A cytokine composition for immunotherapy and a method for preparing the same

Abstract

The application discloses a targeted cytokine composition for immunotherapy and a preparation method thereof. The fusion protein comprises, from N-terminal to C-terminal, an anti-PD-L1 single-domain antibody, a hinge region, a double-mutated human IL-15, a hinge region, an anti-NKG2D single-domain antibody and a double-mutated human IgG1 Fc fragment. In vitro experiments show that the fusion protein can simultaneously bind to double targets, significantly promotes NKG2D-positive CD8-positive T cell proliferation and has weak effect on regulatory T cells, and presents synergistic effect in a tumor-immune cell co-culture system. In vivo pharmacodynamic research shows that in a humanized mouse model, the fusion protein significantly inhibits the growth of colon cancer, the number of CD8-positive T cell and NK cell infiltrations in the tumor is obviously increased, and the effect is better than that of non-targeting and single-targeting controls. The fusion protein provided by the application has long-acting circulation, tumor targeting enrichment and immune cell subpopulation precise regulation functions, and can be used for preparing an antitumor drug.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and immunotherapy, and specifically relates to a cytokine composition for immunotherapy and its preparation method. Background Technology

[0002] Cytokines play a central mediating role in the regulation of the body's immune response. Among them, interleukin-15 (IL-15) is a key cytokine regulating the proliferation, survival, and effector function of natural killer (NK) cells and CD8+ T cells, playing a crucial role in anti-tumor immune responses. IL-15 exerts its biological function through its unique trans-presentation mechanism: it first binds to the IL-15 receptor α (IL-15Rα) on the surface of antigen-presenting cells to form a complex. This complex is then further presented to the IL-2 / 15Rβγc receptor complex on the surface of neighboring NK cells or T cells, thereby activating downstream signaling pathways and initiating immune effects. Compared to interleukin-2 (IL-2), IL-15 does not preferentially proliferate regulatory T cells (Tregs), effectively avoiding the inhibitory effect of Tregs on anti-tumor immune responses, and is therefore considered to have greater potential for anti-tumor applications.

[0003] However, IL-15 faces numerous bottlenecks in its clinical translation, severely limiting its therapeutic efficacy. On the one hand, natural IL-15 is metabolized rapidly in vivo with an extremely short half-life, making it difficult to maintain effective therapeutic concentrations and resulting in limited duration of efficacy. On the other hand, systemic administration of IL-15 easily triggers non-specific immune activation, producing dose-limiting toxicities such as fever and cytokine storms, greatly restricting its clinical dosage and therapeutic safety. To overcome these technical limitations, researchers in this field have developed various IL-15 modification strategies, mainly including: fusing IL-15 with the sushi domain of IL-15Rα, fusing with antibody Fc fragments to prolong its in vivo half-life; and fusing IL-15 with targeted antibody fragments to construct targeted immune cytokines. However, existing modification technologies still have significant shortcomings: traditional antibody-cytokine fusion proteins have large molecular weights, making it difficult to penetrate the tumor tissue barrier, resulting in low enrichment efficiency at the tumor site; and simple Fc fragment fusion strategies can only prolong the half-life of IL-15, failing to address the toxicity problem caused by its systemic non-specific activation. Therefore, how to prolong the in vivo half-life of IL-15 while achieving precise targeted delivery to tumor sites and reducing systemic toxicity remains a key technical challenge that urgently needs to be addressed in this field.

[0004] In recent years, obtaining IL-15 mutants with altered affinity for IL-15Rα through the introduction of specific amino acid mutations has become a research hotspot in the field of IL-15 modification. Among them, the N72D mutation is a proven effective mutation type. This mutation can significantly enhance the affinity of IL-15 for the IL-2 / 15Rβγc receptor complex, while reducing the dependence of IL-15 on the trans-presentation of IL-15Rα. This allows IL-15 to effectively activate effector immune cells only under local high-concentration conditions, thereby significantly reducing the toxic risks associated with systemic immune activation. This modification strategy provides a feasible approach for achieving "cis-targeted" activation of cytokines.

[0005] Single-domain antibodies (VHHs, also known as nanobodies) have become ideal targeted delivery vectors due to their advantages such as small molecular weight (approximately 15 kDa), high thermal and pH stability, ease of genetic engineering, low in vivo immunogenicity, and strong tissue penetration. Previous studies have confirmed that high-affinity VHHs targeting tumor-associated antigens (such as PD-L1) and immune cell activation receptors (such as NKG2D) can be screened from large-capacity antibody libraries using phage display technology. Based on this, combining VHHs targeting tumor tissues with VHHs targeting effector immune cells into an IL-15 mutant to construct a dual-targeting fusion protein holds promise for precisely enriching the biological activity of IL-15 on the surface of effector immune cells in the tumor microenvironment, significantly enhancing the anti-tumor immune response while minimizing its systemic toxicity.

[0006] Based on the above technical background, this invention fuses VHH targeting PD-L1 and VHH targeting NKG2D with IL-15 mutants and Fc fragments to construct a cytokine composition that combines long-acting in vivo circulation, dual-target enrichment, and precise regulation of immune cell subsets. Through systematic in vitro and in vivo animal experiments, the synergistic anti-tumor effect of this composition has been verified, providing a new technical solution for the clinical translation of IL-15. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention provides a fusion protein, which comprises the following elements sequentially from the amino terminus to the carboxyl terminus:

[0008] The first single-domain antibody specifically recognizes the tumor-associated antigen PD-L1. The high-affinity anti-PD-L1 single-domain antibody P07, obtained through phage display library screening, has the amino acid sequence shown in SEQ ID NO:2 and can effectively bind to PD-L1 and block the interaction between PD-1 and PD-L1.

[0009] The first hinge region serves as a linker peptide to maintain the spatial conformation and flexibility of each functional domain. This hinge region is composed of glycine and serine, specifically (GGGGS)3, whose amino acid sequence is shown in SEQ ID NO:3.

[0010] A mutant human IL-15 contains N72D and N79D double amino acid substitutions. The N72D mutation enhances the binding affinity of IL-15 to the IL-2 / 15Rβ receptor while reducing its dependence on the IL-15Rα receptor; the N79D mutation removes the N-glycosylation modification at this site, reducing protein glycosylation heterogeneity and improving protein expression stability. The amino acid sequence of this mutant IL-15 is shown in SEQ ID NO:1.

[0011] The second hinge region has the same composition as the first hinge region, and its amino acid sequence is shown in SEQ ID NO:3.

[0012] The second single-domain antibody specifically recognizes the NKG2D activation receptor on the surface of immune cells. The high-affinity anti-NKG2D single-domain antibody N05, obtained through phage display library screening, has the amino acid sequence shown in SEQ ID NO:4 and can effectively bind to the NKG2D receptor on the surface of NK cells and activated CD8-positive T cells.

