CD276 mini binding protein as well as mutant and application thereof

Abstract

The invention relates to a CD276 mini binding protein as well as a mutant and application thereof. The invention also relates to a chimeric antigen receptor containing the CD276 mini binding protein or the mutant thereof as an extracellular antigen binding domain and a chimeric antigen receptor T cell. The binding force of the CD276 binding protein mutant HM9 and CD276 reaches 36.4 pM, and the binding protein is small in protein molecular weight, good in stability and high in affinity and can be massively expressed in escherichia coli. Compared with a traditional scFc-based CAR-T treatment strategy, the CAR-T cell constructed by the invention has higher tumor killing activity, lower depletion level and faster division and amplification capability.

Description

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-related protein. It was first cloned and identified from a cDNA library derived from human dendritic cells in 2001. The human CD276 gene is located on chromosome 15. It is mostly expressed as a membrane protein and is a type I transmembrane protein with 316 amino acids, consisting of an extracellular domain, a transmembrane domain, and a short intracellular domain. Studies have shown that CD276 expression is abnormally elevated in tumor cells, and the adhesion ability of cell adhesion proteins is increased, leading to enhanced migration and invasion of tumor cells; in addition, CD276 can lead to increased NF-κB activity, resulting in increased expression of VEGF and IL-8, further promoting tumor-related angiogenesis and tumor invasion. Since CD276 is abnormally highly expressed in a variety of cancer cells including medulloblastoma and glioblastoma, but has limited distribution in normal tissues, it is an effective safety target. At present, a variety of B7-H3 targeted immunotherapy strategies, including monoclonal antibodies, bispecific antibodies, ADCs, and CAR-T, have been developed and tested in clinical trials, and have shown good application prospects, making it a very attractive target. Designing and developing more efficient protein drugs and immunotherapy strategies targeting CD276 has great value.

[0003] Protein design is based on the principles of biophysics and biochemistry. By designing the amino acid sequence of proteins, they can spontaneously fold to form the required new three-dimensional structure and have new functions. The design of functional proteins, especially the design of new binding proteins with high affinity and high specificity for any natural protein, has very important applications in diagnosis, treatment and prevention. Compared with protein drugs represented by monoclonal antibodies, artificially designed binding proteins have obvious advantages in the following aspects: small protein molecular weight, good tissue penetration, strong chemical activity, good stability, easy industrialization, and can be quickly designed for specific epitopes of the target, and the binding mode can be precisely controlled. It has become possible to design binding proteins based entirely on the three-dimensional structural information of the target protein. New protein design algorithms and tools provide new tools and R&D models for basic protein research and protein drug development. The development of protein drugs and immunotherapy strategies targeting CD276 based on the latest protein design algorithms has important application prospects and value.

[0004] Chimeric antigen receptor (CAR)-T therapy is considered to be another major revolutionary breakthrough in the field of cancer immunotherapy after immune checkpoint, and has achieved great success in the field of hematological malignancies. CAR-T treatment involves collecting and isolating T cells from a patient's blood, and genetically engineering 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 antigen chimeric receptor (CAR) in CAR-T therapy is mainly composed of the single-chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding to the antigen. The scFv is formed by linking the light chain (VL) and heavy chain (VH) of the monoclonal antibody with a polypeptide, retaining the specificity and affinity of the antibody for the antigen. However, the heavy and light chains of the scFv are also prone to misaggregation and misfolding, with poor structural stability, which not only affects the ability of T cells to recognize antigen cells, but also causes antigen-independent CAR aggregation and T cell activation, leading to premature exhaustion of T cells, greatly reducing the therapeutic effect of CAR-T therapy. Given the abnormally high expression of CD276 in many solid tumor cells, developing a binding protein targeting CD276 based on de novo protein design and developing a new generation of CAR-T treatment strategies targeting CD276 have great research and clinical application value. Summary of the Invention

[0005] In view of the above technical problems, on the one hand, the present invention provides a CD276 mini-binding protein or a mutant thereof,

[0006] wherein the CD276 mini-binding protein has the amino acid sequence shown in SEQ ID No.: 1, and

