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WO2025129565A1 - Miniprotéine de liaison à cd276, mutant de celle-ci et utilisation associée - Google Patents

Miniprotéine de liaison à cd276, mutant de celle-ci et utilisation associée Download PDF

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WO2025129565A1
WO2025129565A1 PCT/CN2023/140615 CN2023140615W WO2025129565A1 WO 2025129565 A1 WO2025129565 A1 WO 2025129565A1 CN 2023140615 W CN2023140615 W CN 2023140615W WO 2025129565 A1 WO2025129565 A1 WO 2025129565A1
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amino acid
mutated
mutant
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seq
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曹龙兴
谢琦
金启涵
夏真
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Westlake Laboratory Of Life Sciences And Biomedicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • 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.
  • CD276 expression is abnormally elevated in tumor cells, and the adhesion ability of cell adhesion proteins increases accordingly, leading to enhanced migration and invasion of tumor cells; in addition, CD276 can lead to increased NF- ⁇ B 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 and safe target.
  • B7-H3 targeted immunotherapy strategies including monoclonal antibodies, bispecific antibodies, ADCs, and CAR-Ts, 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 is of great value.
  • 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.
  • 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.
  • CAR-T therapy is considered to be another revolutionary breakthrough in the field of tumor immunotherapy after immune checkpoints, and has achieved great success in the field of hematological tumors.
  • CAR-T therapy is to collect and separate T cells from the patient's blood, and then bioengineer them to express chimeric antigen receptors (CARs) that specifically recognize tumor surface antigens, thereby enhancing their targeting and killing capabilities against cancer cells.
  • CARs chimeric antigen receptors
  • the extracellular domain of the key antigen chimeric receptor (CAR) in CAR-T therapy is mainly composed of a single-chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding to antigens.
  • scFv is composed of the light chain (VL) and heavy chain (VH) of a monoclonal antibody connected by a polypeptide, retaining the antibody's specificity and affinity for the antigen.
  • VL light chain
  • VH heavy chain
  • the heavy and light chains of scFv are prone to mis-aggregation and folding, and have 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, which greatly reduces the therapeutic effect of CAR-T therapy.
  • Given the abnormally high expression of CD276 in many solid tumor cells developing a new generation of CAR-T treatment strategies targeting CD276 based on protein de novo design has significant research and clinical application value.
  • the present invention provides a CD276 mini-binding protein or a mutant thereof,
  • the CD276 mini-binding protein has an amino acid sequence shown in SEQ ID No.: 1, and
  • the mutant is selected from:
  • 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
  • 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
  • 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
  • Amino acid number 5 mutates to any one of A, D, E, H, I, N, Q, S, T, and V; or amino acid number 6 mutates to any one of F, L, M, W, and Y; or
  • 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
  • the amino acid number 8 is mutated to any one of I, M, or V; or
  • Amino acid 9 mutates to any one of F, H, I, L, and Y; or
  • 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
  • Amino acid 12 mutates to T;
  • Amino acid 13 mutates to any one of A, F, G, S, or T; or
  • 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
  • Amino acid 15 is mutated to any one of A, D, G, H, N, S, T, or V; or
  • Amino acid 16 mutates to L;
  • Amino acid 17 mutates to any one of E, F, I, L, M, T, or V; or
  • Amino acid 18 is mutated to any one of A, H, K, N, Q, R, or T; or
  • Amino acid 19 is mutated to any one of A, F, G, H, I, L, M, N, Q, R, S, T, or V; or
  • Amino acid 20 is mutated to any one of A, D, E, G, H, K, N, Q, R, S, or T; or
  • the amino acid 21 is mutated to any one of D, E, K, N, Q; or
  • Amino acid 22 is mutated to any one of A, F, H, K, L, M, N, Q, R, S, T, V, or Y; or
  • 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
  • the amino acid 24 is mutated to any one of L, M, V, and W; or
  • the amino acid 25 is mutated to either L or M; or
  • 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
  • the amino acid 27 is mutated to any one of H, I, P, or V; or
  • the amino acid 28 is mutated to any one of A, C, I, L, M, S, or V; or
  • the amino acid 31 is mutated to either G or S; or
  • 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
  • Amino acid number 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
  • Amino acid 47 is mutated to any one of I, L, M, Q, and V; or
  • Amino acid number 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
  • Amino acid number 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
  • Amino acid 51 is mutated to any one of A, C, G, I, L, M, S, T, or V; or
  • Amino acid number 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
  • Amino acid number 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
  • amino acid sequence of the CD276 mini binding protein mutant is SEQ ID No.: 2.
