CD276 binding miniprotein, mutant thereof, and use thereof

Abstract

Provided are a CD276 binding miniprotein, a mutant thereof, and a use thereof. Further provided are a chimeric antigen receptor comprising the CD276 binding miniprotein or the mutant thereof as an extracellular antigen binding domain, and a chimeric antigen receptor T cell. The binding affinity of a CD276 binding protein mutant HM9 for CD276 reaches 36.4 pM, and the binding protein has a small molecular weight, good stability and high affinity, and can be massively expressed in Escherichia coli. Compared with traditional scFC-based CAR-T treatment strategies, the constructed CAR-T cell has higher tumor killing activity, a lower exhaustion level, and a faster division and expansion capability.

Description

A CD276 mini-binding protein and its mutant and application Technical Field

[0001] The present application belongs to the field of tumor immunotherapy drugs, and specifically relates to a newly designed CD276 mini-binding protein and its mutants and applications. Background Art

[0002] B7 homolog 3 (B7-H3), more commonly known as CD276, is a co-stimulatory / co-inhibitory regulatory protein that plays a dual role in the immune system and is also an immune checkpoint-associated protein. It was first cloned and identified in 2001 from a cDNA library derived from human dendritic cells. The human CD276 gene is located on chromosome 15 and is mostly expressed as a membrane protein. It is a type I transmembrane protein containing 316 amino acids and composed of an extracellular domain, a transmembrane domain, and a short intracellular domain. Studies have shown that CD276 expression is abnormally elevated in tumor cells, which in turn increases the adhesion capacity of cell adhesion proteins, leading to enhanced tumor cell migration and invasion. Furthermore, CD276 can lead to increased NF-κB activity, resulting in elevated VEGF and IL-8 expression, further promoting tumor-associated angiogenesis and tumor invasion. Because CD276 is abnormally highly expressed in various cancer cells, including medulloblastoma and glioblastoma, but has limited distribution in normal tissues, it is an effective and safe target. Currently, various B7-H3-targeted immunotherapy strategies, including monoclonal antibodies, bispecific antibodies, ADCs, and CAR-T cells, have been developed and tested in clinical trials, showing promising application prospects and making it a very attractive target. Designing and developing more effective protein drugs and immunotherapy strategies targeting CD276 is of great value.

[0003] Protein design, based on biophysical and biochemical principles, involves engineering the amino acid sequence of a protein to enable it to spontaneously fold into a desired novel three-dimensional structure and acquire new functions. The design of functional proteins, particularly novel binding proteins with high affinity and specificity for any naturally occurring protein, holds significant applications in diagnosis, treatment, and prevention. Compared to protein-based drugs, such as monoclonal antibodies, artificially designed binding proteins offer significant advantages in terms of small molecular weight, excellent tissue penetration, strong chemical activity, excellent stability, and ease of industrialization. They allow for rapid design of specific target epitopes and precise control of binding patterns. Designing binding proteins based solely on the target protein's three-dimensional structural information has become possible. New protein design algorithms and tools are providing new tools and models for basic protein research and protein drug discovery. The development of protein drugs and immunotherapy strategies targeting CD276 based on the latest protein design algorithms holds significant potential and promise.

[0004] CAR-T therapy is considered a revolutionary breakthrough in cancer immunotherapy following immune checkpoint therapy and has achieved significant success in the treatment of hematologic malignancies. CAR-T therapy involves collecting and isolating T cells from a patient's blood and bioengineering them to express a chimeric antigen receptor (CAR) that specifically recognizes tumor surface antigens, enhancing their ability to target and kill cancer cells. The extracellular domain of the key chimeric antigen receptor (CAR) in CAR-T therapy is primarily composed of a single-chain variable fragment (scFv) of a monoclonal antibody, responsible for antigen recognition and binding. ScFvs are composed of the light (VL) and heavy (VH) chains of a monoclonal antibody linked by a polypeptide, retaining the antibody's specificity and affinity for the antigen. However, the heavy and light chains of scFvs are prone to mis-aggregation and folding, resulting in poor structural stability. This not only impairs T cell recognition of antigens but can also cause antigen-independent CAR aggregation and T cell activation, leading to premature T cell exhaustion, significantly reducing the therapeutic efficacy of CAR-T therapy. Given the abnormally high expression of CD276 in many solid tumor cells, the development of binding proteins targeting CD276 based on protein de novo design and the development of a new generation of CAR-T treatment strategies targeting CD276 have significant research and clinical application value.

