Use of antibody mutation methods in therapeutic antibody drugs

Abstract

The present invention provides a bifunctional molecule comprising an Fc mutant antibody linked to a cytokine, and the use of the bifunctional molecule in the manufacture of a therapeutic antibody drug. These Fc mutant monoclonal antibodies, monoclonal antibody / antigen complexes, or Fc fusion proteins can significantly reduce toxicity and side effects in vivo while maintaining the biological activity of the original antibody in vivo and in vivo.

Description

[Technical Field]

[0001] The present invention belongs to the field of pharmaceutical technology and specifically relates to the use of antibody mutation methods in therapeutic antibody drugs. [Background technology]

[0002] Fc fusion proteins are a new type of recombinant protein that uses genetic engineering to combine the Fc (fragment crystallizable) fragment of immunoglobulin with a functional protein molecule with certain biological activity, thereby conferring the properties of an antibody to the original functional protein. Monoclonal antibodies and antibody-Fc fusion proteins have been widely used in clinical trials with great success. As of September 2014, nine human IgG-Fc fusion protein drugs have been approved for clinical use by the US Food and Drug Administration (FDA).

[0003] However, among the large number of monoclonal antibodies / antibody fusion proteins currently under research, some products have shown good therapeutic effects, but they also exhibit toxic and side effects, including liver and lung dysfunction, and severe cases can lead to the failure or discontinuation of clinical studies of the products under research. Summary of the Invention

[0004] On the other hand, our research has yielded the following findings:

[0005] The liver and lung dysfunction observed above is related to the distribution of monoclonal antibody / antibody Fc fusion protein in the liver and lung.

[0006] Some highly active monoclonal antibodies / antibody Fc fusion proteins are distributed in the liver and lungs, causing inflammatory reactions in the liver and lungs, leading to toxicity and side effects.

[0007] Furthermore, the distribution of these monoclonal antibody / antibody Fc fusion proteins in the liver and lungs is related to the Fc.

[0008] Binding of Fc to FcRn (an IgG antibody receptor located on the surface of the cell membrane), which is abundant in liver and lung tissues, is the reason why these monoclonal antibody / antibody-Fc fusion proteins are distributed in the liver and lung.

[0009] Reducing Fc binding to FcRn can effectively reduce the hepato-pulmonary toxicity of monoclonal antibodies / antibody fusion proteins.

[0010] The reduction in Fc binding to FcRn has no or only an acceptable effect on the in vivo and in vivo biological activity of the monoclonal antibody / antibody fusion protein.

[0011] For the above reasons, it is an object of the present invention to provide the use of antibody mutation methods in therapeutic antibody drugs.

[0012] To achieve the object of the present invention, the technical solution of the present invention is as follows:

[0013] It is a dual-function molecule characterized by linking an Fc mutant antibody and a cytokine.

[0014] In particular, said Fc mutants are fragments of: Selected from H310A / H435Q, I253A, S254A, R255A, K288A, L309A, H310A, S415A, H433A, H435A, H435R, Y436A, H310Q / H433N, M252Y / T256Q, M252F / T256D.

[0015] In particular, the antibody Selected from IgG1, IgG4, HER2, HER3, EGFR, PDL1, CD19, CD20, CD22, CD24, CD33, CD40, CD40L, CD73, CD276, VEGFR, TIGIT, TIM3, LAG3, CXCR3, CXCR5, CCR3, CCR4, CCR9, and PD1.

[0016] In particular, said cytokines are It is selected from IL2 and mutants thereof, IL7, IL12 and mutants thereof, IL15, IL18, IL21, IL2-CD25, Neo 2 / 15, IFNα, IFNα2b and mutants thereof, IFNγ, TNFα, GM-CSF, Flt3, and CCL21.

[0017] A method for amplifying the above-mentioned dual-function molecule includes linking a cytokine and an Fc mutant antibody to form a fusion protein, constructing the fusion protein in an expression vector, transfecting it into cells, and purifying it.

[0018] A therapeutic antibody drug comprising the above-described dual function molecule.

[0019] The present invention also provides the use of the dual function molecule in the manufacture of a medicament.

[0020] In particular, said drug is a therapeutic antibody drug.

[0021] Compared with existing technologies, the dual-functional molecules provided by the present invention are mutated monoclonal antibodies, monoclonal antibody / antigen complexes, or Fc fusion proteins. In vitro and in vivo studies have shown that these Fc-mutated monoclonal antibodies, monoclonal antibody / antigen complexes, or Fc fusion proteins can maintain their pre-mutation biological activity both in vivo and in vivo, while significantly reducing toxicity and side effects in vivo, and have good future application potential.

[0022] The present invention will be further described below in conjunction with the drawings and examples. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the in vitro biological activity of IL2-Fcmu. [Figure 2] FIG. 2 shows the in vivo antitumor activity of IL2-Fcmu. [Figure 3] Figure 3 shows that mIL12sc-Fcmu maintains in vivo antitumor activity. [Figure 4] FIG. 4 shows the in vitro biological activity of hIL15sc-Fcmu. [Figure 5] FIG. 5 shows the in vivo antitumor activity of hIL15sc-Fcmu. [Figure 6] FIG. 6 shows the in vitro biological activity of the human IL2 / hu22F8mu complex. [Figure 7] FIG. 7 shows the in vivo antitumor activity of the human IL2 / hu22F8mu complex. [Figure 8] FIG. 8 shows the in vitro biological activity of the dual function molecule hu147mu-hIL15sc. [Figure 9] FIG. 9 shows the in vivo antitumor activity of the dual-function molecule hu147mu-hIL15sc. [Figure 10] FIG. 10 shows the in vitro biological activity of the dual function molecule hu3E10mu-hIL15sc. [Figure 11] FIG. 11 shows the in vivo antitumor activity of the dual-function molecule hu3E10mu-hIL15sc. [Figure 12] FIG. 12. In vitro biological activity of the dual function molecule hu19H6-IL15sc. [Figure 13] FIG. 13 shows the in vivo antitumor activity of the dual-function molecule hu19H6mu-hIL15sc. [Figure 14] FIG. 14 shows the in vitro biological activity of the dual function molecule hu609mu-IL15sc. [Figure 15] FIG. 15 shows the in vivo antitumor activity of the dual-function molecule hu609mu-IL15. [Figure 16] FIG. 16 shows the in vitro biological activity of the dual function molecule ch158mu-IL15sc. [Figure 17] FIG. 17 shows the in vivo antitumor activity of the dual-function molecule ch158mu-hIL15sc. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be described in more detail below with reference to specific examples, which are not intended to limit the present invention but merely to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following examples generally follow general conditions for those experimental methods for which specific conditions are not specified. Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[0025] Example 1 Use of antibody mutagenesis techniques in IL2-Fc fusion proteins In this example, a fusion protein consisting of human IL2 (proleukin, the amino acid sequence of which is shown in SEQ ID NO: 2) and a mutant human IgG4 Fc is used as an example to demonstrate that the Fc mutant fusion protein IL2-Fcmu maintains biological activity both in vivo and in vivo while significantly reducing toxicity in vivo.