[0013] An immunoglobulin Fc fragment derived from human IgG1, with double mutations of L14A and L15A introduced. The LALA mutation significantly reduces the binding affinity of the Fc fragment to the Fcγ receptor, thereby reducing non-specific immune activation such as antibody-dependent cell-mediated cytotoxicity. The amino acid sequence of this Fc fragment is shown in SEQ ID NO:5.

[0014] The present invention also provides a nucleic acid molecule encoding the above-mentioned fusion protein, the nucleotide sequence of which is shown in SEQ ID NO:6. The present invention further provides a recombinant expression vector containing the nucleic acid molecule, and a host cell containing the expression vector.

[0015] The present invention also provides pharmaceutical compositions comprising the above-described fusion protein and a pharmaceutically acceptable carrier, and the use of the fusion protein in the preparation of a medicament for treating cancer, preferably a solid tumor expressing PD-L1, including but not limited to colon cancer, non-small cell lung cancer and melanoma.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] First, this invention successfully obtained high-affinity anti-PD-L1 single-domain antibody P07 and anti-NKG2D single-domain antibody N05 by constructing a large-capacity natural VHH phage display library and conducting four rounds of biological panning and functional identification. SPR assay results showed that the KD value of P07 binding to human PD-L1 was 1.2 ± 0.2 nM, and the KD value of N05 binding to human NKG2D was 3.5 ± 0.4 nM. After fusing the two single-domain antibodies with an IL-15 mutant and an Fc fragment, the affinity of the fusion protein for PD-L1 and NKG2D was essentially the same as that of the individual single-domain antibodies, indicating that the construction of the fusion protein did not affect the antigen-binding activity of the two single-domain antibodies, enabling them to simultaneously and specifically bind to PD-L1 on the surface of tumor cells and NKG2D on the surface of effector immune cells.

[0018] Second, this invention employs N72D and N79D double mutant IL-15. Functional validation was performed on the IL-15Rα-deficient KT-3 cell line. The results showed that the EC50 value of wild-type IL-15 / Fc was 0.8±0.1 ng / mL, while the EC50 value of mutant IL-15 / Fc was 25.6±2.5 ng / mL, indicating that the mutant IL-15 has a significantly reduced dependence on IL-15Rα. This characteristic means that the IL-15 mutant can only effectively activate effector cells at locally high concentrations, laying the foundation for subsequent enrichment of the tumor microenvironment through dual-target anchoring.

[0019] Third, in the selective proliferation assay of human primary immune cell subsets, the proliferation index of the fusion protein of this invention on NKG2D-positive CD8-positive T cells was 3.8±0.3, significantly higher than that of the wild-type IL-15 / Fc treatment group (2.2±0.2); while the proliferation index on CD4-positive FoxP3-positive regulatory T cells was 1.2±0.1, significantly lower than that of the wild-type IL-15 / Fc treatment group (2.8±0.3). This result indicates that the fusion protein of this invention can specifically promote the proliferation of effector immune cells while having a weak effect on suppressor immune cells, achieving precise regulation of immune cell subsets.

[0020] Fourth, the synergistic effect of dual targeting was verified in a tumor cell and immune cell co-culture system. Results showed that the proliferation rate of NKG2D-positive CD8-positive T cells in the dual-targeting fusion protein treatment group was 64.5±5.2%, and the IFN-γ production was 1850±150 pg / mL, both significantly higher than the single-targeting PD-L1 group, the single-targeting NKG2D group, and the non-targeting control group, while there were no significant differences among the three control groups. This result confirms that only when anti-PD-L1 single-domain antibody and anti-NKG2D single-domain antibody are present simultaneously can IL-15 signaling be effectively enriched at the interface between tumor cells and immune effector cells, exerting a synergistic effect.

[0021] Fifth, the in vivo antitumor effect was evaluated in an NSG humanized mouse model reconstituted from CD34-positive hematopoietic stem cells. After administration, the average tumor volume of the dual-targeting fusion protein group of this invention was 310±70 mm. 3 The value was significantly smaller than that of the PBS control group (1480±230 mm). 3 1020±180mm in the non-targeted group 3 890±150mm in the single-target PD-L1 group 3 The 920±160mm of the single-target NKG2D group 3 The tumor inhibition rate reached 78.9%. Immunohistochemical analysis showed that the CD8-positive T cell infiltration density in the tumor of the dual-targeted group was 85.5 ± 12.5 cells / mm². 2 The NK cell infiltration density was 35.5 ± 6.5 cells / mm². 2 The levels were significantly higher than those in the control groups, indicating that the fusion protein can effectively recruit effector immune cells to the tumor site.

[0022] Sixth, this invention introduces a human IgG1 Fc fragment containing the LALA mutation, which can prolong the in vivo half-life of the fusion protein through an FcRn-mediated recycling mechanism and reduce FcγR binding capacity to decrease non-specific immune activation. During in vivo experiments, no significant decrease in body weight was observed in any group of mice, and no obvious toxic reactions were observed, indicating that the fusion protein of this invention has good safety at therapeutic doses.

[0023] In summary, this invention provides a cytokine fusion protein that combines long-acting circulation, dual-target enrichment, and precise regulation of immune cell subsets. While maintaining strong anti-tumor activity, it reduces the risk of systemic toxicity and has clear clinical application prospects and industrial transformation value. Attached Figure Description

[0024] Figure 1 SDS-PAGE results of the fusion protein.

[0025] Figure 2 Functional validation results of dual-target fusion protein in co-culture system. Detailed Implementation

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0028] Example 1: Screening, identification, and construction of fusion protein expression vectors for single-domain antibodies

[0029] 1.1 Construction of Phage Display Library

[0030] To obtain high-affinity anti-PD-L1 VHH and anti-NKG2D VHH, this study constructed a large-capacity natural VHH phage display library. The specific procedures are as follows:

[0031] Peripheral blood samples from an alpaca immunological library were used. Peripheral blood B lymphocytes were isolated using density gradient centrifugation. Total RNA was extracted from the cells using TRIzol reagent, and the RNA was reverse transcribed into cDNA strictly according to the reverse transcription kit instructions. Nested PCR was used to amplify the VHH coding sequence. The first round of PCR used cDNA as a template, with primers CALL001 (5'-gtcctgctgcttctacaagg-3') and CALL002 (5'-ggtacgtgctgttgaactgttcc-3'). Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 45 s, 30 cycles; final extension at 72℃ for 10 min. The first round of PCR products were analyzed by agarose gel electrophoresis, revealing two bands of approximately 900 bp and 600 bp. The 600 bp band was the target fragment of VHH-CH2, which was recovered from the gel and used as the template for the second round of PCR. The second round of PCR used the recovered 600 bp fragment as a template, with primers VHH-Back (5'-gatgtgcagctgcaggagtctggrggagg-3', forward primer) and VHH-For (5'-ctagtgcggccgctggagacggtgacctgggt-3', reverse primer). Reaction conditions were adjusted to an annealing temperature of 60℃, and the number of cycles was reduced to 12-14 to minimize mutations; otherwise, the process was the same as the first round of PCR. The second round PCR product was approximately 400 bp, representing the complete VHH coding sequence.