[0007] the mutant is selected from:

[0008] i) a mutant with the amino acid sequence of SEQ ID No.: 2;

[0009] ii) the following single-site mutants of SEQ ID No.: 1:

[0010] 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

[0011] 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

[0012] 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

[0013] 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

[0014] The 5th amino acid is mutated to any one of A, D, E, H, I, N, Q, S, T, V; or

[0015] The 6th amino acid is mutated to any one of F, L, M, W, Y; or

[0016] 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

[0017] The 8th amino acid is mutated to any one of I, M, V; or

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

[0019] 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

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

[0021] The 12th amino acid is mutated to T; or

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

[0023] 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

[0024] The 15th amino acid is mutated to any one of A, D, G, H, N, S, T, V; or

[0025] The 16th amino acid is mutated to L; or

[0026] The 17th amino acid is mutated to any one of E, F, I, L, M, T, V; or

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

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

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

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

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

[0032] 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, or Y; or

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

[0034] The 25th amino acid is mutated to any one of L or M; or

[0035] 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, or Y; or

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

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

[0038] 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, or Y; or

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

[0040] The 31st amino acid is mutated to any one of G or S; or

[0041] The 32nd amino acid is mutated to any one of A, C, F, I, L, M, S, T, V, or Y; or

[0042] 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, or Y; or

[0043] The 34th amino acid is mutated to any one of F, H, M, T, W, or Y; or

[0044] The 35th amino acid is mutated to any one of A, F, H, M, N, Q, or Y; or

[0045] The 36th amino acid is mutated to any one of C, D, E, F, H, I, K, L, M, N, R, S, V, W, or Y; or

[0046] The 37th amino acid is mutated into any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, Y; or

[0047] The 38th amino acid is mutated into any one of A, K, M, Q, R, S, V; or

[0048] The 39th amino acid is mutated into any one of H, K, N, R, S, T, V, Y; or

[0049] The 40th amino acid is mutated into any one of A, C, D, E, G, H, K, M, N, Q, R, S, Y; or

[0050] The 41st amino acid is mutated into any one of A, D, E, H, N, Q, S, W; or

[0051] The 42nd amino acid is mutated into any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; or

[0052] The 43rd amino acid is mutated into any one of A, G, I, K, N, R, S, T, V, Y; or

[0053] The 44th amino acid is mutated into any one of A, G, S; or

[0054] The 45th amino acid is mutated into any one of A, D, F, G, H, K, L, M, N, Q, R, S, V, W, Y; or

[0055] The 46th amino acid is mutated into any one of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; or

[0056] The 47th amino acid is mutated into any one of I, L, M, Q, V; or

[0057] The 48th amino acid is mutated into any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or

[0058] The 49th amino acid is mutated into any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or

[0059] The 50th amino acid is mutated into any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or

[0060] The 51st amino acid is mutated into any one of A, C, G, I, L, M, S, T, V; or

[0061] 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

[0062] 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

[0063] 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

[0064] The 55th amino acid is mutated to any one of F, I, L, M, S, T,

[0065] Preferably, the amino acid sequence of the CD276 mini - binding protein mutant is SEQ ID No.: 2.

[0066] On the other hand, the present invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding the CD276 mini - binding protein or its mutant as described above.

[0067] On the other hand, the present invention provides a vector comprising the isolated nucleic acid as described above.

[0068] On the other hand, the present invention provides a chimeric antigen receptor which sequentially comprises, from the N - terminus to the C - terminus:

[0069] (a) A signal peptide;

[0070] (b) An extracellular antigen - binding domain which comprises or consists of the CD276 mini - binding protein or its mutant as described above;

[0071] (c) A hinge region;

[0072] (d) A transmembrane domain;

[0073] (e) An intracellular co - stimulatory signaling domain;

[0074] (f) An intracellular stimulatory signaling domain.

[0075] 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.

[0076] In a specific embodiment, the amino acid sequence of the chimeric antigen receptor is SEQ ID No.: 3.

[0077] In another aspect, the present invention provides an engineered immune effector cell comprising the aforementioned chimeric antigen receptor.