[0026] This Fc mutation technique can be applied to fusion proteins of Fc with other types of IL2, such as wild-type IL2 (amino acid sequence shown in SEQ ID NO: 1), Mutein (amino acid sequence shown in SEQ ID NO: 3), and Superkine (amino acid sequence shown in SEQ ID NO: 4), to achieve similar effects.

[0027] 1.1 Preparation of the fusion protein IL2-Fcwt and the mutant fusion protein IL2-Fcmu Human IL2 (proleukin, amino acid sequence shown in SEQ ID NO: 2) was linked to human IgG4-Fc (amino acid sequence shown in SEQ ID NO: 16) to form the IL2-Fcwt fusion protein (amino acid sequence shown in SEQ ID NO: 18). It was linked to mutant human IgG4-Fc (amino acid sequence shown in SEQ ID NO: 17) to form the IL2-Fcmu fusion protein (amino acid sequence shown in SEQ ID NO: 19). The IL2-Fcwt and IL2-Fcmu genes were each constructed into the pcDNA3.4 expression vector, transfected into Expi-293F cells, and purified with Protein G to obtain the fusion proteins IL2-Fcwt and IL2-Fcmu, which were >95% pure, quantified, aliquoted, and stored frozen at -80°C for use.

[0028] 1.2 In vitro biological activity of IL2-Fcmu In this example, the biological activity of IL2-Fcmu and IL2-Fcwt was demonstrated through a CTLL2 cell proliferation experiment. The method was as follows: CTLL2 cells were cultured at 5 × 10 in 1640 medium containing 10% FBS. 4 IL2-Fcwt and IL2-Fcmu were diluted to 100 ng / ml in 1640 medium containing 10% FBS, and then diluted three-fold to form a gradient of eight steps, which were then added to the culture plates containing the CTLL2 cells. After 72 hours of culture in a CO2 cell culture incubator, the relative cell number in each well was measured using CCK8, and the EC50 was calculated to determine the activity of the samples.

[0029] As shown in Figure 1, both IL2-Fcwt and IL2-Fcmu were able to stimulate the proliferation of CTLL2 cells, with EC50 values ​​of 0.94 ng / ml and 0.91 ng / ml, respectively, indicating that IL2-Fcmu has biological activity consistent with that of IL2-Fcwt.

[0030] 1.3 Significant reduction in the toxicity of IL2-Fcmu in vivo From day 0 to day 2, C57BL / 6 mice (Wetland Health) were intraperitoneally injected twice, with an injection volume of 0.2 ml per injection. IL2-Fcwt was administered at doses of 0.5 mg / kg, 1 mg / kg, and 2 mg / kg, and IL2-Fcmu was administered at doses of 0.5 mg / kg, 1 mg / kg, 2 mg / kg, and 4 mg / kg. The experimental mice were observed for mortality on day 7.

[0031] Table 1. Survival rate of experimental animals in each dose group [Table 1]

[0032] As shown in Table 1, on day 7 of the experiment, all experimental animals in the 2 mg / kg IL2-Fcwt group died (0% survival rate), while 40% of the experimental animals died in the 1 mg / kg group, and no experimental animals died in the 0.5 mg / kg group. In the 4 mg / kg IL2-Fcmu group, 30% of the experimental animals died, but no experimental animals died in any of the other dose groups. These results demonstrate that the Fc-mutated IL2-Fcmu has significantly reduced toxicity to experimental mice.

[0033] 1.4 In vivo antitumor activity of IL2-Fcmu In this example, the in vivo antitumor activity of IL2-Fcwt and IL2-Fcmu was evaluated and compared using a mouse MC38 colorectal cancer cell xenograft tumor model. The experimental method is as follows.

[0034] In vitro cultured mouse colon cancer MC38 cells were collected and the cell suspension was adjusted to a concentration of 1 × 10 7 The hair on the right side of the armpit of C57BL / 6 mice was shaved. Under aseptic conditions, 100 μl of the cell suspension was subcutaneously inoculated into the right side of the armpit of C57BL / 6 mice. The diameter of the subcutaneously inoculated tumors in the mice was measured with a vernier caliper, and the average tumor volume was 100-200 mm. 3At the age of 18 months, the animals were randomly assigned to groups of 6 animals per group. IL2-Fcwt was administered at a dose of 0.5 mg / kg, and IL2-Fcmu at doses of 0.5 mg / kg and 1.0 mg / kg, both administered intraperitoneally. The control group received an equal volume of PBS, administered intraperitoneally three times a week in a volume of 0.2 ml per administration for two consecutive weeks. Throughout the experiment, the diameter of the transplanted tumors was measured twice a week, and the mice were weighed. The formula for calculating tumor volume (TV) was as follows: TV=1 / 2×a×b 2

[0035] where a and b represent length and width, respectively. The relative tumor volume (RTV) was calculated from the measurement results using the formula RTV = Vt / V0. V0 is the tumor volume measured at the time of group division and administration (i.e., d0), and Vt is the tumor volume at each measurement. The evaluation index for antitumor activity is the relative tumor growth rate T / C (%), and the calculation formula is as follows: T / C(%)=(TRTV / CRTV)×100 Tumor inhibition rate TGI(%)=100-T / C(%)

[0036] TRTV is the RTV of the treatment group, and CRTV is the RTV of the negative control group.

[0037] As shown in Figure 2, at a dose of 0.5 mg / kg, IL2-Fcwt and IL2-Fcmu showed similar antitumor activity, with TGIs of 61% and 53%, respectively. Statistical analysis revealed no significant difference in the antitumor activity between the two groups (p>0.05). At a dose of 1.0 mg / kg, IL2-Fcmu achieved a TGI of 88%, which was higher than the 0.5 mg / kg group and significantly higher than the IL2-Fcwt group.

[0038] Example 2 Use of antibody mutagenesis techniques in IL12-Fc fusion proteins In this example, one of the many forms of IL12-Fc fusion protein, i.e., the IL12P40-P35-Fc fusion protein, is used as an example to demonstrate the effects of Fc mutation technology. The Fc-mutated IL12-Fc fusion protein significantly reduced toxicity and side effects in vivo while maintaining biological activity in vivo and in vivo. This effect is consistent with many forms of IL12-Fc fusion protein, including those described in literature / patents, such as Xencor, Inc. Patent US2020 / 0216509 A1.