[0005] Summary of the Invention

[0006] In view of the above technical problems, on the one hand, the present invention provides a CD276 mini-binding protein or a mutant thereof,

[0007] Wherein, the CD276 mini-binding protein has the amino acid sequence shown in SEQ ID No.: 1, and

[0008] The mutant is selected from:

[0009] i) a mutant having an amino acid sequence of SEQ ID No.: 2;

[0010] ii) the following single-site mutants of SEQ ID No.: 1:

[0011] Amino acid 1 is mutated to any one of A, D, E, F, G, H, I, L, M, N, P, R, T, V, W, or Y; or

[0012] Amino acid 2 is mutated to any one of A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or W; or

[0013] Amino acid 3 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, W, or Y; or

[0014] Amino acid 4 is mutated to any one of A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; or

[0015] Amino acid 5 mutates to any one of A, D, E, H, I, N, Q, S, T, and V; or amino acid 6 mutates to any one of F, L, M, W, and Y; or

[0016] Amino acid 7 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0017] Amino acid 8 is mutated to any one of I, M, and V; or

[0018] Amino acid 9 is mutated to any one of F, H, I, L, or Y; or

[0019] Amino acid 10 is mutated to any one of A, D, E, F, H, I, L, M, Q, S, T, V, W, or Y; or

[0020] Amino acid 11 is mutated to any one of A, H, K, N, Q, R, S, T, V, or Y; or

[0021] Amino acid 12 mutates to T; or

[0022] Amino acid 13 is mutated to any one of A, F, G, S, or T; or

[0023] Amino acid 14 is mutated to any one of A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0024] Amino acid 15 mutates to any one of A, D, G, H, N, S, T, or V; or

[0025] Amino acid 16 mutates to L; or

[0026] Amino acid 17 mutates to any one of E, F, I, L, M, T, or V; or

[0027] Amino acid 18 is mutated to any one of A, H, K, N, Q, R, or T; or

[0028] Amino acid 19 is mutated to any one of A, F, G, H, I, L, M, N, Q, R, S, T, or V; or

[0029] Amino acid 20 is mutated to any one of A, D, E, G, H, K, N, Q, R, S, or T; or

[0030] Amino acid 21 is mutated to any one of D, E, K, N, or Q; or

[0031] Amino acid 22 is mutated to any one of A, F, H, K, L, M, N, Q, R, S, T, V, or Y; or

[0032] Amino acid 23 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, or Y; or

[0033] The amino acid 24 is mutated to any one of L, M, V, or W; or

[0034] The amino acid 25 is mutated to either L or M; or

[0035] Amino acid 26 is mutated to any one of A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; or

[0036] Amino acid 27 is mutated to any one of H, I, P, or V; or

[0037] Amino acid 28 is mutated to any one of A, C, I, L, M, S, or V; or

[0038] Amino acid 29 is mutated to any one of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0039] Amino acid 30 is mutated to any one of A, D, E, F, G, H, K, M, N, Q, R, or W; or

[0040] Amino acid 31 is mutated to either G or S; or

[0041] Amino acid number 32 mutates to any one of A, C, F, I, L, M, S, T, V, and Y; or amino acid number 33 mutates to any one of A, C, D, E, G, H, I, K, L, M, N, Q, R, S, T, and Y; or

[0042] Amino acid 34 mutates to any one of F, H, M, T, W, or Y; or

[0043] Amino acid 35 is mutated to any one of A, F, H, M, N, Q, or Y; or

[0044] Amino acid 36 is mutated to any one of C, D, E, F, H, I, K, L, M, N, R, S, V, W, or Y; or

[0045] Amino acid 37 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, or Y; or

[0046] Amino acid 38 is mutated to any one of A, K, M, Q, R, S, or V; or

[0047] Amino acid 39 is mutated to any one of H, K, N, R, S, T, V, or Y; or

[0048] Amino acid 40 is mutated to any one of A, C, D, E, G, H, K, M, N, Q, R, S, or Y; or

[0049] Amino acid 41 is mutated to any one of A, D, E, H, N, Q, S, or W; or

[0050] Amino acid 42 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; or

[0051] Amino acid 43 is mutated to any one of A, G, I, K, N, R, S, T, V, or Y; or