  • 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.
  • the present invention provides a vector comprising the isolated nucleic acid described above.
  • the present invention provides a chimeric antigen receptor, which comprises, from N-terminus to C-terminus:
  • an extracellular antigen-binding domain which comprises or consists of the CD276 mini-binding protein or a mutant thereof as described above;
  • the signal peptide is derived from human CD8 ⁇ , and its amino acid sequence is: SEQ ID No.: 4; the hinge region is derived from human CD8 ⁇ , and its amino acid sequence is: SEQ ID No.: 5; the transmembrane domain is derived from human CD8 ⁇ , and its amino acid sequence is SEQ ID No.: 6; the intracellular co-stimulatory signal transduction domain is derived from human 4-1BB, and its amino acid sequence is SEQ ID No.: 7; the intracellular stimulatory signal transduction domain is derived from human CD3 ⁇ , and its amino acid sequence is SEQ ID No.: 8.
  • amino acid sequence of the chimeric antigen receptor is SEQ ID No.: 3.
  • the present invention provides an engineered immune effector cell comprising the aforementioned chimeric antigen receptor.
  • the immune effector cells are T cells, NK cells, macrophages.
  • the engineered immune effector cells are CAR-T cells.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned engineered immune effector cells and a pharmaceutically acceptable carrier.
  • the present invention provides an antibody comprising the above-mentioned CD276 mini-binding protein or a mutant thereof.
  • 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.
  • the present invention provides use 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.
  • the tumor is selected from glioblastoma (GBM).
  • GBM glioblastoma
  • CAR-T cells targeting CD276 were constructed, and the killing, proliferation and exhaustion of several tumor cell lines with high expression of CD276 by the CAR-T cells were verified at the cellular and animal levels, and compared with CAR-T cells constructed with CD276scFv.
  • 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.
  • Figure 1 Heat map of flow cytometry enrichment analysis of single amino acid mutations in CD276_mb.
  • FIG. 2 PAGE gel image of protein purification process of CD276_mb and HM9 expressed in E. coli BL21 (DE3), in which M represents Blue Plus V Protein Marker (DM141-01, TransGen), O represents whole cell lysate, S represents supernatant, F represents flow-through, W represents washing solution, E represents eluent, and A represents molecular sieve chromatography purified protein solution.
  • M Blue Plus V Protein Marker (DM141-01, TransGen)
  • O represents whole cell lysate
  • S represents supernatant
  • F represents flow-through
  • W washing solution
  • E represents eluent
  • A molecular sieve chromatography purified protein solution.
  • Figure 3 The results of purification of CD276_mb and HM9 using molecular sieve chromatography Superdex75 10/300GL column. Orange is CD276_mb and blue is HM9.
  • FIG4 Affinity analysis of CD276_mb and CD276 measured using BLI.
  • FIG. 5 A graph showing the affinity analysis of HM9 and CD276 measured using BLI.
  • Figure 6 CD276mb CAR-T design diagram.
  • Figure 7 Flow cytometry was used to measure the killing of different tumor cells by HM9CAR-T cells, T cell proliferation, and exhaustion.
  • Figure 8 Flowchart of CAR-T therapy for orthotopic tumor-bearing mice.
  • Figure 9 Graph showing the effects of HM9-4-1BB CAR-T therapy on the survival time (A) and tumor signal generation (B) of orthotopic tumor-bearing mice.
  • CD276 mini-binding protein can be represented as: CD276_mb.
  • Example 1 Heat map of flow enrichment analysis of full mutation of CD276_mb (SEQ ID No.: 1)
  • the sequence of CD276_mb single amino acid full mutation was constructed into 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 electrotransformed into Saccharomyces cerevisiae EBY100 strain and cultured in SDCAA medium supplemented with 2% (w/v) glucose for 48h.
  • 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/ml and induced at 30°C 200RPM for 16h.
  • the cells were collected by centrifugation at 4000g for 1 minute, 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.
  • FITC anti-cMyc fluorescein isothiocyanate
  • SAPE streptavidin-phycoerythrin
  • the concentration of biotinylated CD276 was gradually reduced from 1 ⁇ M to 10nM, and the results of each round of sorting were deeply sequenced to analyze which point mutations would gradually enrich, and the gradually enriched ones were displayed in red, and the gradually reduced ones were displayed in blue.
  • the analysis heat map is shown in Figure 1.