[0039] 2.1 Production of mIL12sc-Fcwt and mIL12sc-Fcmu Human IL12 contains two subunits, P35 (amino acid sequence shown in SEQ ID NO:5) and P40 (amino acid sequence shown in SEQ ID NO:7), while mouse IL12 contains two subunits, P35 (amino acid sequence shown in SEQ ID NO:6) and P40 (amino acid sequence shown in SEQ ID NO:8). Human IL12 P40 and human IL12 P35 were linked via GSGSSRRGGSGSGGSGGGGS to form human single-chain IL12, i.e., huIL12sc (amino acid sequence shown in SEQ ID NO:9). Mouse IL12 P40 and mouse IL12 P35 were linked via GSGSSRRGGSGSGGSGGGGS to form mouse single-chain IL12, i.e., mIL12sc (amino acid sequence shown in SEQ ID NO:10). huIL12sc was linked to human IgG1-Fc (amino acid sequence shown in SEQ ID NO:14) to form the huIL12sc-Fcwt fusion protein (amino acid sequence shown in SEQ ID NO:20). It was linked to a mutated human IgG1 Fc (amino acid sequence shown in SEQ ID NO: 15) to form a huIL12sc-Fcmu fusion protein (amino acid sequence shown in SEQ ID NO: 22). mIL12sc was linked to a human IgG1-Fc (amino acid sequence shown in SEQ ID NO: 14) to form a mIL2sc-Fcwt fusion protein (amino acid sequence shown in SEQ ID NO: 21). It was linked to a mutated human IgG1 Fc (amino acid sequence shown in SEQ ID NO: 15) to form a mIL2sc-Fcmu fusion protein (amino acid sequence shown in SEQ ID NO: 23). The genes of huIL2sc-Fcwt, huIL12sc-Fcmu, mIL12sc-Fcwt, and mIL2sc-Fcmu were constructed into pcDNA3.4 expression vectors, respectively, transfected into Expi-293F cells, and purified by Protein G to obtain the fusion proteins huIL12sc-Fcwt, huIL12sc-Fcmu, mIL12sc-Fcwt, and mIL2sc-Fcmu, which were >95% pure, quantified, aliquoted, and stored frozen at −80°C for further use.

[0040] 2.2 Significant reduction in the in vivo toxicity of mIL12sc-Fcmu The method is the same as in Example 1.3.

[0041] Table 2. Survival rate of experimental animals in each dose group [Table 2]

[0042] As shown in Table 2, on day 7 after the first administration, all experimental animals in the 2 mg / kg mIL12sc-Fcwt group died (0% survival rate), while 40% of the experimental animals died in the 1 mg / kg group, and no experimental animals died in the 0.5 mg / kg group. In the 4 mg / kg mIL12sc-Fcmu group, 80% of the experimental animals died, but no experimental animals died in any of the other dose groups. These results demonstrate that the Fc-mutated mIL12sc-Fcmu has significantly reduced toxicity to experimental mice.

[0043] 2.3 Maintenance of in vivo antitumor activity of mIL12sc-Fcmu The method was the same as in Example 1.3, and both mIL12sc-Fcwt and mIL12sc-Fcmu were administered at a dose of 0.5 mg / kg three times a week for a total of six administrations.

[0044] As shown in Figure 3, both mIL12sc-Fcwt and mIL12sc-Fcmu exhibited strong antitumor activity, with TGIs of 98% and 96%, respectively, which were not significantly different (p>0.05), indicating that the Fc mutation in mIL12sc-Fcmu did not affect its in vivo antitumor activity.

[0045] Example 3 Use of antibody mutagenesis techniques in IL15-Fc fusion proteins In this example, one of the many forms of IL15-Fc fusion protein, i.e., IL15Rsushi-IL15-Fc fusion protein, is used as an example to demonstrate the effects of Fc mutation technology. The Fc-mutated IL15-Fc fusion protein significantly reduces toxicity and side effects in vivo while maintaining biological activity in vivo and in vivo. Many forms of IL15-Fc fusion protein are described in literature / patents, such as US Patent No. 2018 / 0118805 A1 to Xencor, Inc.

[0046] 3.1 Production of fusion proteins hRsushi-hIL15-Fcwt and hRsushi-hIL15-Fcmu Human IL15Rsushi (amino acid sequence shown in SEQ ID NO: 12) and human IL15 (amino acid sequence shown in SEQ ID NO: 11) were linked via GGGGSGGGGSGGGGSGGGGGSGGGGGSGGGGS to form single-chain IL15, i.e., huIL15sc (amino acid sequence shown in SEQ ID NO: 13). hIL15sc was linked to human IgG1 Fc (amino acid sequence shown in SEQ ID NO: 14) to form the fusion protein hIL15sc-Fcwt (amino acid sequence shown in SEQ ID NO: 24). hIL15sc was linked to mutant human IgG1 Fc (amino acid sequence shown in SEQ ID NO: 15) to form the fusion protein hIL15sc-Fcmu (amino acid sequence shown in SEQ ID NO: 25). The above hIL15sc-Fcmu and hIL15sc-Fcwt genes were constructed into the pcDNA3.4 expression vector, transfected into Expi-293F cells, and purified with Protein G to obtain the fusion proteins hIL15sc-Fcmu and hIL15sc-Fcwt. The purities were >95%, and the proteins were quantified, aliquoted, and frozen at -80°C for storage prior to use.

[0047] 3.2 In vitro biological activity of hIL15sc-Fcmu The biological activities of hIL15sc-Fcmu and hIL15sc-Fcwt were measured using a CTLL2 cell proliferation experiment. The method was basically the same as in Example 1.2, and the dilution gradient of IL15sc-Fcwt and IL15sc-Fcmu was 10 times, starting from 10,000 ng / ml.

[0048] As a result, as shown in Figure 4, both IL15sc-Fcwt and IL15sc-Fcmu were able to stimulate the proliferation of CTLL2 cells, with EC50 values ​​of 3.455 ng / ml and 3.516 ng / ml, respectively, indicating that IL15sc-Fcmu has biological activity consistent with that of IL15-Fcwt, as specifically shown in Table 3.1 below.

[0049] Table 3.1 [Table 3A]

[0050] 3.3 Significantly reduced toxicity and side effects of hIL15sc-Fcmu compared to hIL15sc-Fcwt The method is the same as in Example 1.3.

[0051] Table 3. Survival rate of experimental animals in each dose group [Table 3]

[0052] As shown in Table 3, on day 7 of the experiment, all animals in the 2 mg / kg hIL15sc-Fcwt sample group died (0% survival rate), while 20% of animals died in the 1 mg / kg group and no animals died in the 0.5 mg / kg group. In the 4 mg / kg hIL15sc-Fcmu group, 60% of animals died, but no animals died in any of the other groups. These results indicate that the toxicity of hIL15sc-Fcmu to experimental mice was significantly reduced after Fc mutation.

[0053] 3.4 In vivo antitumor activity of hIL15sc-Fcmu The method was the same as in Example 1.3. Both hIL15sc-Fcwt and hIL15sc-Fcmu were administered at a dose of 0.5 mg / kg three times a week for a total of six administrations.

[0054] As shown in Figure 5, hIL15sc-Fcwt and hIL15sc-Fcmu exhibited good antitumor activity, with TGIs of 53% and 58%, respectively. Statistical analysis revealed no significant difference in the antitumor activity between the two groups (p>0.05), indicating that the Fc-mutated hIL15sc-Fcmu maintains good in vivo antitumor activity.