[0052] Amino acid 44 is mutated to any one of A, G, and S; or

[0053] Amino acid 45 is mutated to any one of A, D, F, G, H, K, L, M, N, Q, R, S, V, W, or Y; or

[0054] Amino acid 46 is mutated to any one of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; or

[0055] Amino acid 47 is mutated to any one of I, L, M, Q, or V; or

[0056] Amino acid 48 is mutated to any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0057] Amino acid 49 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0058] Amino acid 50 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0059] Amino acid 51 is mutated to any one of A, C, G, I, L, M, S, T, or V; or

[0060] Amino acid 52 is mutated to any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0061] Amino acid 53 is mutated to any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0062] Amino acid 54 is mutated to any one of A, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0063] The amino acid 55 is mutated to any one of F, I, L, M, S, or T.

[0064] Preferably, the amino acid sequence of the CD276 mini-binding protein mutant is SEQ ID No.: 2.

[0065] In another aspect, the present invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding the CD276 mini-binding protein or a mutant thereof as described above.

[0066] In another aspect, the present invention provides a vector comprising the isolated nucleic acid described above.

[0067] In another aspect, the present invention provides a chimeric antigen receptor comprising, from N-terminus to C-terminus:

[0068] (a) Signal peptide;

[0069] (b) an extracellular antigen-binding domain comprising or consisting of the CD276 mini-binding protein or a mutant thereof as described above;

[0070] (c) hinge region;

[0071] (d) transmembrane domain;

[0072] (e) intracellular co-stimulatory signaling domain;

[0073] (f) Intracellular stimulatory signaling domain.

[0074] In a specific embodiment, the signal peptide is derived from human CD8α, and its amino acid sequence is: SEQ ID No.: 4; the hinge region is derived from human CD8α, and its amino acid sequence is: SEQ ID No.: 5; the transmembrane domain is derived from human CD8α, and its amino acid sequence is SEQ ID No.: 6; the intracellular co-stimulatory signaling domain is derived from human 4-1BB, and its amino acid sequence is SEQ ID No.: 7; the intracellular stimulatory signaling domain is derived from human CD3ζ, and its amino acid sequence is SEQ ID No.: 8.

[0075] In a specific embodiment, the amino acid sequence of the chimeric antigen receptor is SEQ ID No.: 3.

[0076] In yet another aspect, the present invention provides an engineered immune effector cell comprising the aforementioned chimeric antigen receptor.

[0077] In a specific embodiment, the immune effector cells are T cells, NK cells, macrophages.

[0078] In a specific embodiment, the engineered immune effector cells are CAR-T cells.

[0079] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned engineered immune effector cells and a pharmaceutically acceptable carrier.

[0080] In another aspect, the present invention provides an antibody comprising the above-mentioned CD276 mini-binding protein or a mutant thereof.

[0081] On the other hand, the present invention provides the use of the above-mentioned CD276 mini-binding protein or its mutant in the preparation of monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, and engineered immune effector cells.

[0082] In another aspect, the present invention provides uses of the CD276 mini-binding protein or its mutant, the engineered immune effector cell, the pharmaceutical composition, and the antibody in the preparation of anti-tumor drugs.

[0083] In a specific embodiment, the tumor is selected from glioblastoma (GBM). Beneficial effects

[0084] This application precisely designs binding proteins for the CD276 protein, which is abnormally highly expressed on the surface of various solid tumor cancer cells, optimizes its affinity and stability, and identifies its biochemical properties. A binding protein, HM9, was precisely designed for CD276 (B7-H3), and its affinity was optimized by yeast surface display, ultimately achieving a binding affinity of 36.4 pM for CD276. This binding protein has a small molecular weight, good stability, high affinity, and can be expressed in large quantities in Escherichia coli.

[0085] Furthermore, based on the artificially designed binding protein HM9 targeting CD276, CAR-T cells targeting CD276 were constructed. The killing, proliferation, and depletion of several tumor cell lines that highly express CD276 were verified at the cellular and animal levels. The results were compared with CAR-T cells constructed with CD276scFv. Compared with traditional scFc-based CAR-T treatment strategies, CAR-T cells based on the artificially designed binding protein HM9 have higher tumor killing activity, lower depletion levels, and faster division and expansion capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1: Heat map of flow cytometric enrichment analysis of single amino acid mutations in CD276_mb.

