with papain, each Fab comprising the variable region of the heavy chain and the variable region of the light chain, as well as the constant region of the light chain and the first constant region (CH1) of the heavy chain. Thus, the term "Fab" as used herein refers to an antibody fragment comprising the VL region and constant region (CL) of the light chain, as well as the VH region and first constant region (CH1) of the heavy chain. A Fab' fragment differs from a Fab in that it has a few residues added from the antibody hinge region to the carboxyl terminus of the CH1 region of the heavy chain, which contains one or more cysteines. Fab'-SH is a Fab' fragment in which the cysteine ​​residue in the constant region of the heavy chain possesses a free thiol group. F(ab')2 is an antibody fragment having two antigen-binding sites (two Fabs) and a portion of the Fc region, and is produced by pepsin treatment of an intact antibody.

[0029] The term “fusion protein” refers to a protein product obtained by linking the coding regions of two or more genes by genetic recombination, chemical methods or other suitable methods, and by recombinantly expressing these genes under the control of the same regulatory sequence. In the fusion proteins of this disclosure, the coding regions of two or more genes may be fused at one or more positions by sequences encoding a peptide linker or linker peptide. The term “fusion protein” of this disclosure further includes an antibody / Fc fusion protein construct / complex, or components of an antibody / Fc fusion protein construct / complex formed by non-covalent means.

[0030] The term "first / second linker" refers to a peptide that links IL-15 to another protein molecule or protein fragment to ensure the correct folding and stability of the protein in this disclosure. Examples of such other molecules include, but are not limited to, IL-15Rα, Fc, Fc mutations, and antibodies.

[0031] The terms “PD1 antibody” and “αPD-1” refer to antibodies against programmed death protein 1 (PD1). Exemplary antibodies include, but are not limited to, those listed in U.S. Patent Nos. 7,029674, 7,488802, 7,521051, 8,008449, 8,354509, 8,617546, and 8,709417. Specific embodiments of the antibodies include BGB-A317, nivolumab (Bristol-Myers Squibb), labrolizumab (Merck), and pembrolizumab (Merck). [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 shows schematic diagrams of the fusion protein structures. Figure 1A shows a schematic diagram of the Fc-G4S-IL-15-Rα fusion protein, Figure 1B shows a schematic diagram of the αPD-1 Fab Fc-G4S-IL-15-Rα fusion protein, Figure 1C shows a schematic diagram of the Fc-G4S-Rα-IL-15 fusion protein, and Figure 1D shows a schematic diagram of the αPD-1 Fab Fc-G4S-Rα-IL-15 fusion protein.

[0033] [Figure 2] Figure 2 shows a comparison of the biological activity of several fusion proteins with different first linkers.

[0034] [Figure 3] Figure 3 shows a comparison of the receptor binding abilities of several fusion proteins with different first linkers.

[0035] [Figure 4]Figure 4 shows a comparison of the binding affinity of Fc-G4S-IL-15-Rα, Fc-(G4S)3-IL-15-Rα, αPD-1 Fab Fc-(G4S)3-IL-15-Rα, and aEGFR Fab Fc-(G4S)3-IL-15-Rα fusion proteins to CD8+ T cells.

[0036] [Figure 5] Figure 5 shows the results of intraperitoneal administration of Fc-G4S-IL-15-Rα and Fc-G4S-Rα-IL-15 to MC38 tumor model mice.

[0037] [Figure 6] Figure 6 shows the changes in body weight of MC38 tumor model mice treated with intraperitoneal administration of Fc-G4S-IL-15-Rα and Fc-G4S-Rα-IL-15.

[0038] [Figure 7] Figure 7 shows the survival curves of MC38 tumor model mice treated with intraperitoneal administration of Fc-G4S-IL-15-Rα and Fc-G4S-Rα-IL-15.

[0039] [Figure 8] Figure 8 shows the peripheral blood lymphocyte counts of MC38 tumor model mice treated with intraperitoneal administration of Fc-G4S-IL-15-Rα and Fc-G4S-Rα-IL-15. The vertical axis in the figure is "# / μl", indicating the number of cells per μl of blood.

[0040] [Figure 9] Figure 9 shows the results of intraperitoneal administration of αPD-1 Fab Fc-G4S-IL-15-Rα and Fc-G4S-IL-15-Rα to MC38 tumor model mice.