[0055] Example 4 Use of Fc Mutation Technology in IL2 / Monoclonal Antibody Conjugates In this example, we use the human IL2 antibody hu22F8 we have developed as an example to demonstrate that a conjugate (IL2 / hu22F8mu) consisting of the Fc-mutated antibody hu22F8mu and human IL2 proleukin (amino acid sequence shown in SEQ ID NO: 2) maintains the in vivo and in vivo biological activity of a conjugate (IL2 / hu22F8wt) consisting of wild-type antibody hu22F8wt and human IL2, while significantly reducing its in vivo toxicity. This Fc mutation technology can be applied to other types of IL2 / monoclonal antibody conjugates to achieve similar results, such as the IL2 monoclonal antibody NARA1 described in the literature.

[0056] 4.1 Production of humanized IL2 monoclonal antibody hu22F8mu The amino acid sequence of the heavy chain of humanized IL2 monoclonal antibody hu22F8 is shown in SEQ ID NO: 27, the amino acid sequence of the heavy chain of Fc-mutated humanized IL2 monoclonal antibody hu22F8mu is shown in SEQ ID NO: 26, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO: 28, and the amino acid sequence of human IL2 is shown in SEQ ID NO: 2.

[0057] The heavy chain, mutant heavy chain, and light chain genes were constructed in the pcDNA3.4 vector, matched and transfected into Expi-293F cells. Purification with Protein G yielded the antibodies hu22F8 and hu22F8mu. SDS-PAGE electrophoresis and SEC-HPLC confirmed that the molecular weight of each antibody was approximately 150 kD, with a purity of >95%. The antibodies were quantified, aliquoted, and frozen at -80°C for storage prior to use.

[0058] 4.2 In vitro biological activity of the human IL2 / hu22F8mu complex In this example, the biological activity of the IL2 / hu22F8mu complex was demonstrated through a CTLL2 cell proliferation experiment. The method was essentially the same as in Example 1.2. IL2 was mixed with hu22F8mu or hu22F8wt at a mass ratio of 1:7 and incubated at room temperature for 30 minutes to form the complex IL2 / hu22F8mu or IL2 / hu22F8wt. Calculated as IL2, IL2 / hu22F8mu and IL2 / hu22F8wt were diluted to 50ng / ml, and then three-fold dilutions (8 gradients) were applied to culture plates containing CTLL2 cells.

[0059] As shown in Figure 6, both IL2 / hu22F8mu and IL2 / hu22F8wt were able to stimulate the proliferation of CTLL2 cells, with EC50 values ​​of approximately 0.7419 ng / ml and 0.7485 ng / ml, respectively, indicating that IL2 / hu22F8mu has biological activity consistent with that of IL2 / hu22F8wt.

[0060] 4.3 Significantly reduced in vivo toxicity of the human IL2 / hu22F8mu complex The IL2 / hu22F8wt and IL2 / hu22F8mu complexes were prepared as follows: IL2 was mixed with hu22F8wt or hu22F8mu at a mass ratio of 1:7, and the mixture was left standing at room temperature for 30 minutes before administration. The remaining procedures were the same as in Example 1.3, and the dose was calculated based on IL2.

[0061] Table 4. Survival rate of experimental animals in each antibody dose group [Table 4]

[0062] As shown in Table 4, on day 7 of the experiment, all animals in the 2 mg / kg IL2 / hu22F8wt sample group died (0% survival rate), while 60% of the animals in the 1 mg / kg group died. No animals died in the 0.5 mg / kg group. No animals died in the 4 mg / kg group, the highest dose of IL2 / hu22F8mu, nor in any of the other dose groups. These results indicated that the toxicity of IL2 / hu22F8mu to experimental mice was significantly reduced after Fc mutation.

[0063] 4.4 In vivo antitumor activity of human IL2 / hu22F8mu complex In this example, the in vivo antitumor activity of IL2 / hu22F8mu complex and IL2 / hu22F8wt complex was evaluated and compared using a mouse MC38 colorectal cancer cell xenograft tumor model. The experimental method was basically the same as in Example 1.4.

[0064] The IL2 / hu22F8mu complex was prepared as described above, and the doses calculated as IL2 were 1 mg / kg and 0.5 mg / kg.

[0065] The IL2 / hu22F8wt complex was prepared as described above, and the dose calculated as IL2 was 0.5 mg / kg.

[0066] All of the above were administered by intraperitoneal injection three times a week.

[0067] As shown in Figure 7, the IL2 / hu22F8mu complex exhibited excellent antitumor activity similar to that of the IL2 / hu22F8wt complex. After three weekly doses, the TGIs of the IL2 / hu22F8mu 1 mg / kg and 0.5 mg / kg groups reached 58% and 59%, respectively. The TGI of the IL2 / hu22F8wt 0.5 mg / kg group reached 63%. Statistical analysis showed that there was no significant difference in the antitumor activity between the two groups (p>0.05).

[0068] This result is consistent with the results of Example 4.3, ie, the toxicity of the IL2 / hu22F8mu complex was clearly reduced compared to IL2 / hu22F8wt.

[0069] Example 5 Use of antibody mutagenesis techniques in CD276 monoclonal antibody-IL15 bifunctional molecules Using our humanized CD276 monoclonal antibody hu147 as an example, we demonstrate that in a bifunctional molecule consisting of a CD276 monoclonal antibody and IL15, mutations in the Fc of the monoclonal antibody can reduce the toxicity of such bifunctional molecules in the body, thereby achieving better safety during application and allowing for increased dosages to achieve better therapeutic effects, a finding that is also applicable to other CD276 monoclonal antibodies.

[0070] 5.1 Preparation of the dual-function molecules hu147wt-hIL15sc and hu147mu-hIL15sc The heavy chain of humanized CD276 monoclonal antibody hu147wt (amino acid sequence shown in SEQ ID NO:29) was linked to huIL15sc (constructed as in Example 3, amino acid sequence shown in SEQ ID NO:13) via GGGGSGGGGSGGGGS to form the heavy chain of hu147wt-IL15sc (amino acid sequence shown in SEQ ID NO:30). The heavy chain of Fc-mutated humanized CD276 monoclonal antibody hu147mu (amino acid sequence shown in SEQ ID NO:31) was linked to huIL15sc via GGGSGGGGSGGGGGS to form the heavy chain of hu147mu-hIL15sc (amino acid sequence shown in SEQ ID NO:32). The amino acid sequence of the light chain of hu147wt / mu is shown in SEQ ID NO:33. They were constructed into the pcDNA3.4 expression vector, matched and co-transfected into Expi-293F cells, and purified by Protein G to obtain the dual-function molecules hu147wt-hIL15sc and hu147mu-hIL15sc, which were >95% pure, quantified, aliquoted, and stored frozen at −80°C for further use.