[0087] Figure 2: PAGE gel image of the protein purification process of CD276_mb and HM9 expressed in E. coli BL21 (DE3). In the figure, M represents Blue Plus V Protein Marker (DM141-01, TransGen), O represents whole cell lysate, S represents supernatant, F represents flow-through, W represents wash solution, E represents eluate, and A represents protein solution purified by molecular sieve chromatography.

[0088] Figure 3: Purification results of CD276_mb and HM9 using molecular sieve chromatography Superdex75 10 / 300GL column, orange is CD276_mb, blue is HM9.

[0089] Figure 4: Affinity analysis of CD276_mb and CD276 measured using BLI.

[0090] FIG5 shows a graph showing the affinity analysis of HM9 and CD276 determined using BLI.

[0091] Figure 6: CD276mb CAR-T design diagram.

[0092] Figure 7: Flow cytometry was used to measure the killing, T cell proliferation, and exhaustion of different tumor cells by HM9CAR-T cells.

[0093] Figure 8: Flowchart of CAR-T therapy for orthotopic tumor-bearing mice.

[0094] FIG9 : Graphs showing the effects of HM9-4-1BB CAR-T therapy on survival time (A) and tumor signal generation (B) in orthotopic tumor-bearing mice. DETAILED DESCRIPTION

[0095] The technical solution of the present application is described in detail below through specific embodiments so that those skilled in the art can better understand the technical solution of the present application. However, the scope of protection of the present application is not limited thereto, but also includes any equivalent and replacement forms that can be made by those skilled in the art based on the present application.

[0096] the term

[0097] In this application, CD276 mini-binding protein can be represented as: CD276_mb.

[0098] Example 1: Heat map of flow cytometric enrichment analysis of full mutations of CD276_mb (SEQ ID No.: 1) The sequence of the full mutation of single amino acids in CD276_mb was constructed into the pETCON3 plasmid, and the N-terminus was fused with Aga1p-Factor Xa site-HA-GS Linker (SEQ ID No.: 9), and the C-terminus was fused with cMyc tag (SEQ ID No.: 10). The constructed plasmid was then electroporated into the Saccharomyces cerevisiae EBY100 strain and cultured in SDCAA medium supplemented with 2% (w / v) glucose for 48 h. The yeast cells were transferred to SGCAA medium supplemented with 0.2% (w / v) glucose to an initial cell density of 0.5×10 7 Cells were collected by centrifugation at 4000 g for 1 minute and washed with PBS supplemented with 0.1% (w / v) BSA, incubated with biotinylated CD276 for 30 minutes, washed again, incubated with anti-cMyc fluorescein isothiocyanate (FITC, 130-116-653, Miltenyi Biotech) and streptavidin-phycoerythrin (SAPE, SA10044, Thermo Fisher) for 30 minutes, washed, and analyzed by flow cytometry. The concentration of biotinylated CD276 was gradually reduced from 1 μM to 10 nM. The results of each round of sorting were deeply sequenced to analyze which point mutations were gradually enriched. The gradually enriched ones were shown in red, and the gradually reduced ones were shown in blue. The analysis heat map is shown in Figure 1.

[0099] As can be seen from Figure 1, some single amino acid mutations are significantly enriched, indicating that these mutations may increase the affinity of CD276_mb for CD276. These single-site mutations include but are not limited to the following:

[0100] Amino acid 1 is mutated to any one of A, D, E, F, G, H, I, L, M, N, P, R, T, V, W, or Y; or

[0101] Amino acid 2 is mutated to any one of A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or W; or

[0102] Amino acid 3 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, W, or Y; or

[0103] Amino acid 4 is mutated to any one of A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; or

[0104] Amino acid 5 is mutated to any one of A, D, E, H, I, N, Q, S, T, or V; or

[0105] The amino acid 6 is mutated to any one of F, L, M, W, or Y; or

[0106] Amino acid 7 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0107] Amino acid 8 is mutated to any one of I, M, and V; or

[0108] Amino acid 9 is mutated to any one of F, H, I, L, or Y; or

[0109] Amino acid 10 is mutated to any one of A, D, E, F, H, I, L, M, Q, S, T, V, W, or Y; or

[0110] Amino acid 11 is mutated to any one of A, H, K, N, Q, R, S, T, V, or Y; or