  • 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
  • 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
  • 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
  • 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
  • Amino acid 5 is mutated to any one of A, D, E, H, I, N, Q, S, T, and V; or
  • the amino acid 6 is mutated to any one of F, L, M, W, and Y; or
  • 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
  • the amino acid number 8 is mutated to any one of I, M, or V; or
  • the amino acid number 9 is mutated to any one of F, H, I, L, and Y; or
  • 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
  • Amino acid 11 is mutated to any one of A, H, K, N, Q, R, S, T, V, and Y; or
  • Amino acid 16 mutates to L;
  • Amino acid 17 mutates to any one of E, F, I, L, M, T, or V; or
  • Amino acid 18 is mutated to any one of A, H, K, N, Q, R, or T; or
  • Amino acid 19 is mutated to any one of A, F, G, H, I, L, M, N, Q, R, S, T, or V; or
  • Amino acid 20 is mutated to any one of A, D, E, G, H, K, N, Q, R, S, or T; or
  • the amino acid 21 is mutated to any one of D, E, K, N, Q; or
  • the amino acid 24 is mutated to any one of L, M, V, and W; or
  • the amino acid 25 is mutated to either L or M; or
  • 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
  • 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
  • Amino acid number 30 is mutated to any one of A, D, E, F, G, H, K, M, N, Q, R, or W; or
  • the amino acid 31 is mutated to either G or S; or
  • Amino acid 35 is mutated to any one of A, F, H, M, N, Q, and Y; or
  • Amino acid number 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
  • Amino acid 41 is mutated to any one of A, D, E, H, N, Q, S, or W; or
  • Amino acid number 45 is mutated to any one of A, D, F, G, H, K, L, M, N, Q, R, S, V, W, or Y; or
  • Amino acid 47 is mutated to any one of I, L, M, Q, and V; or
  • Amino acid number 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
  • Amino acid number 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
  • Amino acid number 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
  • Amino acid 51 is mutated to any one of A, C, G, I, L, M, S, T, or V; or
  • Amino acid number 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
  • Amino acid number 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
  • Amino acid number 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
  • the amino acid at position 55 is mutated to any one of F, I, L, M, S, and T.
  • amino acid 4 to mutate from R to W amino acid 5 to mutate from E to V, I; amino acid 6 to mutate from L to F, W; amino acid 19 to mutate from L to Q; amino acid 24 to mutate from L to M; amino acid 32 to mutate from L to V, A; amino acid 35 to mutate from F to H, Y; amino acid 48 to mutate from F to R, L, M.
  • mutations were arranged 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 100nM to 1nM. The last round of screening resulted in the mini-binding protein mutant, HM9 (SEQ ID No.: 2).
  • 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 at the N-terminus (SEQ ID No.11: MGSSHHHHSSGLVPRGS).
  • the cloned plasmid was transformed into E. coli BL21(DE3) (EC1002, WEIDIBio), cultured in 1L LB broth at 37°C 220RPM until OD600 reached 0.6-0.8, IPTG (206-703-0, MACKLIN) was added at a final concentration of 250 ⁇ M, and cultured at 37°C 220RPM overnight.
  • the cells were collected by centrifugation at 8000g for 5 minutes, resuspended in 25ml protein buffer (25mM Tris-HCL (pH 8.0) + 150mM NaCl), and lysed by ultrasonication 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. The whole cell lysate, supernatant, flow-through, washing solution and eluate were sampled and run on 15% SurePAGE gel (M00719, GenScript). The protein gel image is shown in Figure 2.
  • the target protein was further purified using molecular sieve chromatography Superdex75 10/300GL column (29148721, Cytiva) in PBS buffer (pH 8.0). The chromatogram is shown in Figure 3.
  • 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 stable properties.
  • Example 3 Determination of the affinity of CD276_mb to CD276 and the affinity of HM9 to CD276 using BLI
  • the concentration of the purified protein was determined using a Bradford protein concentration assay kit (P0006, Beyotime). 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM were used to determine the affinity of CD276_mb and HM9 to CD276 by BLI experiments.
  • Example 4 Design of CD276_mb CAR-T and use of flow cytometry to measure the killing, T cell proliferation, and exhaustion of HM9CAR-T cells on different tumor cells.
  • the HM9 sequence was integrated into the CAR structure to form a complete HM9CAR coding sequence of CD8 ⁇ signal peptide-HM9-CD8 ⁇ hinge-CD8 ⁇ transmembrane domain-4-1BB costimulatory domain-CD3 ⁇ stimulatory domain.