[0078] In a specific embodiment, the immune effector cell is a T cell, an NK cell, or a macrophage.

[0079] In a specific embodiment, the engineered immune effector cell is a CAR-T cell.

[0080] In yet another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned engineered immune effector cell and a pharmaceutically acceptable carrier.

[0081] In still another aspect, the present invention provides an antibody comprising the aforementioned CD276 mini-binding protein or a mutant thereof.

[0082] In another aspect, the present invention provides the use of the aforementioned CD276 mini-binding protein or a mutant thereof in the preparation of monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, and engineered immune effector cells.

[0083] In yet another aspect, the present invention provides the use of the aforementioned CD276 mini-binding protein or a mutant thereof, the aforementioned engineered immune effector cell, the aforementioned pharmaceutical composition, and the aforementioned antibody in the preparation of anti-tumor drugs.

[0084] In a specific embodiment, the tumor is selected from glioblastoma (GBM).

[0085] Beneficial effects

[0086] The present application precisely designs a binding protein for the CD276 protein that is abnormally highly expressed on the surface of cancer cells in multiple solid tumors, 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 the method of yeast surface display, and finally its binding force to CD276 reached 36.4 pM. This binding protein has a small molecular weight, good stability, high affinity, and can be expressed in large quantities in Escherichia coli.

[0087] Furthermore, based on the artificially designed binding protein HM9 targeting CD276, CAR-T cells against CD276 were constructed, and the killing, proliferation, and exhaustion of these CAR-T cells against several tumor cell lines with high CD276 expression were verified at the cellular and animal levels, and compared with CAR-T cells constructed with CD276 scFv. Compared with the traditional CAR-T treatment strategy based on scFc, the CAR-T cells based on the artificially designed binding protein HM9 have higher tumor killing activity, lower exhaustion level, and faster division and amplification ability. Description of the Drawings

[0088] Figure 1 : Heat map of flow cytometry enrichment analysis of single amino acid mutations of CD276_mb.

[0089] Figure 2 : PAGE gel diagram of the protein purification process of expressing CD276_mb and HM9 in Escherichia 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.

[0090] Figure 3 : Results diagram of purifying CD276_mb and HM9 using a Superdex75 10 / 300GL chromatography column for molecular sieve chromatography. Orange is CD276_mb and blue is HM9.

[0091] Figure 4 : Affinity analysis diagram of CD276_mb and CD276 measured by BLI.

[0092] Figure 5 : Affinity analysis diagram of HM9 and CD276 measured by BLI.

[0093] Figure 6 : Design diagram of CD276 mb CAR-T.

[0094] Figure 7 : Detection of the killing, T cell proliferation, and exhaustion of HM9 CAR-T cells against different tumor cells using flow cytometry.

[0095] Figure 8 : Flow chart of CAR-T treatment for in situ tumor-bearing mice.

[0096] Figure 9 : Diagram showing the effects of HM9-4-1BB CAR-T treatment on the survival time (A) and tumor signal generation (B) of in situ tumor-bearing mice. Detailed implementation manners

[0097] The technical solutions of the present application will be described in detail below through specific embodiments, so that those skilled in the art can better understand the technical solutions of the present application. However, the protection scope of the present application is not limited thereto, and also includes any equivalent and substitution forms that those skilled in the art can make based on the present application.

[0098] Term

[0099] In the present application, the CD276 mini-binding protein can be expressed as: CD276_mb.