[0111] Amino acid 12 mutates to T; or

[0112] Amino acid 13 is mutated to any one of A, F, G, S, or T; or

[0113] Amino acid 14 is mutated to any one of A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0114] Amino acid 15 mutates to any one of A, D, G, H, N, S, T, or V; or

[0115] Amino acid 16 mutates to L; or

[0116] Amino acid 17 mutates to any one of E, F, I, L, M, T, or V; or

[0117] Amino acid 18 is mutated to any one of A, H, K, N, Q, R, or T; or

[0118] Amino acid 19 is mutated to any one of A, F, G, H, I, L, M, N, Q, R, S, T, or V; or

[0119] Amino acid 20 is mutated to any one of A, D, E, G, H, K, N, Q, R, S, or T; or

[0120] Amino acid 21 is mutated to any one of D, E, K, N, or Q; or

[0121] Amino acid 22 is mutated to any one of A, F, H, K, L, M, N, Q, R, S, T, V, or Y; or

[0122] Amino acid 23 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, or Y; or

[0123] The amino acid 24 is mutated to any one of L, M, V, or W; or

[0124] The amino acid 25 is mutated to either L or M; or

[0125] Amino acid 26 is mutated to any one of A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; or

[0126] Amino acid 27 is mutated to any one of H, I, P, or V; or

[0127] Amino acid 28 is mutated to any one of A, C, I, L, M, S, or V; or

[0128] Amino acid 29 is mutated to any one of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0129] Amino acid 30 is mutated to any one of A, D, E, F, G, H, K, M, N, Q, R, or W; or

[0130] Amino acid 31 is mutated to either G or S; or

[0131] Amino acid 32 is mutated to any one of A, C, F, I, L, M, S, T, V, or Y; or

[0132] Amino acid 33 is mutated to any one of A, C, D, E, G, H, I, K, L, M, N, Q, R, S, T, or Y; or

[0133] Amino acid 34 mutates to any one of F, H, M, T, W, or Y; or

[0134] Amino acid 35 is mutated to any one of A, F, H, M, N, Q, or Y; or

[0135] Amino acid 36 is mutated to any one of C, D, E, F, H, I, K, L, M, N, R, S, V, W, or Y; or

[0136] Amino acid 37 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, or Y; or

[0137] Amino acid 38 is mutated to any one of A, K, M, Q, R, S, or V; or

[0138] Amino acid 39 is mutated to any one of H, K, N, R, S, T, V, or Y; or

[0139] Amino acid 40 is mutated to any one of A, C, D, E, G, H, K, M, N, Q, R, S, or Y; or

[0140] Amino acid 41 is mutated to any one of A, D, E, H, N, Q, S, or W; or

[0141] Amino acid 42 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; or

[0142] Amino acid 43 is mutated to any one of A, G, I, K, N, R, S, T, V, or Y; or

[0143] Amino acid 44 is mutated to any one of A, G, and S; or

[0144] Amino acid 45 is mutated to any one of A, D, F, G, H, K, L, M, N, Q, R, S, V, W, or Y; or

[0145] Amino acid 46 is mutated to any one of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; or

[0146] Amino acid 47 is mutated to any one of I, L, M, Q, or V; or

[0147] Amino acid 48 is mutated to any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0148] Amino acid 49 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0149] Amino acid 50 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0150] Amino acid 51 is mutated to any one of A, C, G, I, L, M, S, T, or V; or

[0151] Amino acid 52 is mutated to any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0152] Amino acid 53 is mutated to any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0153] Amino acid 54 is mutated to any one of A, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; or

[0154] The amino acid at position 55 is mutated to any one of F, I, L, M, S, and T.

[0155] From these, amino acid 4 was mutated from R to W; amino acid 5 from E to V and I; amino acid 6 from L to F and W; amino acid 19 from L to Q; amino acid 24 from L to M; amino acid 32 from L to V and A; amino acid 35 from F to H and Y; and amino acid 48 from F to R, L, and M. These mutations were permuted and combined, and a new round of screening was performed using the screening method described above. The concentration of biotinylated CD276 was gradually reduced from 100 nM to 1 nM. The final round of screening resulted in the mini-binding protein mutant, HM9 (SEQ ID No.: 2).