  • the HM9CAR sequence was cloned into the pCDH-CMV-MCS-EF1-copGFP (CD511B-1, systembio) vector, and the structural model of CAR (SEQ ID No.: 3) is shown in Figure 6.
  • 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 signal transduction domain is SEQ ID No.: 7; and the amino acid sequence of the intracellular stimulatory signal transduction domain is SEQ ID No.: 8.
  • CD276_mb and CD276scFv were constructed into CAR structures in the same way as experimental controls.
  • the constructed CAR plasmid was transfected with psPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) into HEK293T (CRL-3216, ATCC) cells for lentivirus production and used to infect T cells isolated from normal human PBMCs, transforming them into biologically functional CAR-T cells, namely HM9-4-1BB CAR-T cells.
  • the CAR-T constructed in the present application has higher tumor killing activity against different tumor cells, lower exhaustion level and faster division and amplification ability. Its significantly enhanced cell proliferation ability and low exhaustion characteristics indicate that HM9-4-1BB CAR-T cells 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, avoiding premature exhaustion and causing T cell dysfunction.

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Abstract

L'invention concerne une miniprotéine de liaison à CD276, un mutant de celle-ci, et une utilisation associée. L'invention concerne en outre un récepteur antigénique chimérique comprenant la miniprotéine de liaison à CD276 ou son mutant en tant que domaine de liaison à l'antigène extracellulaire et un lymphocyte T à récepteur antigénique chimérique. L'affinité de liaison pour CD276 d'un mutant HM9 de protéine de liaison à CD276 atteint 36,4 pM, et la protéine de liaison présente un faible poids moléculaire, une bonne stabilité et une affinité élevée, et peut être massivement exprimée dans Escherichia coli. Par comparaison avec des stratégies de traitement CAR-T à base de scFC classiques, la cellule CAR-T construite présente une activité de destruction tumorale plus élevée, un niveau d'épuisement inférieur et une capacité de division et d'expansion plus rapide.
PCT/CN2023/140615 2023-12-21 2023-12-21 Miniprotéine de liaison à cd276, mutant de celle-ci et utilisation associée Pending WO2025129565A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170240637A1 (en) * 2014-08-27 2017-08-24 Memorial Sloan Kettering Cancer Center Antibodies, compositions, and uses
WO2020140094A1 (fr) * 2018-12-27 2020-07-02 Gigagen, Inc. Protéines de liaison anti-b7-h3 et méthodes d'utilisation de celles-ci
WO2021127428A1 (fr) * 2019-12-18 2021-06-24 Albert Einstein College Of Medicine Récepteurs antigéniques chimériques ciblant b7-h3 (cd276) et méthodes associées
US20210340257A1 (en) * 2018-08-23 2021-11-04 Lotfi Abou-Elkacem Affibody proteins specific for b7-h3 (cd276)
US20220017620A1 (en) * 2018-11-09 2022-01-20 Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd. Anti-b7-h3 antibody, preparation method therefor, conjugate and application thereof
US20230007977A1 (en) * 2019-11-18 2023-01-12 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Antibodies targeting, and other modulators of, the cd276 antigen, and uses thereof
WO2023001154A1 (fr) * 2021-07-20 2023-01-26 广州爱思迈生物医药科技有限公司 Anticorps b7-h3 et son utilisation

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170240637A1 (en) * 2014-08-27 2017-08-24 Memorial Sloan Kettering Cancer Center Antibodies, compositions, and uses
US20210340257A1 (en) * 2018-08-23 2021-11-04 Lotfi Abou-Elkacem Affibody proteins specific for b7-h3 (cd276)
US20220017620A1 (en) * 2018-11-09 2022-01-20 Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd. Anti-b7-h3 antibody, preparation method therefor, conjugate and application thereof
WO2020140094A1 (fr) * 2018-12-27 2020-07-02 Gigagen, Inc. Protéines de liaison anti-b7-h3 et méthodes d'utilisation de celles-ci
US20230007977A1 (en) * 2019-11-18 2023-01-12 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Antibodies targeting, and other modulators of, the cd276 antigen, and uses thereof
WO2021127428A1 (fr) * 2019-12-18 2021-06-24 Albert Einstein College Of Medicine Récepteurs antigéniques chimériques ciblant b7-h3 (cd276) et méthodes associées
WO2023001154A1 (fr) * 2021-07-20 2023-01-26 广州爱思迈生物医药科技有限公司 Anticorps b7-h3 et son utilisation

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