[0100] Example 1: Heat map of flow cytometry enrichment analysis of full mutations of CD276_mb (SEQ ID No.: 1) The sequences of full single amino acid mutations of CD276_mb were constructed into the pETCON3 plasmid, with the N-terminus fused to Aga1p-Factor Xa site-HA-GS Linker (SEQ ID No.: 9) and the C-terminus fused to the cMyc tag (SEQ ID No.: 10). Subsequently, the constructed plasmid was electrotransformed into the Saccharomyces cerevisiae strain EBY100 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 make the initial cell density 0.5×10 7 cells / ml, and induced at 30 °C and 200 RPM for 16 h. The cells were collected by centrifugation at 4000 g for 1 minute, washed with PBS supplemented with 0.1% (w / v) BSA, incubated with biotinylated CD276 for 30 minutes, washed again, incubated with fluorescein isothiocyanate (FITC, 130-116-653, Miltenyi Biotech) against cMyc and streptavidin-phycoerythrin (SAPE, SA10044, Thermo Fisher) for 30 minutes, washed and analyzed by flow cytometry. The concentration of biotinylated CD276 was gradually decreased from 1 μM to 10 nM, and deep sequencing was performed on the results of each round of sorting to analyze which point mutations would be gradually enriched. Those that were gradually enriched were shown in red, and those that were gradually decreased were shown in blue. The analysis heat map is shown in Figure 1 .

[0101] From Figure 1 it can be seen that some single amino acid mutations were significantly enriched, that is, these mutations may be able to enhance the affinity between CD276_mb and CD276. These single-site mutations include but are not limited to the following situations:

[0102] 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

[0103] The amino acid at position 2 is mutated to any one of A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W; or

[0104] The amino acid at position 3 is mutated to any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, W, Y; or

[0105] The amino acid at position 4 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

[0106] The amino acid at position 5 is mutated to any one of A, D, E, H, I, N, Q, S, T, V; or

[0107] The amino acid at position 6 is mutated to any one of F, L, M, W, Y; or

[0108] The amino acid at position 7 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

[0109] The amino acid at position 8 is mutated to any one of I, M, V; or

[0110] The amino acid at position 9 is mutated to any one of F, H, I, L, Y; or

[0111] The amino acid at position 10 is mutated to any one of A, D, E, F, H, I, L, M, Q, S, T, V, W, Y; or

[0112] The amino acid at position 11 is mutated to any one of A, H, K, N, Q, R, S, T, V, Y; or

[0113] The amino acid at position 12 is mutated to T; or

[0114] The amino acid at position 13 is mutated to any one of A, F, G, S, T; or

[0115] The amino acid at position 14 is mutated to any one of A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; or

[0116] The amino acid at position 15 is mutated to any one of A, D, G, H, N, S, T, V; or

[0117] The amino acid at position 16 is mutated to L; or

[0118] The amino acid at position 17 is mutated to any one of E, F, I, L, M, T, V; or

[0119] The 18th amino acid is mutated to any one of A, H, K, N, Q, R, T; or

[0120] The 19th amino acid is mutated to any one of A, F, G, H, I, L, M, N, Q, R, S, T, V; or

[0121] The 20th amino acid is mutated to any one of A, D, E, G, H, K, N, Q, R, S, T; or

[0122] The 21st amino acid is mutated to any one of D, E, K, N, Q; or

[0123] The 22nd amino acid is mutated to any one of A, F, H, K, L, M, N, Q, R, S, T, V, Y; or

[0124] 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

[0125] The 24th amino acid is mutated to any one of L, M, V, W; or

[0126] The 25th amino acid is mutated to any one of L, M; or

[0127] 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

[0128] The 27th amino acid is mutated to any one of H, I, P, V; or

[0129] The 28th amino acid is mutated to any one of A, C, I, L, M, S, V; or

[0130] 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

[0131] The 30th amino acid is mutated to any one of A, D, E, F, G, H, K, M, N, Q, R, W; or

[0132] The 31st amino acid is mutated to any one of G, S; or

[0133] The 32nd amino acid is mutated to any one of A, C, F, I, L, M, S, T, V, Y; or

[0134] 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

[0135] The 34th amino acid is mutated into any one of F, H, M, T, W, and Y; or

[0136] The 35th amino acid is mutated into any one of A, F, H, M, N, Q, and Y; or

[0137] The 36th amino acid is mutated into any one of C, D, E, F, H, I, K, L, M, N, R, S, V, W, and Y; or

[0138] The 37th amino acid is mutated into any one of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, and Y; or

[0139] The 38th amino acid is mutated into any one of A, K, M, Q, R, S, and V; or

[0140] The 39th amino acid is mutated into any one of H, K, N, R, S, T, V, and Y; or