[0156] Example 2: Expression and purification of CD276_mb and HM9

[0157] The genes encoding CD276_mb and HM9 were cloned into the pET-28a(+) E. coli expression vector containing an N-terminal 6×His tag and a thrombin cleavage site (SEQ ID No. 11: MGSSHHHHSSGLVPRGS). The cloned plasmids were transformed into E. coli BL21(DE3) (EC1002, WEIDIBio) and cultured in 1 L of LB broth at 37°C, 220 rpm, until the OD600 reached 0.6-0.8. IPTG (206-703-0, MACKLIN) was added to a final concentration of 250 μM and cultured overnight at 37°C, 220 rpm. Cells were harvested by centrifugation at 8000 g for 5 minutes, resuspended in 25 ml of protein buffer (25 mM Tris-HCl (pH 8.0) + 150 mM NaCl), and lysed by sonication for 10 minutes. Whole-cell lysates were separated by centrifugation at 14,000 RPM for 30 minutes. The target protein was purified from the supernatant using Ni-NTA resin. The whole-cell lysate, supernatant, flow-through, wash solution, and eluate were sampled and run on a 15% SurePAGE gel (M00719, GenScript). The protein gel image is shown in Figure 2.

[0158] The target protein was further purified using molecular sieve chromatography on a Superdex75 10 / 300GL column (29148721, Cytiva) in PBS buffer (pH 8.0). The chromatogram is shown in FIG3 .

[0159] As can be seen from Figure 3, both CD276_mb and HM9 can be expressed at high levels in E. coli BL21 (DE3), can be purified by Ni-NTA resin, and their configurations are monomeric without high polymer form and stable properties.

[0160] Example 3: Determination of the affinity of CD276_mb to CD276 and the affinity of HM9 to CD276 using BLI

[0161] The concentration of the purified protein was determined using a Bradford protein assay kit (P0006, Beyotime). The affinity of CD276_mb and HM9 for CD276 was determined by BLI assay at 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM.

[0162] The results are shown in Figures 4 and 5. As can be seen from the figures, the binding force between CD276_mb and CD276 after fitting is about 12.0 nM; the binding force between HM9 and CD276 after fitting is about 36.4 pM, which confirms that the HM9 obtained in this application has strong binding activity with CD276.

[0163] Example 4: CD276_mb CAR-T design and flow cytometry determination of HM9CAR-T cell killing of different tumor cells, T cell proliferation, and exhaustion.

[0164] The HM9 sequence was integrated into the CAR structure to form a complete HM9CAR coding sequence of CD8α signal peptide-HM9-CD8α hinge-CD8α transmembrane domain-4-1BB costimulatory domain-CD3ζ stimulatory domain. The HM9CAR sequence was cloned into the pCDH-CMV-MCS-EF1-copGFP (CD511B-1, systembio) vector. The structural schematic diagram of CAR (SEQ ID No.: 3) is shown in Figure 6. Among them, the amino acid sequence of the signal peptide is: SEQ ID No.: 4; the amino acid sequence of the hinge region is: SEQ ID No.: 5; the amino acid sequence of the transmembrane domain is SEQ ID No.: 6; the amino acid sequence of the intracellular costimulatory signaling domain is SEQ ID No.: 7; and the amino acid sequence of the intracellular stimulatory signaling domain is SEQ ID No.: 8.

[0165] At the same time, different CD276_mb and CD276scFv (clone 376.96, SEQ ID No.: 12) were constructed into CAR structures in the same manner as experimental controls.

[0166] The constructed CAR plasmid was transfected into HEK293T (CRL-3216, ATCC) cells with psPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) for lentiviral production and used to infect T cells isolated from normal human PBMCs, converting them into biologically functional CAR-T cells, namely HM9-4-1BB CAR-T cells.

[0167] HM9-4-1BB CAR-T cells were co-cultured with CD276-positive patient-derived glioblastoma cells GSC3565 and MGG4 at different effector cell (E) / target cell (T) ratios (1:1, 1:4, 1:16) for 48 hours, and the remaining cells were subjected to multicolor flow cytometric staining analysis (CD3-APC (OKT3, BioLegend); PD-1-PE (EH12.2H7, BioLegend); Fixable Viability Dye eFluor TM780 (65-0865-18, Thermo) and absolute cell population counts were performed using Precision Count Beads (424902, BioLegend). Killing % = tumor number (target well) / tumor number (tumor culture well only), proliferation fold = T cell number (target well) / T cell number (T cell culture well only), PD-1+ % = PD-1 positive cell number / T cell number. Flow cytometry was used to determine the tumor killing ability, cell proliferation ability, and anti-exhaustion ability of HM9-4-1BB CAR-T and scFv-4-1BB CAR-T cells against different glioblastoma tumor cells, GSC3565 and MGG6. The results are shown in Figure 7.