[0032] Overlap extension PCR was used to transplant the naturally derived CDR1, CDR2, and CDR3 variable region sequences into a codon-optimized, thermostable, and anti-aggregation VHH scaffold. The optimized scaffold significantly improved the soluble expression efficiency of the subsequent VHH protein. The amplified VHH fragment was digested with Sfi I restriction endonuclease at 37°C for 4 h. The digestion product was purified by agarose gel electrophoresis and cloned into the phage vector pMECS, which had undergone the same digestion treatment. The ligation product was then electroporated into E. coli TG1 competent cells (electroporation conditions: 1.8 kV, 200 Ω, 25 μF).

[0033] Transformed bacteria were plated on LB agar containing ampicillin (100 μg / mL) and incubated overnight at 37°C. Single colonies were picked and expanded to construct an initial VHH phage display library. The library size was determined to be 2.5 × 10^10 using the plate count method. Twenty single colonies were randomly selected for Sanger sequencing. The sequencing results were compared and analyzed using DNAMAN software, confirming that the VHH sequences in the library had good diversity and no obvious sequence bias, and could be used for subsequent affinity screening.

[0034] 1.2 Screening and Identification of Anti-PD-L1 VHH

[0035] Using recombinant human PD-L1-Fc fusion protein (MCE, catalog number: HY-P73717) as the target, the constructed VHH phage display library was subjected to four rounds of biological panning to gradually improve the screening specificity. The specific procedure was as follows: PD-L1-Fc protein was diluted to 2 μg / mL with coating buffer (0.05M NaHCO3, pH 9.6), and 100 μL was added to each well of a 96-well microplate. The plate was incubated overnight at 4°C. The coating buffer was discarded, and the plate was washed three times with PBST buffer (PBS + 0.1% Tween-20). 200 μL of 5% skim milk powder (prepared with PBS) was added to each well, and the plate was blocked at 37°C for 1 h. The blocking buffer was discarded, and the plate was washed three times. The phage library (10^12 cells in the first round) was then added. PFU), incubate at 37℃ for 2 h; wash unbound phages with PBST (wash 5 times in rounds 1-2, wash 10 times in rounds 3-4, and increase the Tween-20 concentration in PBST to 0.5% in round 4), elute specifically bound phages with 100 mM triethylamine, and immediately neutralize to neutral with an equal volume of 1 M Tris-HCl (pH 7.4); infect logarithmic-phase Escherichia coli TG1 with the eluted phages, culture at 37℃ with shaking for 1 h, spread on LB-ampicillin plates, culture overnight, collect bacterial colonies, and extract phage particles for the next round of panning.

[0036] After the fourth round of selection, 96 single clones were randomly selected and inoculated into LB-ampicin liquid medium. They were cultured at 37°C with shaking until OD600≈0.6, then IPTG was added to a final concentration of 1mM to induce VHH protein expression, and cultured for another 4 hours. Positive clones were identified using a phage ELISA method: 2 μg / mL PD-L1-Fc was coated onto the ELISA plate and incubated overnight at 4°C; after blocking, culture supernatant (containing phage-VHH fusion protein) was added, and incubation was performed at 37°C for 1 hour; after washing, HRP-labeled anti-M13 phage antibody was added, and incubation was performed at 37°C for 30 minutes; TMB chromogenic solution was used for color development for 15 minutes, and the reaction was terminated with 2M H2SO4. The OD450 value was measured using an ELISA reader. BSA (2 μg / mL) coated wells were used as negative controls. An OD450 / negative control ratio >5 was set as the criterion for positive clones, resulting in 42 positive clones.

[0037] Forty-two positive clones were sequenced, and after removing repetitive sequences, 16 unique VHH sequences (named P01-P16) were obtained. Each VHH coding sequence was cloned into the prokaryotic expression vector pET-22b(+) to construct a recombinant expression plasmid, which was then transformed into *E. coli* BL21(DE3) competent cells. Positive clones were selected, cultured in LB-ampicin liquid medium, and induced with IPTG. The cells were then collected, sonicated, and the supernatant was obtained. The VHH protein was purified using a Ni-NTA affinity chromatography column (elution buffer: 50 mM Tris-HCl, 500 mM imidazole, pH 8.0). SDS-PAGE electrophoresis was used to identify protein purity, and the results showed that all VHH proteins had a purity >90%, suitable for subsequent affinity assays.

[0038] The affinity of each VHH protein for human PD-L1 was determined using SPR technology: Human PD-L1 protein was immobilized on the surface of a CM5 chip via amino-coupling, with an immobilization level of approximately 1000 RU; the mobile phase was PBS (pH 7.4) at a flow rate of 30 μL / min; VHH protein was serially diluted to 0 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, and 40 nM, and sequentially flowed through the chip, with a binding time of 120 s and a dissociation time of 300 s; the chip regeneration buffer was 10 mM Gly-HCl (pH 2.0), with each regeneration lasting 10 s. The kinetic curves were fitted using a 1:1 binding model using Biacore Evaluation software, and the dissociation constant (KD), binding rate constant (kon), and dissociation rate constant (koff) were calculated.

[0039] Simultaneously, a competitive ELISA method was used to detect the ability of each VHH to block PD-1 / PD-L1 interaction: ELISA plates were coated with 2 μg / mL PD-L1-Fc and incubated overnight at 4°C; after blocking, a mixture of biotin-labeled PD-1-Fc (100 ng / mL) and serially diluted VHH protein (0-1000 nM) was added and incubated at room temperature for 1 h; after washing, HRP-labeled streptavidin was added and incubated at room temperature for 30 min; after TMB color development, the OD450 value was measured, and the half-maximal inhibitory concentration (IC50) was calculated.

[0040] The results are shown in Table 1. The KD values ​​of the 16 VHHs with human PD-L1 ranged from 0.7 nM to 85 nM. Among them, VHHs PO3, PO7, and P12 showed the highest affinity (KD < 5 nM) and could effectively block the binding of PD-1 to PD-L1 (IC50 values ​​were 0.8 nM, 0.5 nM, and 1.2 nM, respectively). Considering affinity, PD-1 / PD-L1 blocking activity, and sequence diversity, P07 (KD = 1.2 ± 0.2 nM, IC50 = 0.5 ± 0.1 nM) was finally selected as the first single-domain antibody for the fusion protein of this invention.