[0071] 5.2 In vitro biological activity of the dual-function molecule hu147mu-hIL15sc The experimental method was the same as in Example 1.2, and the biological activities of hu147mu-IL15sc and hu147wt-IL15sc were measured in a CTLL2 cell proliferation experiment.

[0072] As shown in Figure 8, both hu147mu-IL15sc and hu147wt-IL15sc were able to stimulate the proliferation of CTLL2 cells, with EC50 values ​​of 103.0 ng / ml and 91.0 ng / ml, respectively, indicating that hu147mu-IL15sc has biological activity consistent with that of hu147wt-IL15sc.

[0073] 5.3 Significantly reduced in vivo toxicity of the dual-function molecule hu147mu-hIL15sc The method is the same as in Example 1.3.

[0074] Table 5. Survival rate of experimental animals in each dose group [Table 5]

[0075] As shown in Table 5, by day 7 of the experiment, all animals in the 4.0 mg / kg and 2.0 mg / kg hu147wt-hIL15sc sample groups had died (0% survival rate), while 60% of animals in the 1 mg / kg group had died, and no animals in the 0.5 mg / kg group had died. At the highest dose of 4 mg / kg, hu147mu-hIL15sc, 70% of animals died, while no animals died in any of the other dose groups. These results indicate that the toxicity of hu147mu-hIL15sc to experimental mice was significantly reduced after Fc mutation.

[0076] 5.4 In vivo antitumor activity of the dual-function molecule hu147mu-hIL15sc The method was the same as in Example 1.3, and both hu147mu-hIL15sc and hu147wt-hIL15sc were administered at a dose of 0.5 mg / kg three times a week for a total of six administrations.

[0077] As shown in Figure 9, both hu147mu-hIL15sc and hu147wt-hIL15sc exhibited strong antitumor activity, with TGIs of 69% and 67%, respectively, which were not significantly different (p>0.05), indicating that the Fc mutation in hu147mu-hIL15sc did not affect its in vivo antitumor activity.

[0078] Example 6 Use of antibody mutagenesis techniques in CD73 monoclonal antibody-IL15 bifunctional molecules Using our humanized CD73 monoclonal antibody hu3E10 as an example, we demonstrate that in a bifunctional molecule consisting of a CD73 monoclonal antibody and IL15, mutations in the Fc of the monoclonal antibody can reduce the toxicity of such a bifunctional molecule in the body, thereby achieving better safety during application and allowing for increased dosages to achieve better therapeutic effects, a finding that is also applicable to other CD73 monoclonal antibodies.

[0079] 6.1 Preparation of the bifunctional molecules hu3E10wt-IL15sc and hu3E10mu-IL15sc The heavy chain of humanized CD73 monoclonal antibody hu3E10wt (amino acid sequence shown in SEQ ID NO: 34) was linked to huIL15sc (constructed as in Example 3, amino acid sequence shown in SEQ ID NO: 13) via GGGGSGGGGSGGGGS to form the heavy chain of hu3E10wt-IL15sc (amino acid sequence shown in SEQ ID NO: 35). The heavy chain of Fc-mutated humanized CD73 monoclonal antibody hu3E10mu (amino acid sequence shown in SEQ ID NO: 36) was linked to huIL15sc via GGGGSGGGGSGGGGS to form the heavy chain of hu3E10mu-IL15sc (amino acid sequence shown in SEQ ID NO: 37). The amino acid sequence of the light chain of hu3E10wt / mu is shown in SEQ ID NO: 38. They were constructed into the pcDNA3.4 expression vector, matched and co-transfected into Expi-293F cells, and purified by Protein G to obtain the dual-function molecules hu3E10wt-hIL15sc and hu3E10mu-hIL15sc, which were >95% pure, quantified, aliquoted, and stored frozen at −80°C for further use.

[0080] 6.2 In vitro biological activity of the dual-function molecule hu3E10mu-hIL15sc The experimental method was the same as in Example 1.2, and the biological activities of hu3E10mu-IL15sc and hu3E10wt-IL15sc were measured in a CTLL2 cell proliferation experiment.

[0081] As shown in Figure 10, both hu3E10mu-IL15sc and hu3E10wt-IL15sc were able to stimulate the proliferation of CTLL2 cells, with EC50 values ​​of 104.8 ng / ml and 99.3 ng / ml, respectively, indicating that hu3E10mu-IL15sc has biological activity consistent with that of hu3E10wt-IL15sc.

[0082] 6.3 Significantly reduced in vivo toxicity of the dual-function molecule hu3E10mu-hIL15sc The method is the same as in Example 1.3.

[0083] Table 6. Survival rate of experimental animals in each dose group [Table 6]

[0084] As shown in Table 6, by day 7 of the experiment, all animals in the 4.0 mg / kg and 2.0 mg / kg hu3E10wt-hIL15sc sample groups had died (0% survival rate), while 50% of the animals in the 1 mg / kg group had died, and no animals in the 0.5 mg / kg group had died. At the highest dose of 4 mg / kg, hu3E10mu-hIL15sc, 90% of the animals died, while no animals died in any of the other dose groups. These results indicate that the toxicity of hu3E10mu-hIL15sc to experimental mice was significantly reduced after Fc mutation.

[0085] 6.4 In vivo antitumor activity of the dual-function molecule hu3E10mu-hIL15sc The method was the same as in Example 1.3, and both hu3E10mu-hIL15sc and hu3E10wt-hIL15sc were administered at a dose of 0.5 mg / kg three times a week for a total of six administrations.

[0086] As shown in Figure 11, both hu3E10mu-hIL15sc and hu3E10wt-hIL15sc exhibited strong antitumor activity, with TGIs of 70% and 75%, respectively, which were not significantly different (p>0.05), indicating that the Fc mutation in hu3E10mu-hIL15sc did not affect its in vivo antitumor activity.

[0087] Example 7 Use of antibody mutagenesis techniques in HER2 monoclonal antibody-IL15 bifunctional molecules Using the HER2 monoclonal antibody hu19H6 we have developed as an example, we demonstrate that in a bifunctional molecule consisting of a HER2 monoclonal antibody and IL15, mutations in the Fc of the monoclonal antibody can reduce the toxicity of such a bifunctional molecule in the body, thereby achieving better safety during application and increasing the dosage used to achieve better therapeutic effects, which also applies to other HER2 monoclonal antibodies.

[0088] 7.1 Production of the bifunctional molecules hu19H6-IL15 and hu19H6mu-IL5 The heavy chain of humanized HER2 monoclonal antibody hu19H6wt (amino acid sequence shown in SEQ ID NO: 39) was linked to huIL15sc (constructed as in Example 3, amino acid sequence shown in SEQ ID NO: 13) via GGGGSGGGGSGGGGS to form the heavy chain of hu19H6wt-IL15sc (amino acid sequence shown in SEQ ID NO: 40). The heavy chain of Fc-mutated humanized HER2 monoclonal antibody hu19H6mu (amino acid sequence shown in SEQ ID NO: 41) was linked to huIL15sc via GGGGSGGGGSGGGGS to form the heavy chain of hu19H6mu-IL15sc (amino acid sequence shown in SEQ ID NO: 42). The amino acid sequence of the light chain of hu19H6wt / mu is shown in SEQ ID NO: 43. They were constructed into the pcDNA3.4 expression vector, matched and co-transfected into Expi-293F cells, and purified by Protein G to obtain the dual-function molecules hu19H6wt-hIL15sc and hu19H6mu-hIL15sc, which were >95% pure, quantified, aliquoted, and stored frozen at −80°C for further use.