[0041] [Figure 10] Figure 10 shows the results of intraperitoneal administration of αPD-1 Fab Fc-G4S-IL-15-Rα and αPD-1 Fab mixed Fc-G4S-IL-15-Rα to MC38 tumor model mice.

[0042] [Figure 11] Figure 11 shows the results of intraperitoneal administration of αPD-1 Fab Fc-G4S-IL-15-Rα and αPD-1 Fab Fc-G4S-Rα-IL-15 to MC38 tumor model mice.

[0043] [Figure 12] Figure 12 shows the changes in body weight of MC38 tumor model mice treated with intraperitoneal administration of αPD-1 Fab Fc-G4S-IL-15-Rα and αPD-1 Fab Fc-G4S-Rα-IL-15.

[0044] [Figure 13] Figure 13 shows the peripheral blood lymphocyte counts of MC38 tumor model mice treated with intraperitoneal administration of αPD-1 Fab Fc-G4S-IL-15-Rα and αPD-1 Fab Fc-G4S-Rα-IL-15.

[0045] [Figure 14] Figure 14 shows the results for MC38 small tumor model mice treated with intraperitoneal administration of αPD-1-IL-15 fusion proteins containing different first linker and Fc fragments on day 7.

[0046] [Figure 15] Figure 15 shows the changes in body weight of MC38 microtumor model mice treated with intraperitoneal administration of αPD-1-IL-15 fusion proteins having different first linker and Fc fragments on day 7.

[0047] [Figure 16] Figure 16 shows the results for MC38 large tumor model mice treated with intraperitoneal administration of αPD-1-IL-15 fusion proteins containing different first linker and Fc fragments on day 14.

[0048] [Figure 17]Figure 17 shows the results for MC38 large tumor model mice treated with intraperitoneal administration of αPD-1-IL-15 fusion proteins with different first linkers on day 14.

[0049] [Figure 18] Figure 18 shows the changes in body weight of MC38 large tumor model mice treated with intraperitoneal administration of αPD-1-IL-15 fusion proteins having different first linkers on day 14.

[0050] [Figure 19] Figure 19 shows the peripheral blood lymphocyte count of MC38 large tumor model mice treated with intraperitoneal administration of αPD-1-IL-15 fusion proteins having different first linkers on day 14.

[0051] [Figure 20] Figure 20 shows the results of intratumoral administration of αPD-1 Fab-Fc-(G4S)3-IL-15-Rα to B16 tumor model mice.

[0052] [Figure 21] Figure 21 shows the results of systemic administration of anti-human PD-1 Fab-Fc-G4S-human IL-15-Rα and anti-human PD-1 Fab-Fc-(G4S)3-human IL-15-Rα to A549 lung cancer tumor CD34+ humanized mice.

[0053] [Figure 22] Figure 22 shows the SDS-PAGE electrophoresis images of αPD-1 Fab-Fc-(G4S)3-Rα-IL-15, Fc-G4S-Rα-IL-15, αPD-1 Fab-Fc-(G4S)3-IL-15-Rα, and Fc-(G4S)3-IL-15-Rα fusion proteins.

[0054] [Figure 23] Figure 23 shows the SDS-PAGE electrophoresis images of the anti-human PD-1 Fab-Fc-G4S-human IL-15-Rα and anti-human PD-1 Fab-Fc-(G4S)3-human IL-15-Rα fusion proteins. [Examples]