[0041] Table 1: Affinity to anti-PD-L1 VHH and PD-1 / PD-L1 blocking activity

[0042]

[0043] 1.3 Screening and Identification of Anti-NKG2D VHH

[0044] Using the same screening strategy as for anti-PD-L1 VHH, the VHH phage display library was subjected to four rounds of biological panning with recombinant human NKG2D-Fc fusion protein (MCE, catalog number: HY-P78498) as the target. The panning conditions, elution and phage amplification methods were the same as in Section 1.2.

[0045] After the fourth round of selection, 96 single clones were randomly selected, and positive clones were identified by phage ELISA (NKG2D-Fc as the coating antigen, BSA as the negative control, and OD450 / negative control >5 was considered positive), resulting in 35 positive clones. These 35 positive clones were sequenced, and after removing repetitive sequences, 14 unique sequences VHH (named N01-N14) were obtained.

[0046] Fourteen VHHs were cloned into the pET-22b(+) vector, transformed into BL21(DE3) competent cells, and induced to express by IPTG. The VHH proteins were then purified by Ni-NTA affinity chromatography (purity >90%). The affinity of each VHH for human NKG2D was determined using SPR technology, following the same method as described in Section 1.2. The results showed that VHHs N05 and N09 exhibited the highest affinity, with KD values ​​of 3.5 ± 0.4 nM and 4.2 ± 0.5 nM, respectively.

[0047] The binding ability of each VHH to human primary NK cells was detected by flow cytometry: peripheral blood was collected from healthy donors, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. NK cells were purified using an NK cell isolation kit. The NK cell concentration was adjusted to 1×10^6 cells / mL, VHH protein (final concentration 10 μg / mL) was added, and the cells were incubated at 4°C for 30 min. The cells were washed twice with PBS. FITC-labeled anti-His tag monoclonal antibody was added, and the cells were incubated at 4°C for 30 min. After washing, the cells were resuspended in PBS. The mean fluorescence intensity (MFI) of NK cells (CD3-CD56+) was detected by flow cytometry. NK cells without VHH protein were used as a blank control, and the MFI fold increase was calculated.

[0048] The results (Table 2) showed that N05 exhibited the strongest binding signal to NK cells, with an MFI fold increase of 25.3 ± 2.5 times, significantly higher than N09 (18.5 ± 1.8 times) and other VHH clones. Considering both affinity and cell binding ability, N05 (KD = 3.5 ± 0.4 nM) was selected as the second single-domain antibody for the fusion protein of this invention.

[0049] Table 2: Affinity to anti-NKG2D VHH and binding ability to NK cells

[0050]

[0051] 1.4 Design and Construction of Mutant IL-15

[0052] Based on previous studies, the N72 asparagine mutation in IL-15 to aspartic acid (N72D) significantly enhances its binding affinity to the IL-2 / 15Rβ receptor while reducing its dependence on the IL-15Rα receptor; the N79 asparagine mutation to aspartic acid (N79D) removes the N-glycosylation modification at this site, reducing protein glycosylation heterogeneity and improving protein expression stability and in vitro activity. Based on this, this invention designed and synthesized the entire human IL-15 gene (SEQ ID NO:1) containing both N72D and N79D mutations. The gene synthesis was completed by Suzhou Genewise Biotechnology Co., Ltd.

[0053] To verify the functional characteristics of the mutants, expression vectors for wild-type IL-15 / Fc fusion protein and mutant IL-15 / Fc fusion protein were constructed: the coding sequences of wild-type human IL-15 gene and double mutant IL-15 gene with human IgG1 Fc fragment were linked through a flexible hinge region (GGGGS)3 and cloned into the eukaryotic expression vector pCDNA3.4 to construct recombinant expression plasmids pCDNA3.4-WT-IL15-Fc and pCDNA3.4-Mut-IL15-Fc.

[0054] Two recombinant plasmids were transfected into Expi293F cells, and protein expression and purification were performed according to the method in Example 2 to obtain fusion proteins with a purity >95%. Functional validation was performed using the IL-15Rα-deficient human T-cell leukemia cell line KT-3 (see Example 3.2). This cell line expresses only the IL-2 / 15Rβγc receptor complex and does not express IL-15Rα, allowing for specific detection of the dependence of IL-15 mutants on IL-2 / 15Rβγc.

[0055] 1.5 Construction of fusion protein expression vector

[0056] Based on the above screening and identification results, the structural sequence of the fusion protein was determined to be: anti-PD-L1 VHH (P07) - flexible hinge region (GGGGS) 3 - mutant IL-15 - flexible hinge region (GGGGS) 3 - anti-NKG2D VHH (N05) - human IgG1 Fc fragment (containing L14A and L15A mutations (numbered according to the amino acid sequence of SEQ ID NO:5) can significantly reduce the binding ability of the Fc fragment to FcγR and reduce non-specific immune activation).

[0057] The coding sequences of the above fusion fragments were synthesized from the whole gene, and the corresponding sequences of each fragment are as follows: anti-PD-L1 VHH (P07, SEQ ID NO:2), flexible hinge region (GGGGS)3 (SEQ ID NO:3), double mutant IL-15 (SEQ ID NO:1), flexible hinge region (GGGGS)3 (SEQ ID NO:3), anti-NKG2D VHH (N05, SEQ ID NO:4), and human IgG1 Fc fragment (SEQ ID NO:5). A Hind III restriction endonuclease site was introduced at the 5' end of the synthesized gene, and an EcoR I restriction endonuclease site was introduced at the 3' end to facilitate subsequent cloning into the expression vector.

[0058] The fusion gene fragment synthesized by double digestion with Hind III and EcoRI and the eukaryotic expression vector pCDNA3.4 were digested at 37°C for 4 h, and the digestion products were purified by agarose gel electrophoresis. Following the T4 DNA ligase instructions, the digested fusion gene fragment was ligated into the pCDNA3.4 vector and incubated overnight at 16°C.

[0059] The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar containing ampicillin (100 μg / mL) and incubated overnight at 37°C. Single clones were picked, and positive clones were identified by colony PCR (primers Hind III-F and EcoRI IR). PCR-positive clones were sent for sequencing verification. The recombinant expression plasmid, whose sequencing results were completely consistent with the designed sequence, was named pCDNA3.4-DB-IL15m-Fc. The nucleotide sequence of the full-length fusion protein encoded by this plasmid is shown in SEQ ID NO:6.