[0089] 7.2 In vitro biological activity of the dual-function molecule hu19H6-IL15sc The experimental method was the same as in Example 1.2, and the biological activities of hu19H6mu-IL15sc and hu19H6wt-IL15sc were measured in a CTLL2 cell proliferation experiment.

[0090] As shown in Figure 12, both hu19H6mu-IL15sc and hu19H6wt-IL15sc were able to stimulate the proliferation of CTLL2 cells, with EC50 values ​​of 89.24 ng / ml and 90.24 ng / ml, respectively, indicating that hu19H6mu-IL15sc has biological activity consistent with that of hu19H6wt-IL15sc.

[0091] 7.3 Significantly reduced in vivo toxicity of the dual-function molecule hu19H6mu-IL5 The method is the same as in Example 1.3.

[0092] Table 7. Survival rate of experimental animals in each dose group [Table 7]

[0093] As shown in Table 7, by day 7 of the experiment, all animals in the 4.0 mg / kg group administered hu19H6wt-hIL15sc sample died, 90% of animals in the 2.0 mg / kg group died (survival rate: 10%), 20% of animals in the 1 mg / kg group died, and no animals died in the 0.5 mg / kg group. At the highest dose of 4 mg / kg, hu19H6mu-hIL15sc, 80% of animals died, while no animals died in any of the other dose groups. These results indicate that the toxicity of hu19H6mu-hIL15sc to experimental mice was significantly reduced after Fc mutation.

[0094] 7.4 In vivo antitumor activity of the dual-function molecule hu19H6mu-hIL15sc The method was the same as in Example 1.3, and both hu19H6mu-hIL15sc and hu19H6wt-hILsc5 were administered at a dose of 0.5 mg / kg three times a week for a total of six times.

[0095] As shown in Figure 13, both hu19H6mu-hIL15sc and hu19H6wt-hIL15sc exhibited strong antitumor activity, with TGIs of 93% and 95%, respectively, which were not significantly different (p>0.05), indicating that the Fc mutation in hu19H6mu-hIL15sc did not affect in vivo antitumor activity.

[0096] Example 8 Use of antibody mutagenesis techniques in PD1 monoclonal antibody-IL15 bifunctional molecules Using the PD1 monoclonal antibody hu609 we have developed as an example, we demonstrate that in a bifunctional molecule consisting of a PD1 monoclonal antibody and IL15, mutations in the Fc of the monoclonal antibody can reduce the toxicity of such a bifunctional molecule in the body, thereby achieving better safety during application and increasing the dosage used, thereby achieving better therapeutic effects.

[0097] 8.1 Production of the dual-function molecules hu609-IL15 and hu609mu-IL15 The heavy chain of humanized PD1 monoclonal antibody hu609wt (amino acid sequence shown in SEQ ID NO: 44) was linked to huIL15sc (constructed as in Example 3, amino acid sequence shown in SEQ ID NO: 13) via GGGGSGGGGGSGGGG to form the heavy chain of hu609wt-IL15sc (amino acid sequence shown in SEQ ID NO: 45). The heavy chain of Fc-mutated humanized PD1 monoclonal antibody hu609mu (amino acid sequence shown in SEQ ID NO: 46) was linked to huIL15sc via GGGGSGGGGGSGGGG to form the heavy chain of hu609mu-IL15sc (amino acid sequence shown in SEQ ID NO: 47). The amino acid sequence of the light chain of hu609wt / mu is shown in SEQ ID NO: 48. They were constructed into the pcDNA3.4 expression vector, matched and co-transfected into Expi-293F cells, and purified by Protein G to obtain the dual-function molecules hu609wt-hIL15sc and hu609mu-hIL15sc, which were >95% pure, quantified, aliquoted, and stored frozen at −80°C for further use.

[0098] 8.2 In vitro biological activity of the dual-function molecule hu609mu-IL15sc The experimental method was the same as in Example 1.2, and the biological activities of hu609mu-IL15sc and hu609wt-IL15sc were measured in a CTLL2 cell proliferation experiment.

[0099] As shown in Figure 14, both hu609mu-IL15sc and hu609wt-IL15sc were able to stimulate the proliferation of CTLL2 cells, with EC50 values ​​of 85.11 ng / ml and 81.33 ng / ml, respectively, indicating that hu609mu-IL15sc has biological activity consistent with that of hu609wt-IL15sc.

[0100] 8.3 Significantly reduced in vivo toxicity of the dual-function molecule hu609mu-IL15sc The method is the same as in Example 1.3.

[0101] Table 8. Survival rate of experimental animals in each dose group [Table 8]

[0102] As a result, as shown in Table 8, on the 7th day of the experiment, all experimental animals died in the 4.0 mg / kg and 2.0 mg / kg groups of hu609wt-hIL15sc sample, while 40% of the experimental animals died in the 1 mg / kg dose group, and no experimental animals died in the 0.5 mg / kg dose group.

[0103] The highest dose of hu19H6mu-hIL15sc (4 mg / kg) resulted in 90% mortality, while no other doses resulted in mortality. These results indicate that the toxicity of hu609mu-hIL15sc to experimental mice was significantly reduced after Fc mutation.

[0104] 8.4 In vivo antitumor activity of the dual-function molecule hu609mu-IL15sc The method was the same as in Example 1.3, and both hu609mu-IL15sc and hu609-IL15sc were administered at a dose of 0.5 mg / kg three times a week for a total of six administrations.

[0105] As shown in Figure 15, both hu609mu-hIL15sc and hu609-hIL15sc exhibited strong antitumor activity, with TGIs of 89.6% and 89.3%, respectively, which were not significantly different (p>0.05), indicating that the Fc-mutated hu609mu-IL5 did not affect in vivo antitumor activity.

[0106] Example 9 Use of antibody mutagenesis technology in HER3 monoclonal antibody-IL15sc bifunctional molecules Using the HER3 chimeric monoclonal antibody ch158 we have developed as an example, we demonstrate that in a bifunctional molecule consisting of a HER3 monoclonal antibody and IL15, mutations in the monoclonal antibody Fc can reduce the toxicity of such bifunctional molecules in the body, thereby achieving better safety during application and increasing the dosage used to achieve better therapeutic effects, which also applies to other HER3 monoclonal antibodies.