[0055] [Example 1] Design and construction of a fusion protein 1. Design and construction of the following eight types of fusion proteins (1) Fc-G4S-IL-15-Rα and Fc-G4S-Rα-IL-15 fusion proteins Fc-G4S-IL-15-IL-15Rα sushi (hereinafter referred to as Fc-G4S-IL-15-Rα) and Fc-G4S-IL-15Rα sushi-IL-15 (hereinafter referred to as Fc-G4S-Rα-IL-15) have the configurations shown in Figures 1A and 1C. IL-15Rα sushi is the sushi domain of the IL-15 receptor subunit α and has the amino acid sequence shown in SEQ ID NO: 3 or 17. The second linker between IL-15Rα sushi and IL-15 has the amino acid sequence shown in SEQ ID NO: 6. The first linker between IL-15Rα sushi or IL-15 and Fc is (G4S). n The integer multiple iterations of GGGGS are represented by Fc-G4S-IL-15-Rα, where Fc-G4S-Rα-IL-15 has the amino acid sequence shown in SEQ ID NO: 8, and Fc-G4S-Rα-IL-15 has the amino acid sequence shown in SEQ ID NO: 7. (2) Fc-(G4S)3-IL-15-Rα and Fc-(G4S)5-IL-15-Rα fusion protein obtained by further modifying the first linker of Fc-G4S-IL-15-Rα The first linkers of Fc-(G4S)3-IL-15-Rα and Fc-(G4S)5-IL-15-Rα are (GGGGS)3 and (GGGGS)5, respectively. Fc-(G4S)3-IL-15-Rα has the amino acid sequence shown in SEQ ID NO: 11, and Fc-(G4S)5-IL-15-Rα has the amino acid sequence shown in SEQ ID NO: 13. (3) αPD-1 Fab Fc-G4S-IL-15-Rα and αPD-1 Fab Fc-G4S-Rα-IL-15 obtained by further fusing anti-PD-1 Fab to Fc-G4S-IL-15Rα and Fc-G4S-Rα-IL-15 αPD-1 Fab Fc-G4S-IL-15-Rα and αPD-1 Fab Fc-G4S-Rα-IL-15 have the structures shown in Figures 1B and 1C. Anti-PD-1 Fab has a light chain with the amino acid sequence shown in SEQ ID NOs. 4, 18, or 19 and a heavy chain with the amino acid sequence shown in SEQ ID NOs. 5, 20, or 21. αPD-1 Fab Fc-G4S-IL-15-Rα has the amino acid sequence shown in SEQ ID NOs. 10, and αPD-1 Fab Fc-G4S-Rα-IL-15 has the amino acid sequence shown in SEQ ID NOs. 9. (4) αPD-1 Fab Fc-(G4S)3-IL-15-Rα and αPD-1 Fab Fc-(G4S)5-IL-15-Rα fusion protein obtained by further modifying the first linker of αPD-1 Fab Fc-G4S-IL-15-Rα αPD-1 Fab Fc-(G4S)3-IL-15-Rα and αPD-1 Fab Fc-(G4S)5-IL-15-Rα have a first linker which is (GGGGS)3 and (GGGGS)5, respectively. αPD-1 Fab Fc-(G4S)3-IL-15-Rα has the amino acid sequence shown in SEQ ID NO: 12, and αPD-1 Fab Fc-(G4S)5-IL-15-Rα has the amino acid sequence shown in SEQ ID NO: 14.

[0056] 2. Construction, transfection, expression, recovery, and purification of fusion proteins

[0057] The genes for the four forms of the fusion protein described above were constructed in eukaryotic expression vectors and transiently expressed in 293F cells using single or multiple plasmids. The recovered cell supernatant was purified with protein A. The purified protein was quantified by ELISA and Nanodrop, and its purity was detected by SDS-PAGE (4 μg was packed into each sample).

[0058] The specific protocol was as follows:

[0059] 2.1 Plasmid Construction

[0060] The pEE12.4-IgGκ-hIgG1 Fc plasmid, containing the mouse IgGκ signal peptide and the constant region sequence of human IgG1, was obtained from the inventors' laboratory. All genes involved in this application (IL-15, IL-15 Rα, and αPD-1 Fab) were synthesized by a third-party commercial synthesis company and then inserted into the pEE12.4 expression vector by enzymatic digestion and ligation or homologous recombination. The plasmid was extracted using a plasmid extraction kit from TIANGEN and stored at -20°C.

[0061] 2.2 Transient transfection for rapid serum-free expression of the target protein

[0062] (1) 293F cells were suspended and cultured in SMM293TII medium at 37°C, CO28%, and 135 rpm until the cell density reached 4-4.5 × 10⁶. 6 The solution could be used for transfection once it reached 200 ml with a cell / ml concentration.

[0063] (2) The cells were collected by centrifugation, washed once with Freestyle 293 medium, and resuspended in 200 ml of Freestyle 293 medium.