[0060] Example 2: Expression and purification of fusion protein

[0061] 2.1 Transient transfection: After resuscitation, Expi293F cells were seeded in Expi293 Expression Medium and cultured at 37°C, 8% CO2, and with shaking at 125 rpm. Transfection was performed when the cell density reached 3 × 10^6 cells / mL and cell viability > 95%. The recombinant plasmid used for transfection was pCDNA3.4-DB-IL15m-Fc, and the transfection reagent was polyethyleneimine (PEI, Polysciences), with a plasmid DNA to PEI mass ratio of 1:3. Add 100 μg of plasmid DNA to every 100 mL of cell culture and dilute with 5 mL of Opti-MEM medium; simultaneously add 300 μg of PEI and dilute with 5 mL of Opti-MEM medium, and incubate at room temperature for 5 min; slowly add the diluted PEI to the diluted plasmid DNA, mix gently, and incubate at room temperature for 20 min to form a PEI-DNA complex; slowly add the complex dropwise to the cell culture, shake gently, and continue to incubate at 37°C, 8% CO2, and 125 rpm with shaking.

[0062] 2.2 Culture and Harvest: On day 3 post-transfection, 5% (w / v) glucose solution and 0.5% (v / v) GlutaMAX reagent were added to the cell culture to maintain nutrient supply, and the culture was continued for another 4 days (total culture time 7 days). During the culture period, samples were taken daily, and cell viability was counted using trypan blue staining to ensure that cell viability was maintained above 70%. After the culture was completed, the cell culture supernatant was collected, centrifuged at 8000 rpm and 4℃ for 30 min to remove cell pellet; the supernatant was then vacuum filtered through a 0.22 μm filter membrane to further remove cell debris and impurities, and the clear supernatant was collected and stored at 4℃ for later use.

[0063] 2.3 Protein Purification: A two-step purification method of affinity chromatography-gel filtration chromatography was used to obtain high-purity fusion proteins. Step 1: Affinity Chromatography: Clarified cell supernatant was loaded onto a 5 mL HiTrapProtein A HP affinity chromatography column pre-equilibrated with PBS (pH 7.4) at a flow rate of 2 mL / min. After loading, the column was washed with PBS at a flow rate of 3 mL / min until the elution peak returned to baseline, removing unbound contaminants. The target protein was eluted with 0.1 M citrate buffer (pH 3.0) at a flow rate of 1 mL / min, collecting 1 mL of eluent from each tube. Immediately, 100 μL of 1 M Tris-HCl (pH 9.0) was added to each tube of eluent to rapidly neutralize to pH 7.4 to prevent protein denaturation. The second step is gel filtration chromatography: The target protein eluent obtained by affinity chromatography is concentrated to 2 mL using an ultrafiltration centrifuge tube (10 kDa); the concentrated protein sample is loaded onto a Superdex 200 Increase 10 / 300 GL gel filtration chromatography column pre-equilibrated with PBS, with PBS as the mobile phase and a flow rate of 0.5 mL / min, and 0.5 mL of eluent is collected from each tube; the protein purity of each elution peak is identified by SDS-PAGE electrophoresis, and the eluent corresponding to the monomeric protein peak is collected.

[0064] After purification, the concentration of the fusion protein was determined using a BCA protein quantification kit, and the purity was identified by SDS-PAGE electrophoresis. The results showed ( Figure 1 The resulting fusion protein had a purity of >95% (molecular weight of approximately 70 kDa) and no obvious contaminating protein bands; the protein yield was approximately 25 mg / L of cell culture supernatant, which met the requirements for subsequent in vitro and in vivo functional experiments.

[0065] Example 3: In vitro functional identification of fusion proteins

[0066] 3.1 Antigen binding capacity test (SPR)

[0067] The binding ability of the fusion protein to human PD-L1 and human NKG2D antigens was detected using the Biacore T200 system to verify whether the antigen-binding activity of the two VHH domains in the fusion protein was retained. The specific methods are as follows:

[0068] Human PD-L1 and human NKG2D proteins were diluted to 10 μg / mL with 10 mM sodium acetate buffer (pH 5.0) and immobilized in different channels of the CM5 chip via amino coupling, with immobilization levels of 1200 RU (PD-L1) and 1500 RU (NKG2D). The mobile phase was PBS (pH 7.4) at a flow rate of 30 μL / min. The fusion protein purified in Example 2 was serially diluted to 0 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM and flowed sequentially through two channels of the chip, with a binding time of 120 s and a dissociation time of 300 s. The chip regeneration solution was 10 mM Gly-HCl (pH 2.0), with each regeneration lasting 10 s and repeated 3 times to ensure good regeneration results.

[0069] Biacore Evaluation software was used to fit the kinetic curves using a 1:1 binding model, and the KD, kon, and koff values ​​of the fusion protein with the two antigens were calculated. The results showed that the KD value of the fusion protein binding to human PD-L1 was 1.2 ± 0.2 nM (kon = 12.8 ± 1.0 × 10⁵ M). -1 s -1 koff = 1.54 ± 0.2 × 10 -4 s -1 The KD value for binding with human NKG2D is 3.5 ± 0.4 nM (kon = 9.6 ± 0.8 × 10^5 M). -1 s -1 koff = 3.36 ± 0.3 × 10 -4 s -1 The affinity of the fusion protein is basically consistent with that of the individual P07 and N05 VHH in Example 1, indicating that the construction of the fusion protein did not affect the antigen-binding activity of the two VHH domains, and it can specifically bind to PD-L1 and NKG2D antigens at the same time.

[0070] 3.2 Functional validation of IL-15 mutants (IL-15Rα-dependent reduction)

[0071] Using the IL-15Rα-deficient KT-3 cell line, we verified the reducing effect of double-mutant IL-15 on IL-15Rα receptor dependence. The specific experimental method is as follows:

[0072] KT-3 cells were routinely cultured in RPMI-1640 medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) and incubated at 37°C in a 5% CO2 incubator. Cells were passaged regularly to maintain a viability of >95%. Before the experiment, KT-3 cells were adjusted to 1×10^4 cells / well and seeded into 100 μL per well in 96-well cell culture plates, incubated overnight at 37°C to allow cell adhesion.

[0073] The wild-type IL-15 / Fc (WT) and mutant IL-15 / Fc (Mut) fusion proteins prepared in Example 1.4 were serially diluted with culture medium to 0.1 ng / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively. 100 μL of the diluted protein solution was added to each well, with three replicates for each concentration. The PBS-treated group served as a blank control. Cells were incubated at 37°C in a 5% CO2 incubator for 72 h.

[0074] After culture, 10 μL of CCK-8 reagent was added to each well, gently shaken, and cultured at 37°C for another 4 hours. The OD450 value of each well was measured using a microplate reader to reflect the cell proliferation level. The EC50 values ​​of the two proteins were calculated using GraphPad Prism software to compare their ability to induce KT-3 cell proliferation, and to assess the dependence of the mutant on IL-15Rα.