[0141] The 40th amino acid is mutated into any one of A, C, D, E, G, H, K, M, N, Q, R, S, and Y; or

[0142] The 41st amino acid is mutated into any one of A, D, E, H, N, Q, S, and W; or

[0143] The 42nd amino acid is mutated into any one of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; or

[0144] The 43rd amino acid is mutated into any one of A, G, I, K, N, R, S, T, V, and Y; or

[0145] The 44th amino acid is mutated into any one of A, G, and S; or

[0146] The 45th amino acid is mutated into any one of A, D, F, G, H, K, L, M, N, Q, R, S, V, W, and Y; or

[0147] The 46th amino acid is mutated into any one of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; or

[0148] The 47th amino acid is mutated into any one of I, L, M, Q, and V; or

[0149] The 48th amino acid is mutated into any one of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; or

[0150] 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

[0151] 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

[0152] The 51st amino acid is mutated to any one of A, C, G, I, L, M, S, T, V; or

[0153] 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

[0154] 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

[0155] 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

[0156] The 55th amino acid is mutated to any one of F, I, L, M, S, T.

[0157] Among them, it was selected that the 4th amino acid was mutated from R to W; the 5th amino acid was mutated from E to V, I; the 6th amino acid was mutated from L to F, W; the 19th amino acid was mutated from L to Q; the 24th amino acid was mutated from L to M; the 32nd amino acid was mutated from L to V, A; the 35th amino acid was mutated from F to H, Y; the 48th amino acid was mutated from F to R, L, M. These mutations were permutated and combined, and a new round of screening was carried out using the screening method described above. The concentration of biotinylated CD276 gradually decreased from 100 nM to 1 nM, and the mini - binding protein mutant - HM9 (SEQ ID No.: 2) was obtained in the last round of screening.

[0158] Example 2: Expression, isolation and purification of CD276_mb and HM9

[0159] The genes encoding CD276_mb and HM9 were cloned into the pET-28a(+) E. coli expression vector with a 6×His tag and a thrombin cleavage site (SEQ ID No.11: MGSSHHHHSSGLVPRGS) at the N-terminus. The cloned plasmid was transformed into E. coli BL21(DE3) (EC1002, WEIDIBio), and cultured in 1 L of LB broth at 37 °C with 220 RPM until the OD600 reached 0.6 - 0.8. IPTG (206 - 703 - 0, MACKLIN) with a final concentration of 250 μM was added, and the culture was continued overnight at 37 °C with 220 RPM. The cells were collected 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. The whole cell lysate was separated by centrifugation at 14000 RPM for 30 minutes. The target protein was purified from the supernatant using Ni-NTA resin. Samples of the whole cell lysate, supernatant, flow-through, wash, and eluate were run on a 15% SurePAGE gel (M00719, GenScript). The protein gel image is shown in Figure 2 。

[0160] The target protein was further purified using a Superdex75 10 / 300GL column (29148721, Cytiva) for size exclusion chromatography in PBS buffer (pH 8.0). The chromatogram is shown in Figure 3 。

[0161] It can be seen from Figure 3 that both CD276_mb and HM9 can be expressed at a high level in E. coli BL21(DE3), can be purified by Ni-NTA resin, and their configurations are monomers, without high polymer forms, and are stable in nature.

[0162] Example 3: Measuring the affinity of CD276_mb for CD276 and the affinity of HM9 for CD276 using BLI

[0163] The concentration of the purified protein was measured using a Bradford protein concentration assay kit (P0006, Beyotime). The affinities of CD276_mb and HM9 for CD276 were measured by BLI experiments at 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM.

[0164] The measurement results are shown in Figure 4 and 5As shown. It can be seen from the figure that 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 obtained HM9 in this application has strong binding activity with CD276.

[0165] Example 4: Design of CD276_mb CAR-T and determination of the killing of different tumor cells, T cell proliferation, and exhaustion of HM9 CAR-T cells using a flow cytometer.