[0064] (3) 360 μg of plasmid was diluted in 6 ml of Freestyle 293 medium. Cells were co-transfected with the two plasmids to obtain αPD-1 fusion proteins. The ratio of plasmid expressing αPD-1 light chain to plasmid expressing αPD-1 heavy chain was 2:1 (240 μg plasmid expressing αPD-1 light chain + 120 μg plasmid expressing αPD-1 heavy chain). A 0.22 μm filter membrane was used for filtration and sterilization.

[0065] (4) 720 μg of polyethyleneimine (PEI) was diluted in 6 ml of Freestyle 293 medium and sterilized by filtration through a 0.22 μm filter membrane.

[0066] (5) 5 ml of PEI was added to the plasmid drop by drop while vortexing, and then left to stand for 5-10 minutes.

[0067] (6) The cell suspension was added to the plasmid / PEI mixture and cultured at 37°C, CO28%, and 85 rpm.

[0068] (7) After 4 hours, 200 ml of EX-CELL™ 293 medium and 2 mM L-glutamine were added to the cell suspension, and the cells were cultured further at a rotation speed of 135 rpm.

[0069] (8) After 24 hours, 3.8 mM valproic acid (VPA), a cell proliferation inhibitor, was added. Six days after transfection, the cells were collected by centrifugation at 8000 rpm and 4°C for 2 hours, and the cell supernatant was collected and further purified.

[0070] 2.3 Purification of target protein using Protein A

[0071] (1) Sample preparation: The collected cell supernatant was filtered through a 0.22 μm filter membrane to remove cell fragments, and a final concentration of 0.05% NaN3 was added.

[0072] (2) The protein A chromatography column was rinsed and equilibrated with 10 times the column volume of redistilled water and PBS, respectively.

[0073] (3) The sample was repeatedly packed using a constant flow pump at a flow rate 10 times the column volume / hour.

[0074] (4) The column was washed with PBS at 10 times its volume to remove impurity proteins.

[0075] (5) Elution was performed using 0.1 M glycine (pH 2.7), the eluate was collected, the eluted protein was pooled, and neutralized with an appropriate amount of neutralizing buffer (1 M Tris, pH 9.0) (the pH was adjusted to the extent that there was no agglomerating precipitate when observing the protein solution, and the pH was approximately 4).

[0076] Protein concentrations and purity were measured by SDS-PAGE electrophoresis, NanoDrop2000, and ELISA. Aliquots were stored at -80°C, but the aliquots needed to be slowly thawed at 4°C and could not be repeatedly frozen and thawed.

[0077] Figure 22 shows the SDS-PAGE electrophoresis results for αPD-1 Fab-Fc-(G4S)3-Rα-IL-15, Fc-G4S-Rα-IL-15, αPD-1 Fab Fc-(G4S)3-IL-15-Rα, and Fc-(G4S)3-IL-15-Rα fusion proteins. Figure 23 shows the SDS-PAGE electrophoresis results for anti-human PD-1 Fab Fc-G4S-human IL-15-Rα and anti-human PD-1 Fab Fc-(G4S)3-human IL-15-Rα fusion proteins. [Example 2] Study of the biological function of αPD-1-IL-15 fusion protein

[0078] 1. Function to promote lymphocyte production

[0079] The lymphocyte proliferation-promoting test (CCK8 test) includes the following steps:

[0080] (1) CTLL2 cells were cultured in 1640 complete medium containing 100 U / ml of commercially available recombinant IL2 cytokine.

[0081] (2) Perform the test and wash the cells 2-3 times with complete medium that does not contain IL2, then 2 × 10 4 Diluted to / ml

[0082] (3) Samples of Fc-G4S-IL-15-Rα, Fc-G4S-Rα-IL-15, Fc-(G4S)3-IL-15-Rα, αPD-1 Fc-(G4S)3-IL-15-Rα, and αPD-1 Fc-(G4S)5-IL-15-Rα were diluted in IL2-free complete medium at an initial concentration of 5 μg / ml, a 5-fold dilution, and a 10-fold dilution gradient.

[0083] (4) 100 μl of cell suspension and 100 μl of sample were added to a 96-well cell culture plate and thoroughly mixed by pipetting with a pipette tip.