[0168] As can be seen from Figure 7, compared with scFv-4-1BB CAR-T cells, the CAR-T constructed in the present application has higher tumor killing activity, lower exhaustion level and faster division and amplification ability against different tumor cells. Its significantly enhanced cell proliferation ability and low exhaustion characteristics indicate that HM9-4-1BB CAR-T can retain the normally activated cytotoxic state and perform tumor killing function for a longer period of time when facing the challenge of solid tumors compared with scFv-4-1BB CAR-T cells, avoiding premature exhaustion and causing T cell dysfunction.

[0169] Example 5: Therapeutic Effect of HM9-4-1BB CAR-T on Tumor-Bearing Mice

[0170] Patient-derived glioblastoma cells (10,000 cells / 3 μl PBS) were injected into the cranium of NOD / SCID / IL2Rg- / - (NSG) mice (005557, JAX Lab) using a stereotaxic injection device at Bregma coordinates x = +2.0 mm, y = -2.0 mm, and z = -3.5 mm. Ten days after tumor implantation, these mice were treated with orthotopic CAR-T cell injections (1,000,000 cells / 3 μl PBS). See Figure 8 for a flow chart.

[0171] After tumor injection, the occurrence and development of the tumor and the time point when the mice developed neurological symptoms were recorded, and the survival curve of tumor-bearing mice was drawn.

[0172] The results are analyzed in Figure 9. The data in Figure 9 show that mice treated with HM9-4-1BB CAR-T survived for more than 80 days without detectable tumor signal generation, while mice treated with scFv-4-1BB CAR-T had prolonged survival of tumor-bearing mice but developed tumors to varying degrees. This result confirms that the HM9-4-1BB CAR-T constructed in this application exhibits significantly superior effects in tumor treatment compared to the prior art scFv-4-1BB CAR-T.

Claims

1. A CD276 minibody or a mutant thereof, Among them, wherein the CD276 minibody has the amino acid sequence shown in SEQ ID No.: 1, and the mutant is selected from: i) a mutant with the amino acid sequence of SEQ ID No.: 2; ii) the following single-site mutants of SEQ ID No.: 1: the 1st amino acid is mutated to any one of A, D, E, F, G, H, I, L, M, N, P, R, T, V, W, Y; or the 2nd amino acid is mutated to any one of A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W; or the 3rd amino acid is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, W, Y; or the 4th amino acid is mutated to any one of A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; or the 5th amino acid is mutated to any one of A, D, E, H, I, N, Q, S, T, V; or the 6th amino acid is mutated to any one of F, L, M, W, Y; or the 7th amino acid is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or the 8th amino acid is mutated to any one of I, M, V; or the 9th amino acid is mutated to any one of F, H, I, L, Y; or the 10th amino acid is mutated to any one of A, D, E, F, H, I, L, M, Q, S, T, V, W, Y; or the 11th amino acid is mutated to any one of A, H, K, N, Q, R, S, T, V, Y; or the 12th amino acid is mutated to T; or the 13th amino acid is mutated to any one of A, F, G, S, T; or the 14th amino acid is mutated to any one of A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or the 15th amino acid is mutated to any one of A, D, G, H, N, S, T, V; or the 16th amino acid is mutated to L; or the 17th amino acid is mutated to any one of E, F, I, L, M, T, V; or the 18th amino acid is mutated to any one of A, H, K, N, Q, R, T; or the 19th amino acid is mutated to any one of A, F, G, H, I, L, M, N, Q, R, S, T, V; or the 20th amino acid is mutated to any one of A, D, E, G, H, K, N, Q, R, S, T; or the 21st amino acid is mutated to any one of D, E, K, N, Q; or the 22nd amino acid is mutated to any one of A, F, H, K, L, M, N, Q, R, S, T, V, Y; or the 23rd amino acid is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y; or the 24th amino acid is mutated to any one of L, M, V, W; or the 25th amino acid is mutated to any one of L, M; or The 26th amino acid is mutated to any one of A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; or The 27th amino acid is mutated to any one of H, I, P, V; or The 28th amino acid is mutated to any one of A, C, I, L, M, S, V; or The 29th amino acid is mutated to any one of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or The 30th amino acid is mutated to any one of A, D, E, F, G, H, K, M, N, Q, R, W; or The 31st amino acid is mutated to any one of G, S; or The 32nd amino acid is mutated to any one of A, C, F, I, L, M, S, T, V, Y; or The 33rd amino acid is mutated to any one of A, C, D, E, G, H, I, K, L, M, N, Q, R, S, T, Y; or The 34th amino acid is mutated to any one of F, H, M, T, W, Y; or The 35th amino acid is mutated to any one of A, F, H, M, N, Q, Y; or The 36th amino acid is mutated to any one of C, D, E, F, H, I, K, L, M, N, R, S, V, W, Y; or The 37th amino acid is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, Y; or The 38th amino acid is mutated to any one of A, K, M, Q, R, S, V; or The 39th amino acid is mutated to any one of H, K, N, R, S, T, V, Y; or The 40th amino acid is mutated to any one of A, C, D, E, G, H, K, M, N, Q, R, S, Y; or The 41st amino acid is mutated to any one of A, D, E, H, N, Q, S, W; or The 42nd amino acid is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; or The 43rd amino acid is mutated to any one of A, G, I, K, N, R, S, T, V, Y; or The 44th amino acid is mutated to any one of A, G, S; or The 45th amino acid is mutated to any one of A, D, F, G, H, K, L, M, N, Q, R, S, V, W, Y; or The 46th amino acid is mutated to any one of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; or The 47th amino acid is mutated to any one of I, L, M, Q, V; or The 48th amino acid is mutated to any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or The 49th amino acid is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or The 50th amino acid is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or The 51st amino acid is mutated to any one of A, C, G, I, L, M, S, T, V; or The 52nd amino acid is mutated to any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or The 53rd amino acid is mutated to any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or The 54th amino acid is mutated to any one of A, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or The 55th amino acid is mutated to any one of F, I, L, M, S, T, Preferably, the amino acid sequence of the CD276 mini-binding protein mutant is SEQ ID No.:

2.

2. An isolated nucleic acid comprising a nucleic acid sequence encoding the CD276 mini-binding protein or a mutant thereof as claimed in claim 1.

3. A vector comprising the isolated nucleic acid as claimed in claim 2.

4. A chimeric antigen receptor comprising, in sequence from the N-terminus to the C-terminus: (a) A signal peptide; (b) An extracellular antigen-binding domain comprising or consisting of the CD276 mini-binding protein or a mutant thereof as claimed in claim 1; (c) A hinge region; (d) A transmembrane domain; (e) An intracellular co-stimulatory signaling domain; (f) An intracellular stimulatory signaling domain.

5. The chimeric antigen receptor according to claim 4, wherein, The signal peptide is derived from human CD8α and has an amino acid sequence of: SEQ ID No.: 4; the hinge region is derived from human CD8α and has an amino acid sequence of: SEQ ID No.: 5; the transmembrane domain is derived from human CD8α and has an amino acid sequence of SEQ ID No.: 6; the intracellular co-stimulatory signaling domain is derived from human 4-1BB and has an amino acid sequence of SEQ ID No.: 7; and the intracellular stimulatory signaling domain is derived from human CD3ζ and has an amino acid sequence of SEQ ID No.: 8, Preferably, the amino acid sequence of the chimeric antigen receptor is SEQ ID No.:

3.

6. An engineered immune effector cell comprising the chimeric antigen receptor as claimed in claim 4 or 5, Preferably, the immune effector cell is a T cell, NK cell, macrophage, and more preferably, the engineered immune effector cell is a CAR-T cell.

7. A pharmaceutical composition comprising the aforementioned engineered immune effector cell and a pharmaceutically acceptable carrier.

8. An antibody comprising the CD276 mini-binding protein or a mutant thereof as claimed in claim 1.

9. Use of the CD276 mini-binding protein or a mutant thereof as claimed in claim 1 in the preparation of a monoclonal antibody, bispecific antibody, antibody-drug conjugate, engineered immune effector cell.

10. Use of the CD276 mini-binding protein or a mutant thereof as claimed in claim 1, the engineered immune effector cell as claimed in claim 6, the pharmaceutical composition as claimed in claim 7, or the antibody as claimed in claim 8 in the preparation of an anti-tumor drug, Preferably, the tumor is glioblastoma.

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