[0166] Integrate the HM9 sequence into the CAR structure to form a complete HM9 CAR coding sequence of CD8α signal peptide - HM9 - CD8α hinge - CD8α transmembrane domain - 4 - 1BB co - stimulatory domain - CD3ζ stimulatory domain. Clone the HM9 CAR sequence into the pCDH - CMV - MCS - EF1 - copGFP (CD511B - 1, systembio) vector. The structural pattern 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 co - stimulatory signal transduction domain is SEQ ID No.: 7; the amino acid sequence of the intracellular stimulatory signal transduction domain is SEQ ID No.: 8.

[0167] At the same time, different CD276_mb and CD276 scFv (clone 376.96, SEQ ID No.: 12) were constructed into the CAR structure in the same way as experimental controls.

[0168] The constructed CAR plasmids were transfected into HEK293T (CRL - 3216, ATCC) cells together with psPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) for lentivirus production and used to infect T cells isolated from normal human PBMCs to transform them into biologically functional CAR - T cells, namely HM9 - 4 - 1BB CAR - T cells.

[0169] The HM9-4-1BB CAR-T cells were co-cultured with CD276-positive patient-derived glioblastoma cells GSC3565 and MGG4 for 48 hours at different effector cell (E) / target cell (T) ratios (1:1, 1:4, 1:16), and the remaining cells were subjected to multi-color flow cytometry staining analysis (CD3-APC (OKT3, BioLegend); PD-1-PE (EH12.2H7, BioLegend); Fixable Viability Dye eFluor TM 780 (65-0865-18, Thermo)) and absolute counting of cell populations was performed using Precision Count Beads (424902, BioLegend). Killing % = number of tumors (target wells) / number of tumors (tumor-only culture wells), proliferation fold = number of T cells (target wells) / number of T cells (T cell-only culture wells), % of PD-1+ = number of PD-1-positive cells / number of T cells. Flow cytometry experiments were used to measure 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 .

[0170] From Figure 7 It can be seen that compared with scFv-4-1BB CAR-T cells, the CAR-T constructed in this application has higher tumor killing activity, lower exhaustion levels, and faster proliferation and expansion ability against different tumor cells. Its significantly enhanced cell proliferation ability and low exhaustion characteristics indicate that HM9-4-1BB CAR-T can maintain a normal activated cytotoxic state and perform tumor killing functions more persistently compared to scFv-4-1BB CAR-T cells when facing solid tumor challenges, avoiding premature exhaustion and causing T cell dysfunction.

[0171] Example 5: Therapeutic effect of HM9-4-1BB CAR-T on tumor-bearing mice

[0172] Patient-derived glioblastoma cells (10,000 cells / 3 μl PBS) were injected intracranially into NOD / SCID / IL2Rg− / − (NSG) mice (005557, JAX Lab) using a stereotactic injector at coordinates Bregma x = +2.0 mm, y = -2.0 mm, z = -3.5 mm. Ten days after tumor implantation, in-situ CAR-T cell (1,000,000 cells / 3 μl PBS) injection treatment was performed. The flow chart is shown in Figure 8 .

[0173] After tumor injection, record the tumor development and the time points when the mice showed neurological symptoms, and plot the survival curve of tumor-bearing mice.

[0174] The results analysis is shown in Figure 9 。 Figure 9 The data showed that the survival time of the mice treated with HM9-4-1BB CAR-T exceeded 80 days and no detectable tumor signals were generated. Although the survival time of the tumor-bearing mice treated with scFv-4-1BB CAR-T was prolonged, tumor development occurred to varying degrees. This result confirmed that compared with the existing scFv-4-1BB CAR-T, the HM9-4-1BB CAR-T constructed in this application showed significantly excellent effects in tumor treatment.

Claims

1. A CD276 mini-binding protein or a mutant thereof, Among them, wherein the CD276 mini-binding protein 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 its mutant 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 its mutant 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 further 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 its mutant as claimed in claim 1.

9. Use of the CD276 mini-binding protein or its mutant as claimed in claim 1 in the preparation of monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, and engineered immune effector cells.

10. Use of the CD276 mini-binding protein or its mutant 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 anti-tumor drugs, Preferably, the tumor is glioblastoma.

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