[0084] (5) After 72 hours of incubation, 20 μl of CCK8 was added to the cells, and after a further 3 hours of incubation, the OD values ​​at 450 nm and 630 nm were detected using a microplate reader.

[0085] The detection results are shown in Figure 2, and the following can be observed. (1) The biological activity of Fc-G4S-Rα-IL-15 was significantly increased, approximately 10,000 times higher than that of Fc-G4S-IL-15-Rα, indicating that the biological activity of the two forms of IL-15 fusion protein is related to the relative positions of IL-15 and Rα. (2) The biological activity of αPD-1 Fc-(G4S)3-IL-15-Rα was not altered compared to Fc-(G4S)3-IL-15-Rα, indicating that the introduction of αPD-1 antibodies does not alter the biological activity of IL-15 in cells with low PD-1 expression. (3) The biological activity of αPD-1 Fc-(G4S)5-IL-15-Rα is higher than that of αPD-1 Fc-(G4S)3-IL-15-Rα, which indicates that the biological activity of IL-15 in the fusion protein form is also related to the length of the first linker.

[0086] 2. Detection of IL-15 fusion protein binding ability

[0087] The binding ability of Fc-G4S-IL-15-Rα, Fc-G4S-Rα-IL-15, and Fc-(G4S)3-IL-15-Rα to the IL-15 receptor was detected in the CTLL2 reporter cell line, and the results are shown in Figure 3. The binding ability of IL-15 to its receptor is positively correlated with its biological activity, meaning that the low biological activity of Fc-G4S-IL-15-Rα is due to its low affinity for that receptor.

[0088] The binding ability of Fc-G4S-IL-15-Rα, Fc-(G4S)3-IL-15-Rα, αPD-1 Fab Fc-(G4S)3-IL-]5-Rα, and aEGFR Fab Fc-(G4S)3-IL-15-Rα to CD8+ T cells sorted from the tumors of MC38 tumor-bearing mice was detected, and the results are shown in Figure 4, indicating that the PD-1 Fab antibody mediates the binding of the binding protein to CD8+ T cells.

[0089] 3. Detection of the therapeutic effect and toxic side effects of IL-15 and αPD-1-IL-15 fusion protein in tumor model mice

[0090] 3.1. MC38 tumor model (administered 7 days after inoculation)

[0091] Method 1 (dose 15 μg)

[0092] 5×10 5 of MC38 tumor cells were subcutaneously inoculated into the lower left of C57 mice. When the tumor grew to 40 mm 3 , 15 μg of Fc-G4S-IL-15-Rα or Fc-G4S-Rα-IL-15 was intraperitoneally administered 3 times at 2-day intervals, and PBS was similarly administered to the control group. The tumor volume was measured (volume = length × width × height / 2), and the body weight change and survival curve of the mice were recorded.

[0093] The results are shown in Figures 5, 6, 7, and 8. (1) By intraperitoneal administration of Fc-G4S-IL-15-Rα, the tumor was not controlled, the body weight did not change, and all mice survived. This indicates that Fc-G4S-IL-15-Rα has no significant antitumor effect and no toxic side effects. (2) By intraperitoneal administration of Fc-G,-G4S-Rα-IL-15, the tumor was significantly controlled, but the body weight decreased significantly and 40% died. Furthermore, in peripheral blood lymphocytes, CD8+ T, B220+, NK, and NKT cells proliferated. This indicates that Fc-G4S-Rα-IL-15 has a significant antitumor effect and severe toxic side effects.

[0094] Method 2 (Dosage: 15 μg)

[0095] 5 x 10 5 MC38 tumor cells were subcutaneously inoculated into the lower left side of C57 mice. The tumor was 40 mm. 3 Once the tumors had grown, Fc-G4S-IL-15-Rα (15 μg) or αPD-1 Fab Fc-G4S-IL-15-Rα (equomolar amount 30 μg) was administered intraperitoneally three times at two-day intervals, while PBS was administered to the control group in the same manner. Tumor volume was measured (volume = length × width × height / 2).

[0096] The results are shown in Figure 9. Tumors were not controlled in the group administered intraperitoneally with Fc-G4S-IL-15-Rα, but were significantly controlled in the group administered intraperitoneally with αPD-1 Fab Fc-G4S-IL-15-Rα. This indicates that αPD-1 Fab Fc-G4S-IL-15-Rα can enhance the therapeutic effect through the action of αPD-1 antibodies.

