A cart cell and application thereof in preparation of anti-tumor drugs
By constructing bispecific CAR-T cells with optimized VHH tandem sequence, the problems of heterogeneity in target antigen expression and tumor microenvironment in existing technologies have been solved, achieving efficient and safe treatment of glioblastoma and enhancing tumor infiltration and durable killing effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 广州睿笛生物科技有限公司
- Filing Date
- 2026-04-01
- Publication Date
- 2026-06-05
Smart Images
Figure CN122145641A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell immunotherapy in biomedicine, specifically relating to a CAR-T cell and its application in the preparation of anti-tumor drugs. Background Technology
[0002] Chimeric antigen receptor T-cell (CAR-T) therapy has been successfully applied to hematologic malignancies, but its clinical translation in solid tumors such as glioblastoma multiforme (GBM) faces three major obstacles: spatiotemporal heterogeneity and dynamic loss of target antigen expression, relapse resistance mediated by tumor stem cell (CSC) subsets, and the obstruction of the immunosuppressive tumor microenvironment (TME) [Jackson HJ, Brentjens R J. Overcoming antigen escape with CART-cell therapy[J]. Cancer discovery, 2015, 5(12): 1238-1240.]. Clinical trials have shown that single-target CAR-T therapy against the GBM-related antigen EGFRvIII often leads to tumor escape and disease recurrence due to downregulation of the target antigen [O'Rourke DM, Nasrallah MLP, Desai A, et al. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma[J]. Sciencetranslational medicine, 2017, 9(399): eaaa0984.]. Meanwhile, the CSC subset, which highly expresses markers such as CD133, exhibits intrinsic resistance to multiple therapies and is a key driver of tumor regeneration and metastasis [Singh SK, Clarke ID, Terasaki M, et al. Identification of a cancer stem cell in humanbrain tumors[J]. Cancer research, 2003, 63(18): 5821-5828.], and existing therapies often struggle to effectively eliminate it.
[0003] To overcome antigen escape, developing CAR-T cells that simultaneously target multiple antigens has become a clear technological direction. Existing technologies have disclosed CAR-T protocols that target antigens such as EGFRvIII, IL13Rα2, or CD133. However, most of these designs rely on traditional single-chain variable region fragments (scFv) as extracellular antigen recognition domains. The relatively large molecular weight of scFv (approximately 25-30 kDa) may limit its penetration and diffusion efficiency in dense solid tumor tissues. More importantly, scFv has potential protein stability issues, such as misfolding or aggregation, which may affect the uniformity and persistence of functional expression of CAR molecules on the surface of T cells [Jayaraman J, Mellody MP, Hou AJ, et al. CAR-T design: Elements and their synergistic function[J]. EBioMedicine, 2020, 58.].
[0004] Single-domain antibodies, especially VHHs (Variable domain of heavy chain of heavy-chain antibodies, also known as nanobodies) derived from species such as camels or sharks, are the smallest known complete antigen-binding fragments (approximately 15 kDa), providing a new technical pathway to overcome the limitations of scFvs. VHHs have outstanding advantages such as small molecular weight, high stability (tolerant to extreme pH and temperature), good solubility, tissue penetration potential, ease of large-scale production in prokaryotic systems, and low immunogenicity [Muyldermans S. Nanobodies: natural single-domain antibodies[J].Annual review of biochemistry, 2013, 82(1): 775-797.]. However, there are still a series of key technical bottlenecks that have not been fully resolved in applying VHHs to construct highly efficient and safe bispecific CAR-T against GBM: First, obtaining VHHs that have both high affinity and high specificity against CD133 and EGFRvIII (especially the latter, which requires strict differentiation from wild-type EGFR) is itself a highly difficult screening task. Second, in constructing bispecific VHH-CARs, the tandem sequence of the two VHH domains and the design of the linker peptides can unpredictably affect its spatial conformation, antigen-binding kinetics, and downstream signal transduction intensity. The optimal conformation cannot be directly derived from existing knowledge. Although existing literature mentions the general concept of using VHHs to construct CARs, it does not provide specific optimization guidance for the specific target combination of CD133 and EGFRvIII. Third, whether this specifically designed dual VHH-CAR can functionally produce a synergistic antitumor effect that surpasses traditional scFv-CARs and inverse tandem structures, and effectively address antigen heterogeneity, lacks publicly available technical solutions and experimental evidence to support this.