[0075] The results (Table 3) showed that wild-type IL-15 / Fc significantly induced KT-3 cell proliferation at a low concentration (1 ng / mL), with an OD450 value of 1.25 ± 0.10. In contrast, mutant IL-15 / Fc showed weaker proliferation induction at low concentrations, with an OD450 value of only 0.45 ± 0.05 at 1 ng / mL. Only at a high concentration (100 ng / mL) did the proliferation level become comparable to that of wild-type IL-15 (OD450 values: 1.85 ± 0.15 vs 1.92 ± 0.18). EC50 calculations showed that the EC50 of wild-type IL-15 / Fc was 0.8 ± 0.1 ng / mL, while that of mutant IL-15 / Fc was 25.6 ± 2.5 ng / mL, indicating a significantly reduced dependence of the double mutant IL-15 on the IL-15Rα receptor, consistent with the design expectations.

[0076] Table 3: Results of KT-3 cell proliferation assay (OD450, mean ± SD)

[0077]

[0078] 3.3 Selective proliferation experiment of immune cell subsets

[0079] This experiment involved sorting human primary NKG2D+CD8+ T cells (effective immune cells) and CD4+FoxP3+Tregs (suppressive immune cells) to detect the selective proliferation effect of the fusion protein on these two cell subsets, thus verifying the precise regulatory ability of the fusion protein on effector immune cells. The specific methods are as follows:

[0080] Peripheral blood was collected from healthy donors, and PBMCs were separated by density gradient centrifugation. NKG2D+ CD8+ T cells and CD4+ FoxP3+ Tregs were sorted using the MACS magnetic bead sorting system: (1) Sorting of NKG2D+ CD8+ T cells: CD8+ T cells were first sorted using a CD8+ T cell sorting kit, then CD8+ T cells were labeled with NKG2D-PE antibody, and anti-PE magnetic beads were added to obtain NKG2D+ CD8+ T cells; (2) Sorting of CD4+ FoxP3+ Tregs: CD4+ FoxP3+ Tregs were obtained by sorting using a Regulatory T Cell Isolation Kit, following the kit instructions. After sorting, the cell purity was detected by flow cytometry to ensure that the purity of both cell types was >90%.

[0081] The sorted cells were stained with 5 μM CFSE and incubated at 37°C for 15 min. Staining was terminated by adding RPMI-1640 medium containing 10% fetal bovine serum. The cells were washed twice with PBS, resuspended in the medium, and the cell density was adjusted to 1×10^5 cells / mL. Cells were seeded into 96-well cell culture plates at 100 μL per well (1×10^5 cells / well), with three treatment groups: (1) PBS control group; (2) wild-type IL-15 / Fc protein treatment group (final concentration 100 ng / mL); (3) the fusion protein treatment group of this invention (molar concentration equivalent to 100 ng / mL IL-15). Each treatment group was divided into three replicates and cultured at 37°C in a 5% CO2 incubator for 72 h.

[0082] After culture, cells were collected, washed twice with PBS, and CFSE dilution was detected by flow cytometry. CFSE dilution reflects cell proliferation. The cell proliferation index of each treatment group was calculated using the proliferation model in FlowJo software. A higher proliferation index indicates stronger cell proliferation capacity.

[0083] The results (Table 4) showed that in NKG2D+ CD8+ T cells, the proliferation index of the fusion protein treatment group was 3.8±0.3, significantly higher than that of the wild-type IL-15 / Fc treatment group (2.2±0.2), with a statistically significant difference (p<0.01). However, in CD4+ FoxP3+ Tregs, the proliferation index of the fusion protein treatment group was only 1.2±0.1, lower than that of the wild-type IL-15 / Fc treatment group (2.8±0.3), with a statistically significant difference (p<0.001). These results indicate that the fusion protein of this invention can specifically promote the proliferation of effector immune cells (NKG2D+ CD8+ T cells) while inhibiting the proliferation of suppressor immune cells (Tregs), achieving precise regulation of immune cell subsets and thus enhancing anti-tumor immune responses.

[0084] Table 4: Results of immune cell proliferation experiment

[0085]

[0086] Example 4: In vitro validation of dual-target synergistic effect

[0087] 4.1 Construction of the control fusion protein

[0088] To clarify the synergistic effect of dual-targeting structures (anti-PD-L1 VHH and anti-NKG2D VHH) on the anti-tumor function of IL-15 mutants, this study constructed three groups of control fusion proteins, specifically designed as follows:

[0089] (1) Single-target PD-L1 fusion protein: anti-PD-L1 VHH-flexible hinge region-IL-15 double mutant-flexible hinge region-Fc fragment (deleted anti-NKG2D VHH domain, the rest of the sequence is consistent with the target fusion protein).

[0090] (2) Single-target NKG2D fusion protein: - Anti-NKG2D VHH - Flexible hinge region - IL-15 double mutant - Flexible hinge region - Fc fragment (deleted anti-PD-L1 VHH domain, the rest of the sequence is consistent with the target fusion protein).

[0091] (3) Non-target control protein: IL-15 double mutant - flexible hinge region - Fc fragment (two VHH targeting domains are missing, only cytokines and Fc constant regions are retained).

[0092] All the control fusion proteins and the target dual-target fusion proteins of the present invention were prepared according to the eukaryotic expression and purification process described in Example 2. SDS-PAGE electrophoresis confirmed that the purity of all proteins was ≥95%, and they can be used for subsequent in vitro functional verification experiments.

[0093] 4.2 Functional Validation in the Co-cultivation System

[0094] A tumor-immune cell co-culture system was constructed using human primary NKG2D+ CD8+ T cells and PD-L1 positive MC38 colon cancer cells to simulate the interaction between immune cells and tumor cells in the in vivo tumor microenvironment and to verify the synergistic function of the dual-targeting fusion protein.

[0095] The experimental procedure was as follows: peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, and NKG2D+CD8+ T cells were purified using magnetic bead sorting. The purity of the sorted cells was identified by flow cytometry as ≥90%. PD-L1 positive MC38 tumor cells in logarithmic growth phase and purified NKG2D+CD8+ T cells were seeded in 96-well round-bottom culture plates at an effector-to-target ratio of 1:10 (1×10^4 tumor cells / well, 1×10^5 T cells / well), with a final volume of 200 μL per well.

[0096] Different fusion proteins were added to the groups, and the dosage of all groups was adjusted based on the IL-15 molar concentration (uniformly 100 ng / mL) to ensure that the cytokine dosage of each group was consistent. Each treatment group was set up with 3 replicates, and the following 5 experimental groups were set up: ① blank control group (PBS); ② non-targeted IL-15 mutant / Fc group; ③ single-targeted PD-L1-IL-15 mutant / Fc group; ④ single-targeted NKG2D-IL-15 mutant / Fc group; ⑤ the dual-targeted fusion protein group of the present invention.