[0097] Method 3 (Dosage: 15 μg)

[0098] 5 x 10 5 MC38 tumor cells were subcutaneously inoculated into the lower left side of C57 mice. The tumor was 40 mm. 3 Once the tumors had grown, αPD-1 Fab (15 μg) + Fc-G4S-IL-15-Rα (15 μg) or αPD-1 Fab Fc-G4S-IL-15-Rα (equomolar amount 30 μg) was administered intraperitoneally three times at two-day intervals, while PBS was administered to the control group in the same manner. The volume of the tumors was measured (volume = length × width × height / 2).

[0099] The results are shown in Figure 10. Tumors were not controlled in the group administered intraperitoneally with αPD-1 Fab+Fc-G4S-IL-15-Rα, but tumors were significantly controlled in the group administered intraperitoneally with αPD-1 Fab Fc-G4S-IL-15-Rα. This indicates that the antitumor effect of αPD-1 Fab Fc-G4S-IL-15-Rα depends on the co-effect of the αPD-1 Fab and IL-15 fusion protein.

[0100] Method 4 (Dosage: 30 μg)

[0101] 5 x 10 5 MC38 tumor cells were subcutaneously inoculated into the lower left side of C57 mice. The tumor was 40 mm. 3 Once the tumors had grown, αPD-1 Fab Fc-G4S-Rα-IL-15 or αPD-1 Fab Fc-G4S-IL-15-Rα was administered intraperitoneally three times at two-day intervals, while PBS was administered to the control group. Tumor volume was measured (volume = length × width × height / 2), and changes in the mice's body weight were recorded.

[0102] The results are shown in Figures 11, 12, and 13. (1) Intraperitoneal administration of αPD-1 Fab Fc-G4S-IL-15-Rα resulted in good control of tumors, no change in body weight, and no significant proliferation of peripheral blood lymphocytes. This indicates that αPD-1 Fab Fc-G4S-IL-15-Rα has a significant antitumor effect without toxic side effects. (2) Intraperitoneal administration of αPD-1 Fab Fc-G4S-Rα-IL-15 resulted in good control of tumors, but significant weight loss was observed. Furthermore, CD8+T, B220+, NK, and NKT cells proliferated in peripheral blood lymphocytes. This indicates that αPD-1 Fab Fc-G4S-Rα-IL-15 has significant antitumor effects and severe toxic side effects.

[0103] Method 5 (Dosage: 30 μg)

[0104] 5 x 10 5 MC38 tumor cells were subcutaneously inoculated into the lower left side of C57 mice. The tumor was 40 mm. 3Once the tumors had grown, αPD-1 Fab Fc-G4S-IL-15-Rα, αPD-1 Fab mFc-G4S-IL-15-Rα, αPD-1 Fab Fc-(G4S)3-IL-15-Rα, αPD-1 Fab mFc-(G4S)3-IL-15-Rα, and aEGFR Fab Fc-G4S-IL-15-Rα were administered intraperitoneally three times at two-day intervals, while PBS was administered to the control group in the same manner. Tumor volume was measured (volume = length × width × height / 2), and changes in the mice's body weight were recorded.

[0105] The results are shown in Figures 14 and 15. (1) Intraperitoneal administration of αPD-1 Fab Fc-G4S-IL-15-Rα, αPD-1 Fab mFc-G4S-IL-15-Rα, αPD-1 Fab Fc-(G4S)3-IL-15-Rα, and αPD-1 Fab mFc-(G4S)3-IL-15-Rα resulted in good tumor control and no significant change in body weight. This indicates that αPD-1 Fab Fc-G4S-IL-15-Rα, αPD-1 Fab mFc-G4S-IL-15-Rα, αPD-1 Fab Fc-(G4S)3-IL-15-Rα, and αPD-1 Fab mFc-(G4S)3-IL-15-Rα have significant antitumor effects without toxic side effects. (2) Intraperitoneal administration of aEGFR Fab Fc-G4S-IL-15-Rα did not control the tumor and did not change body weight. This indicates that aEGFR antibodies cannot play an antitumor role in place of αPD-1 antibodies.