[0107] 9.1 Preparation of the dual-function molecules ch158-IL15sc and ch158mu-IL15sc The heavy chain of HER3 monoclonal antibody ch158wt (amino acid sequence shown in SEQ ID NO: 49) was linked to huIL15sc (constructed similarly to Example 3, amino acid sequence shown in SEQ ID NO: 13) via GGGGSGGGGGSGGGG to form the heavy chain of ch158wt-IL15sc (amino acid sequence shown in SEQ ID NO: 50). The heavy chain of Fc mutant HER3 monoclonal antibody ch158mu (amino acid sequence shown in SEQ ID NO: 51) was linked to huIL15sc via GGGGSGGGGGSGGGG to form the heavy chain of ch158mu-IL15sc (amino acid sequence shown in SEQ ID NO: 52). The amino acid sequence of the light chain of ch158wt / mu is shown in SEQ ID NO: 53. They were constructed into the pcDNA3.4 expression vector, matched and co-transfected into Expi-293F cells, and purified by Protein G to obtain the dual-function molecules ch158wt-hIL15sc and ch158mu-hIL15sc, which were >95% pure, quantified, aliquoted, and stored frozen at −80°C for further use.

[0108] 9.2 In vitro biological activity of the dual-function molecule ch158mu-IL15sc The experimental method was the same as in Example 1.2, and the biological activities of ch158mu-IL15sc and ch158wt-IL15sc were measured in a CTLL2 cell proliferation experiment.

[0109] As shown in Figure 16, both ch158mu-IL15sc and ch158wt-IL15sc were able to stimulate the proliferation of CTLL2 cells, with EC50 values ​​of 78.05 ng / ml and 84.65 ng / ml, respectively, indicating that ch158mu-IL15sc has biological activity consistent with that of ch158wt-IL15sc.

[0110] 9.3 Significantly reduced in vivo toxicity of the dual-function molecule ch158mu-IL15sc The method is the same as in Example 1.3.

[0111] Table 9. Survival rate of experimental animals in each dose group [Table 9]

[0112] As a result, as shown in Table 9, on the 7th day of the experiment, all experimental animals died in the 4.0 mg / kg and 2.0 mg / kg groups of ch158wt-hIL15sc sample, 40% of the experimental animals died in the 1 mg / kg dosage group, and no experimental animals died in the 0.5 mg / kg dosage group.

[0113] The highest dose of ch158mu-hIL15sc (4 mg / kg) resulted in 90% deaths, while no other doses resulted in deaths. These results indicate that the toxicity of ch158mu-hIL15sc to experimental mice was significantly reduced after Fc mutation.

[0114] 9.4 In vivo antitumor activity of the dual-function molecule ch158mu-hIL15sc The method was the same as in Example 1.3, and both ch158mu-IhL5sc and ch158wt-hILsc5 were administered at a dose of 0.5 mg / kg three times a week for a total of six administrations.

[0115] As shown in Figure 17, both ch158mu-IL15sc and ch158wt-IhL5sc exhibited strong antitumor activity, with TGIs of 72.9% and 72.7%, respectively, which were not significantly different (p>0.05), indicating that the Fc mutation in ch158mu-hIL15sc did not affect its in vivo antitumor activity.

[0116] Example 10: Other tumor-targeting Fc mutant antibodies - IL15sc bifunctional molecules The bifunctional molecules consisting of the above-mentioned Fc mutant antibodies and IL15 family include antibodies related to tumor therapy, such as IL2, PD1, HER2, CD73, CD276, and HER3, and such bifunctional molecules consisting of Fc mutant antibodies and IL15 family maintained antitumor activity while significantly reducing toxicity and side effects in the body.

[0117] The application of Fc mutations in this respect is not limited to antibodies related to the above antigens, but can also be applied to antibodies corresponding to other antigens, such as EGFR, PDL1, CD19, CD20, CD22, CD33, VEGFR, TIGIT, TIM3, LAG3, CXCR3, CXCR5, CCR3, CCR4, CCR9, CD40, CD40L, and CD24. Dual-function molecules consisting of these antibodies and IL15 can also achieve the goal of reducing toxicity and side effects through such Fc mutations.

[0118] Taking Rituximab as an example, the amino acid sequence of the heavy chain of a dual-function molecule consisting of an Fc-mutated CD20 antibody and IL15 is shown in SEQ ID NO: 54, and the amino acid sequence of the light chain is shown in SEQ ID NO: 55.

[0119] Taking cetuximab as an example, the amino acid sequence of the heavy chain of a dual-function molecule consisting of an Fc-mutated EGFR antibody and IL15 is shown in SEQ ID NO: 56, and the amino acid sequence of the light chain is shown in SEQ ID NO: 57.

[0120] Taking atezolizumab, avelumab, and durvalumab as examples, the dual-function molecules consisting of an Fc-mutated PDL1 antibody and IL15 have heavy chain amino acid sequences shown in SEQ ID NOs: 58 / 60 / 62, respectively, and light chain amino acid sequences shown in SEQ ID NOs: 59 / 61 / 63, respectively.

[0121] Taking daratumumab as an example, the amino acid sequence of the heavy chain of the dual-function molecule consisting of an Fc-mutated CD38 antibody and IL15 is shown in SEQ ID NO: 64, and the amino acid sequence of the light chain is shown in SEQ ID NO: 65.

[0122] Taking vedolizumab as an example, the amino acid sequence of the heavy chain of the dual-function molecule consisting of an Fc-mutated integrin α4β7 antibody and IL15 is shown in SEQ ID NO: 66, and the amino acid sequence of the light chain is shown in SEQ ID NO: 67.

[0123] Taking alemtuzumab as an example, the amino acid sequence of the heavy chain of a dual-function molecule consisting of an Fc-mutated CD52 antibody and IL15 is shown in SEQ ID NO: 68, and the amino acid sequence of the light chain is shown in SEQ ID NO: 69.

[0124] Taking Mogamulizumab as an example, the amino acid sequence of the heavy chain of this dual-function molecule consisting of an Fc-mutated CCR4 antibody and IL15 is shown in SEQ ID NO: 70, and the amino acid sequence of the light chain is shown in SEQ ID NO: 71.

[0125] Taking Daclizumab and Basiliximab as examples, the amino acid sequences of the heavy chains of dual-function molecules consisting of an Fc-mutated CD25 antibody and IL15 are shown in SEQ ID NOs: 72 / 74, respectively, and the amino acid sequences of the light chains are shown in SEQ ID NOs: 73 / 75, respectively.

[0126] Taking gemtuzumab as an example, the amino acid sequence of the heavy chain of the dual-function molecule consisting of an Fc-mutated CD33 antibody and IL15 is shown in SEQ ID NO: 76, and the amino acid sequence of the light chain is shown in SEQ ID NO: 77.

[0127] Taking ipilimumab as an example, the amino acid sequence of the heavy chain of a dual-function molecule consisting of an Fc-mutated CTLA4 antibody and IL15 is shown in SEQ ID NO: 78, and the amino acid sequence of the light chain is shown in SEQ ID NO: 79.