[0097] After incubating the culture plates in a 37℃, 5% CO2 incubator for 72 hours, all cells in the wells were collected. The T cell proliferation capacity was detected by CFSE staining dilution method combined with flow cytometry. The proportion of cells with low CFSE expression (i.e., the proportion of proliferating cells) was analyzed using FlowJo software. At the same time, the culture supernatant was collected, and cell debris was removed by centrifugation (1200 rpm, 5 min, 4℃). The secretion level of IFN-γ in the supernatant was measured using a human IFN-γ ELISA kit, strictly following the kit instructions, to reflect the killing and activation function of T cells.

[0098] The experiment was repeated three times. Data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was performed using SPSS 26.0 software. LSD-t test was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.001 was considered extremely statistically significant.

[0099] Experimental results show that ( Figure 2According to Table 5, the proliferation rate of NKG2D+CD8+ T cells in the dual-targeted fusion protein treatment group of this invention was 64.5 ± 5.2%, which was significantly higher than that in the single-targeted PD-L1 group (32.5 ± 3.5%), the single-targeted NKG2D group (35.8 ± 4.0%), and the non-targeted control group (28.5 ± 3.0%), with all differences being highly statistically significant (P<0.001). The IFN-γ secretion level in the supernatant showed a consistent trend, with the IFN-γ production in the dual-targeted group being 1850 ± 150 pg / mL, which was significantly higher than that in the single-targeted PD-L1 group (850 ± 80 pg / mL), the single-targeted NKG2D group (920 ± 90 pg / mL), and the non-targeted group (580 ± 60 pg / mL), with the differences also being highly statistically significant (P<0.001).

[0100] Further analysis showed no significant differences in T cell proliferation rate and IFN-γ secretion level among the single-target PD-L1 group, the single-target NKG2D group, and the non-targeted control group (P>0.05), suggesting that a single targeting structure cannot effectively enrich IL-15 signal. These results indicate that only when anti-PD-L1 VHH and anti-NKG2D VHH are present simultaneously can IL-15 cytokine signal be specifically enriched at the interface between tumor cells and immune effector cells, synergistically activating the proliferation and killing function of NKG2D+ CD8+ T cells, achieving a synergistic effect greater than the sum of its parts (1+1>2).

[0101] Table 5: Results of NKG2D+CD8+ T cell function assay in co-culture system (mean ± SD, n=3)

[0102]

[0103] Example 5: In vivo antitumor pharmacodynamics study

[0104] 5.1 Establishment of animal models

[0105] This study used a humanized NSG mouse model reconstructed from human CD34+ hematopoietic stem cells. This model can effectively reconstruct the human immune system (including effector immune cells such as T cells and NK cells) and accurately assess the anti-tumor activity of the dual-targeting fusion protein in the human immune microenvironment.

[0106] The experimental animals were 6-8 week old female NSG mice, weighing 18-22 g, housed in an SPF-grade animal room (temperature 22-25℃, humidity 50±5%, 12-hour light-dark cycle), with free access to food and water. Before modeling, the mice were irradiated with a sublethal dose of 250 cGy of gamma rays (irradiation source: 60Co, irradiation rate 100 cGy / min) to suppress residual immune cells in the mice and create conditions for the engraftment of human CD34+ hematopoietic stem cells.

[0107] Within 4-6 hours after irradiation, each mouse was injected with 5 × 10^4 human CD34+ hematopoietic stem cells via tail vein injection, with an injection volume of 100 μL. Gentamicin (final concentration 100 μg / mL) was added to the mice's drinking water after injection for two consecutive weeks to prevent bacterial infection. At the same time, the mice's mental state, diet and weight changes were observed regularly, and mice with abnormal conditions were removed in a timely manner.

[0108] Sixteen weeks after hematopoietic stem cell injection, blood was collected from the orbital venous plexus (50-100 μL per mouse). After EDTA anticoagulation, fluorescently labeled antibodies (anti-human CD45-PE, anti-human CD3-FITC, and anti-human CD56-APC) were added, and the mixture was incubated at 4°C in the dark for 30 minutes. After washing twice with PBS, the proportion of human cells in the peripheral blood of mice was detected by flow cytometry.

[0109] The inclusion criteria for successful human immune system reconstruction were: human CD45+ cells accounted for ≥25% of total white blood cells in peripheral blood, human CD3+ T cells accounted for ≥30% of human CD45+ cells, and human CD56+ NK cells accounted for ≥5% of human CD45+ cells. Mice that met the criteria were used for subsequent tumor inoculation experiments.

[0110] Logarithmic growth phase MC38 colon cancer cells were digested and collected, and the cell concentration was adjusted to 5 × 10^6 cells / mL with PBS. 100 μL (containing 5 × 10^5 tumor cells) was subcutaneously injected into the right abdomen of each successfully immunized mouse. Tumor size was measured twice weekly after inoculation. When the tumor volume reached 80-100 mm³ (approximately 7-10 days post-inoculation), mice were randomly assigned to groups and drug administration was initiated, ensuring no significant difference in initial tumor volume among the groups (P>0.05).

[0111] 5.2 Grouping and Dosing

[0112] Eligible humanized mice were randomly divided into 5 groups of 8 mice each. Statistical analysis showed no significant differences in body weight and initial tumor volume among the groups (P>0.05). The grouping and drug administration regimens are as follows:

[0113] ① Solvent control group: Intraperitoneal injection of PBS, twice a week (Monday and Thursday), for 3 consecutive weeks, for a total of 6 times;

[0114] ② Non-targeted IL-15 mutant / Fc control group: The dosage was 10 mg / kg, administered intraperitoneally, with the same frequency and cycle as the solvent control group;

[0115] ③ Single-target PD-L1-IL-15 mutant / Fc group: Dosage 10 mg / kg, intraperitoneal injection, dosing frequency and cycle are the same as solvent control group;

[0116] ④ Single-target NKG2D-IL-15 mutant / Fc group: Dosage 10 mg / kg, intraperitoneal injection, dosing frequency and cycle are the same as solvent control group;

[0117] ⑤ The dual-targeting fusion protein group of this invention: the dosage is 10 mg / kg, intraperitoneal injection, and the dosing frequency and cycle are the same as the solvent control group.

[0118] The dosage volume was adjusted according to the mouse's body weight (10 μL / g) to ensure accurate dosage for each mouse. During the administration period, the mice's mental state, diet, activity and weight changes were observed daily, and their body weight was measured twice a week to assess the drug's toxicity. If the mouse's body weight decreased by more than 20% or obvious symptoms of poisoning appeared, the experiment on that mouse was terminated and recorded.