[0106] 3.2. MC38 tumor model (administered 14 days after inoculation)

[0107] Method 1 (Dosage: 30 μg)

[0108] 5 x 10 5 MC38 cells were subcutaneously inoculated into the lower left side of C57 mice. The tumor was 100 mm. 3When the tumors grew, αPD-1 Fab Fc-G4S-IL-15-Rα, αPD-1 Fab mFc-G4S-IL-15-Rα, αPD-1 Fab Fc-(G4S)3-IL-15-Rα, αPD-1 Fab mFc-(G4S)3-IL-15-Rα, and aEGFR Fab Fc-(G4S)3-IL-15-Rα were administered intraperitoneally three times at two-day intervals, and PBS was administered to the control group. The volume of the tumor was measured (volume = length × width × height / 2).

[0109] The results are shown in Figure 16. (1) Intraperitoneal administration of αPD-1 Fab Fc-(G4S)3-IL-15-Rα and αPD-1 Fab mFc-(G4S)3-IL-15-Rα resulted in good control of tumors. This indicates that when the first linker is (G4S)3, the fusion protein has a better antitumor effect. (2) Intraperitoneal administration of aEGFR Fab Fc-(G4S)3-IL-15-Rα did not control the tumor and did not change body weight. This indicates that aEGFR antibodies cannot play an antitumor role in place of αPD-1 antibodies. (3) There was no significant difference in antitumor effect between αPD-1 Fab Fc-G4S-IL-15-Rα and αPD-1 Fab mFc-G4S-IL-15-Rα administered intraperitoneally, which indicates that mFc does not play a significant role in antitumor treatment.

[0110] Method 2 (Dosage: 30 μg)

[0111] 5 x 10 5 MC38 cells were subcutaneously inoculated into the lower left side of C57 mice. The tumor was 100 mm. 3 Once the tumors had grown, αPD-1 Fab Fc-(G4S)3-IL-15-Rα and αPD-1 Fab Fc-(G4S)5-IL-15-Rα were administered intraperitoneally three times at two-day intervals, while PBS was administered to the control group in the same manner. Tumor volume was measured (volume = length × width × height / 2), and changes in the mice's body weight were recorded.

[0112] The results are shown in Figures 17, 18, and 19. (1) Intraperitoneal administration of αPD-1 Fab Fc-(G4S)3-IL-15-Rα and αPD-1 Fab Fc-(G4S)5-IL-15-Rα resulted in good control of tumors without significant difference. This indicates that the fusion protein with the (G4S)5 first linker did not exhibit a better antitumor effect compared to the fusion protein with the (G4S)3 first linker. (2) Intraperitoneal administration of αPD-1 Fab Fc-(G4S)5-IL-15-Rα resulted in mild weight loss and significant proliferation of peripheral blood lymphocytes in mice. This indicates that fusion proteins with the first linker of (G4S)5 have peripheral toxic side effects.

[0113] 3.3.B16 Tumor model (administration performed 7 days after inoculation)

[0114] (1) 3 × 10 5 B16 cells were subcutaneously inoculated into the lower left side of C57 mice.

[0115] (2) Tumor is 30 mm 3 Once the tumors had grown, αPD-1 Fab Fc-(G4S)3-IL-15-Rα was administered intratumorally three times at two-day intervals, with 30 μg administered each time. The control group was similarly administered PBS.

[0116] (3) The volume of the tumor was measured (volume = length × width × height / 2).

[0117] The results are shown in Figure 20. (1) Intratumoral administration of αPD-1 Fab Fc-(G4S)3-IL-15-Rα controlled the tumor. This indicates that the fusion protein has a certain antitumor effect against B16.

[0118] 3.4. CD34+ Humanized Mouse A549 Lung Cancer Model

[0119] (1) 2 × 10 6A549 cells were subcutaneously inoculated into the lower left subcutaneous region of CD34+ humanized mice.

[0120] (2) Tumor is 30 mm 3 Once the animals had grown, they were systemically administered anti-human PD-1 Fab Fc-G4S-human IL-15-Rα or anti-human PD-1 Fab Fc-(G4S)3-human IL-15Rα at a dose of 10 μg on day 10, and at doses of 20 μg on days 17 and 20. PBS was administered to the control group in a similar manner.