[0128] Taking the antibody described in Patent US 10,479,838B2 as an example, the amino acid sequence of the heavy chain of the dual-function molecule consisting of an Fc-mutated CD40 antibody and IL15 is shown in SEQ ID NO: 80, and the amino acid sequence of the light chain is shown in SEQ ID NO: 81.

[0129] Taking the antibody described in Patent US20180280506A1 as an example, the dual-function molecule consisting of an Fc-mutated TIGIT antibody and IL15 has a heavy chain amino acid sequence shown in SEQ ID NO: 82 and a light chain amino acid sequence shown in SEQ ID NO: 83.

[0130] Taking the antibody described in Patent US 10,465,010 B2 as an example, the dual-function molecule consisting of Fc-mutated GITR antibody and IL15 has a heavy chain amino acid sequence shown in SEQ ID NO: 84 and a light chain amino acid sequence shown in SEQ ID NO: 85.

[0131] Taking the antibody described in Patent US20190284277A1 as an example, the dual-function molecule consisting of an Fc-mutated LAG3 antibody and IL15 has a heavy chain amino acid sequence shown in SEQ ID NO: 86 and a light chain amino acid sequence shown in SEQ ID NO: 87.

[0132] Taking the antibody described in patent US20070148739A1 as an example, the dual-function molecule consisting of an Fc-mutated CD138 antibody and IL15 has a heavy chain amino acid sequence shown in SEQ ID NO: 88 and a light chain amino acid sequence shown in SEQ ID NO: 89.

[0133] Example 11 Other Fc mutations that reduce antibody binding to FcRn The above-mentioned method of reducing the in vivo toxicity of a bifunctional antibody-IL15 molecule by Fc mutation is achieved by reducing the binding of the bifunctional molecule to FcRn through Fc mutation. In the examples, one Fc mutation method, namely, H310A / H435Q mutation, was used, but other mutations that reduce antibody-FcRn binding can also achieve the same purpose. These Fc mutations include an I253A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 90 / 104 / 117), an S254A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 91 / 105 / 118), an R255A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 92 / 106 / 119), a K288A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 93 / 107 / 120), an L309A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 94 / 121), an H310A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 95 / 108 / 122), an S415A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 96 / 109 / 123), an H433A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 97 / 109 / 124), an H510A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 98 / 109 / 125), an H610A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 99 / 100 / 110), an H710A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 101 / 102 / 113), an H810A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 102 / 103 / 114), an H910A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: 103 / 104 / 115), an H911A mutation (the amino acid sequences of the Fc are shown in SEQ ID NOs: These include, but are not limited to, H435A mutation (the amino acid sequences of the Fc are set forth in SEQ ID NOs: 97 / 110 / 124), H435A mutation (the amino acid sequences of the Fc are set forth in SEQ ID NOs: 98 / 111 / 125), H435R mutation (the amino acid sequences of the Fc are set forth in SEQ ID NOs: 99 / 112 / 126), Y436A mutation (the amino acid sequences of the Fc are set forth in SEQ ID NOs: 100 / 113 / 127), H310Q / H433N mutation (the amino acid sequences of the Fc are set forth in SEQ ID NOs: 101 / 114 / 128), M252Y / T256Q mutation (the amino acid sequences of the Fc are set forth in SEQ ID NOs: 102 / 115 / 129), M252F / T256D mutation (the amino acid sequences of the Fc are set forth in SEQ ID NOs: 103 / 116 / 130), etc.

[0134] Example 12: Dual-function molecules consisting of Fc mutant antibodies and cytokines other than IL15 In the above examples, we used bifunctional molecules consisting of IL15 and Fc mutant antibodies as examples to demonstrate that Fc mutations that reduce FcRn binding significantly reduce the toxicity of the bifunctional molecules in vivo. Replacing IL15 in these bifunctional molecules with other cytokines also achieves the same effect, including but not limited to:

[0135] IL2 (amino acid sequence shown in SEQ ID NO: 1) and its various mutants (amino acid sequences shown in SEQ ID NOs: 2 / 3 / 4); IL12 (amino acid sequences shown in SEQ ID NOs: 5 / 7 / 9) and variants thereof (amino acid sequences shown in SEQ ID NOs: 131 / 132); Neo 2 / 15 (amino acid sequence shown in SEQ ID NO: 133); IL7 (amino acid sequence shown in SEQ ID NO: 134); IL18 (amino acid sequence shown in SEQ ID NO: 135); IL21 (amino acid sequence shown in SEQ ID NO: 136); IL2-CD25 (amino acid sequences shown in SEQ ID NOs: 137 / 138 / 139); IFNα (amino acid sequence shown in SEQ ID NO: 140); IFNα2b (the amino acid sequence of which is shown in SEQ ID NO: 141) and variants thereof (the amino acid sequence of which is shown in SEQ ID NO: 142); IFNγ (amino acid sequence shown in SEQ ID NO: 143); TNFα (amino acid sequence shown in SEQ ID NO: 144); GM-CSF (amino acid sequence shown in SEQ ID NO: 145); Flt3 (amino acid sequence shown in SEQ ID NO: 146); CCL21 (amino acid sequence shown in SEQ ID NO: 147).

[0136] Although the present invention has been described in detail in the above embodiments, these are only some of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive steps based on these embodiments, and all of these embodiments fall within the scope of protection of the present invention.

Claims

1. A bifunctional molecule characterized by linking an Fc mutant antibody and a cytokine.

2. The Fc mutant comprises the following fragment: The dual functional molecule of claim 1, characterized in that it is selected from H310A / H435Q, I253A, S254A, R255A, K288A, L309A, H310A, S415A, H433A, H435A, H435R, Y436A, H310Q / H433N, M252Y / T256Q, and M252F / T256D.

3. The antibody The dual function molecule of claim 1, characterized in that it is selected from IgG1, IgG4, HER2, HER3, EGFR, PDL1, CD19, CD20, CD22, CD24, CD33, CD40, CD40L, CD73, CD276, VEGFR, TIGIT, TIM3, LAG3, CXCR3, CXCR5, CCR3, CCR4, CCR9, and PD1.

4. The cytokine is The dual function molecule of claim 1, wherein the dual function molecule is selected from IL2 and mutants thereof, IL7, IL12 and mutants thereof, IL15, IL18, IL21, IL2-CD25, Neo 2 / 15, IFNα, IFNα2b and mutants thereof, IFNγ, TNFα, GM-CSF, Flt3, and CCL21.

5. A method for amplifying a dual-function molecule described in any one of claims 1 to 4, characterized in that the amplification method comprises linking a cytokine and an Fc mutant antibody to form a fusion protein, constructing the fusion protein in an expression vector, transfecting it into cells, and purifying it.

6. A therapeutic antibody drug comprising a dual function molecule according to any one of claims 1 to 4.

7. Use of a dual function molecule according to any one of claims 1 to 4 in the manufacture of a medicament.

8. 8. The use according to claim 7, wherein the drug is a therapeutic antibody drug.

Images