[0119] 5.3 Results Evaluation

[0120] In vivo efficacy assessment indicators include tumor growth inhibition, tumor weight, and immune cell infiltration within tumor tissue. Specific detection methods are as follows:

[0121] (1) Tumor growth monitoring: The long diameter (a) and short diameter (b) of the tumor were measured twice a week. The tumor volume was calculated using the formula V=0.5×a×b². The tumor growth curve was plotted to evaluate the inhibitory effect of different treatment groups on tumor growth.

[0122] (2) Tumor weight detection: After the administration was completed (day 21), all mice were euthanized by cervical dislocation. The tumor tissue on the right abdomen was quickly dissected, and the surface blood and body fluids were dried with filter paper. The tumor weight was then measured with an electronic balance (accuracy 0.1 mg) and the tumor inhibition rate was calculated [Tumor inhibition rate = (average tumor weight of control group - average tumor weight of administration group) / average tumor weight of control group × 100%];

[0123] (3) Detection of immune cell infiltration in tumor tissue: The dissected tumor tissue was divided into two parts. One part was fixed in 4% paraformaldehyde solution, routinely embedded in paraffin, and sectioned (4 μm thick). The infiltration of CD8+ T cells and NK cells in the tumor tissue was detected by immunohistochemical SP method. The primary antibodies were anti-human CD8 antibody and anti-human CD56 antibody, respectively, and the secondary antibody was HRP-labeled goat anti-rabbit IgG antibody. DAB staining, hematoxylin counterstaining, dehydration and clearing, and mounting with neutral resin were performed. The slides were observed and photographed under an optical microscope (×200x). Five fields of view were randomly selected from each slide. The number of CD8+ T cells and NK cells was counted using Image-Pro Plus 6.0 software, and the average infiltration density (cells / mm²) was calculated. 2 ).

[0124] All experimental data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using SPSS 26.0 software. One-way ANOVA combined with LSD-t test was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.001 was considered extremely statistically significant.

[0125] The experimental results (Table 6) showed that at the end of drug administration (day 21), there were significant differences in tumor growth among the groups: the mean tumor volume in the solvent control group (PBS group) was 1480 ± 230 mm. 3 The non-targeted IL-15 mutant / Fc group was 1020 ± 180 mm. 3 The single-target PD-L1 group had a diameter of 890 ± 150 mm. 3 The single-target NKG2D group had a diameter of 920 ± 160 mm. 3 The average tumor volume of the dual-targeting fusion protein group of this invention is only 310 ± 70 mm. 3 The tumor growth inhibition rate of the dual-target fusion protein group was significantly lower than that of all control groups, and the difference was statistically significant (P<0.001). The calculated tumor growth inhibition rate of the dual-target fusion protein group reached 78.9%, which was significantly higher than that of the non-target group (29.7%), the single-target PD-L1 group (39.9%) and the single-target NKG2D group (37.8%).

[0126] The tumor weight results were consistent with the changes in tumor volume (Table 6): the average tumor weight was 1.52 ± 0.25 g in the PBS group, 1.08 ± 0.20 g in the non-targeted group, 0.95 ± 0.16 g in the single-targeted PD-L1 group, 0.98 ± 0.17 g in the single-targeted NKG2D group, and 0.38 ± 0.08 g in the dual-targeted fusion protein group. The tumor weight in the dual-targeted group was significantly lower than that in the other groups (P<0.001).

[0127] Immunohistochemical analysis results (Table 6) showed that the CD8+ T cell infiltration density in tumor tissue of the dual-targeting fusion protein group was 85.5 ± 12.5 cells / mm². 2 The number of cells was significantly higher than that of the single-target PD-L1 group (42.5 ± 8.5 cells / mm). 2 ), single-target NKG2D group (45.8 ± 9.0 cells / mm) 2 ) and the non-targeted group (28.5 ± 6.5 cells / mm) 2 The differences were statistically significant (P<0.001); NK cell infiltration density showed the same trend, with the dual-targeting group at 35.5 ± 6.5 cells / mm². 2 The number of cells was significantly higher than that of the single-target PD-L1 group (15.8 ± 4.0 cells / mm). 2 ), single-target NKG2D group (18.5 ± 4.5 cells / mm) 2 () and non-targeted group (10.5 ± 3.0 cells / mm) 2 (P<0.001).

[0128] During the administration period, the body weight of mice in each group did not decrease significantly (body weight change rate <10%), and no toxic reactions such as lethargy, reduced appetite, or abnormal activity were observed, indicating that the fusion proteins were safe and had no obvious toxicity at a dose of 10 mg / kg.

[0129] The above in vivo experimental results show that the dual-targeting fusion protein of the present invention can effectively recruit human CD8+ T cells and NK cells to infiltrate into tumor tissues and significantly inhibit tumor growth. Its anti-tumor effect is significantly better than that of non-targeting and single-targeting fusion proteins, further verifying the synergistic effect of the dual-targeting structure.

[0130] Table 6: Results of in vivo antitumor drug efficacy experiment (Day 21, mean ± SD, n=8)

[0131]

[0132] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein contains a single-domain antibody targeting PD-L1 and a single-domain antibody targeting NKG2D.

2. The fusion protein according to claim 1, characterized in that, The fusion protein comprises, from the amino terminus to the carboxyl terminus, the following: (a) A first single-domain antibody that specifically recognizes PD-L1; (b) First hinge area; (c) Mutant human interleukin-15; (d) Second hinge area; (e) A second single-domain antibody that specifically recognizes NKG2D; (f) Immunoglobulin Fc fragment.

3. The fusion protein according to claim 2, characterized in that, The amino acid sequence of the first single-domain antibody is shown in SEQ ID NO:2, the amino acid sequence of the second single-domain antibody is shown in SEQ ID NO:4, the amino acid sequence of the mutant human interleukin-15 is shown in SEQ ID NO:1, the amino acid sequence of the immunoglobulin Fc fragment is shown in SEQ ID NO:5, and the amino acid sequences of the first hinge region and the second hinge region are both shown in SEQ ID NO:

3.

4. A single-domain antibody, characterized in that, The single-domain antibody specifically recognizes PD-L1, and its amino acid sequence is shown in SEQ ID NO:

2.

5. A single-domain antibody, characterized in that, The single-domain antibody specifically recognizes NKG2D, and its amino acid sequence is shown in SEQ ID NO:

4.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein according to any one of claims 1 to 3, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

6.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fusion protein according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

8. Use of the fusion protein according to any one of claims 1 to 3 in the preparation of a medicament for treating cancer.

9. The use according to claim 8, characterized in that, The cancer is a solid tumor expressing PD-L1.

10. The use according to claim 9, characterized in that, The solid tumor is colon cancer, non-small cell lung cancer, or melanoma.

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