[0121] (3) The volume of the tumor was measured (volume = length × width × height / 2).

[0122] The results are shown in Figure 21. (1) Tumors were controlled by intraperitoneal administration of anti-human PD-1 Fab Fc-G4S-human IL-15-Rα or anti-human PD-1 Fab Fc-(G4S)3-human IL-15-Rα. This indicates that the fusion protein has an antitumor effect against human-derived A549 lung cancer in humanized mice.

[0123] Finally, it should be noted that the embodiments described above are used solely to help those skilled in the art understand the nature of the Disclosure and are not intended to limit the scope of protection of the Disclosure.

Claims

1. (1) The first structural unit is the sushi domain of the interleukin-15 (IL-15) receptor subunit α, (2) The second structural unit is IL-15, (3) An antibody Fc fragment or mutant Fc fragment, which is a third structural unit located at the N-terminus of the fusion protein, (4) A block comprising a second linker connecting the first structural unit and the second structural unit, wherein the second linker has the amino acid sequence shown in SEQ ID NO: 6, The first linker, when the C-terminus of the fusion protein is the first structural unit, connects the second structural unit and the third structural unit. The first linker is (G ４ S) ｎ It has an amino acid sequence that is an integer multiple repeat of GGGGS, where n is 3, moreover (5) comprising a block of a fourth structural unit linked to the N-terminus of the third structural unit which is the Fab of the therapeutic antibody, The Fab of the therapeutic antibody is a fusion protein, which is an anti-PD-1 Fab (a Fab of PD1 antibody).

2. The fusion protein according to claim 1, wherein the anti-PD-1 Fab comprises a heavy chain and a light chain, the heavy chain being located at the N-terminus of the fusion protein.

3. The IL-15 is mouse-derived or human-derived IL-15 having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

15. The sushi domain of the IL-15 receptor subunit α is a sushi domain of a mouse or human-derived IL-15 receptor subunit α and has the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

17. The fusion protein according to claim 1, wherein the Fc fragment or mutant Fc fragment has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

16.

4. The fusion protein according to claim 3, wherein the anti-PD-1 Fab is a mouse-derived or human-derived anti-PD-1 Fab.

5. The anti-PD-1 Fab has a light chain having the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 18, or SEQ ID NO: 19, and The fusion protein according to claim 3, wherein the anti-PD-1 Fab has a heavy chain having the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 20, or SEQ ID NO:

21.

6. A homodimer composed of the fusion protein according to claim 1, wherein the monomers are linked to each other via dimerization of the third structural unit.

7. (6) Homodimer 6 (anti-PD-1 Fab Fc-(G ４ S) ３ -IL-15-Rα) The monomer comprises, from the N-terminus to the C-terminus, anti-PD-1 Fab, human-derived IgG1-Fc, and a first linker ((G ４ S) ３ ) and a homodimer 6 containing IL-15, a second linker, and the sushi domain of the IL-15 receptor subunit α, The homodimer according to claim 6.

8. The homodimer 6 (anti-PD-1 Fab Fc-(G) ４ S) ３ The homodimer according to claim 7, wherein -IL-15-Rα) is a homodimer 6 having the amino acid sequence shown in SEQ ID NO:

12.

9. A nucleotide fragment encoding the fusion protein according to any one of claims 1 to 5.

10. Use in the manufacture of a pharmaceutical product, wherein the fusion protein described in any one of claims 1 to 5 or the homodimer described in any one of claims 6 to 7 is used, and the pharmaceutical product is an antitumor drug.

11. The use according to claim 10, wherein the pharmaceutical is a pharmaceutical for B-cell lymphoma, colorectal cancer, melanoma, or lung cancer.

12. (1) A step of constructing an expression vector containing a gene encoding a fusion protein, preferably the expression vector being a pEE12.4 expression vector, (2) A step of constructing a host cell containing the expression vector by transiently transfecting the host cell, wherein the host cell is a 293F cell, (3) A step of culturing the host cells and collecting the cell supernatant, (4) A method for preparing a fusion protein according to any one of claims 1 to 5, comprising the step of purifying the fusion protein using a protein A affinity chromatography column.

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