[0005] In summary, the prior art has not yet disclosed a CAR-T cell product that utilizes a specifically screened high-affinity anti-CD133 and anti-EGFRvIII VHH in a specific optimal sequence verified through experiments, integrates a safety switch, and has been proven to have significant synergistic efficacy and good safety. This invention aims to fill this technological gap. Summary of the Invention
[0006] The present invention aims to overcome the shortcomings of the prior art and provide a novel bispecific CAR-T therapy based on VHH. This therapy can not only simultaneously target CD133 and EGFRvIII to overcome antigenic heterogeneity, but also unexpectedly endow CAR-T cells with a better memory phenotype and solid tumor infiltration ability through a unique VHH tandem sequence design.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] In a first aspect, the present invention provides a bispecific antigen-binding molecule comprising the following components in tandem: a first single-domain antibody (VHH) targeting the human CD133 antigen, a flexible linker peptide, and a second single-domain antibody (VHH) targeting the human EGFRvIII antigen. Preferably, the tandem sequence is from the N-terminus to the C-terminus: anti-CD133 VHH - linker peptide - anti-EGFRvIII VHH.
[0009] Secondly, the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain; wherein the extracellular antigen-binding domain is the bispecific antigen-binding molecule described above.
[0010] Thirdly, the present invention provides a polynucleotide encoding the CAR, an expression vector containing the polynucleotide, and an immune effector cell (such as a CAR-T cell) transduced by the vector and expressing the CAR.
[0011] Fourthly, the present invention provides pharmaceutical compositions comprising the aforementioned immune effector cells and their use in the preparation of antitumor drugs, particularly for the treatment of glioblastoma.
[0012] Compared with the prior art, the technical solution of the present invention has the following unexpected beneficial effects:
[0013] 1. Superior Dual-Target Synergy and High Specificity: The CAR constructed in this invention can simultaneously bind to CD133 (KD ~4.21 nM) and EGFRvIII (KD ~2.79 nM) with high affinity, and the EGFRvIII-resistant VHH shows no cross-reactivity with wild-type EGFR, resulting in higher safety. In vitro killing experiments show that, against heterogeneous tumor cells that are double-positive for CD133 and EGFRvIII, the killing efficacy of the CAR-T of this invention (Group A) is significantly superior to that of single-target CAR-T and reverse-sequential tandem CAR-T (Group B), especially at low target-to-target ratios, demonstrating its strong synergistic effect.
[0014] 2. Beneficial Regulation of T Cell Fate: The most inventive finding of this invention lies in the fact that a specific “VHH-CD133-linker-VHH-EGFRvIII” tandem sequence can significantly upregulate the proportion of central memory T cells (T_CM) in CAR-T cells. Compared with dual-target CAR-T cells (Group E) constructed based on traditional scFv, the proportion of T_CM in the CAR-T cells of this invention is significantly higher (P<0.01). The T_CM subset has stronger in vivo persistence and self-renewal capacity, a characteristic that enhances the long-term efficacy and durability of treatment from the perspective of the cell product itself.
[0015] 3. Enhanced Solid Tumor Infiltration: Benefiting from the small molecular weight and compact structure of VHH, the CAR-T cells of this invention exhibited a deeper maximum penetration depth and higher intramolecular fluorescence intensity (P<0.01) than scFv-CAR-T cells in the three-dimensional tumor spheroid penetration experiment. This directly proves that this invention can effectively overcome the physical barrier of solid tumors and enhance the infiltration of effector cells into the tumor parenchyma.
[0016] 4. Breakthrough in vivo antitumor efficacy: In a mouse model of glioblastoma in situ, the CAR-T therapy group (Group A) of this invention exhibited the fastest tumor clearance rate and the strongest growth inhibition effect, with a median survival of over 80 days and a long-term survival rate of 75%, significantly better than all control groups (including the scFv dual-target group, P<0.05). This comprehensively confirms its superior in vivo persistence, invasiveness, and killing power.
[0017] 5. Excellent manufacturability and stability: The VHH used in this invention can be expressed at high yield and high purity in prokaryotic systems (such as Escherichia coli), with good stability, avoiding the common aggregation problem of scFv, laying a solid foundation for large-scale, low-cost production of clinical-grade CAR-T products.
[0018] In summary, this invention creatively optimizes the tandem sequence of VHH to obtain a novel bispecific CAR, which not only achieves synergy at the antigen binding level, but also brings multi-level and synergistic technological breakthroughs in deep functions such as regulating T cell memory phenotype and enhancing tumor invasion, providing a new strategy for the treatment of solid tumors such as glioblastoma. Attached Figure Description
[0019] Figure 1 SDS-PAGE detection results of VHH-CD133 and VHH-EGFRvIII, where 1 and 2 are the purified VHH-CD133 and VHH-EGFRvIII, respectively.
[0020] Figure 2 Results of fluorescence microscopy observation after transfection. Detailed Implementation
[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0022] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.
[0023] Example 1: Screening, expression, and comprehensive characterization of specific single-domain antibodies (VHH)
[0024] 1.1 Construction and High-Specificity Screening of VHH Immunotherapy Library
[0025] (1) Antigen: Immunogen 1, recombinant human CD133 (AC133) protein (ab160218). Immunogen 2, synthesized EGFRvIII specific linear peptide (LEEKKGNYVVTDHC, >95% purity), coupled to KLH via an N-terminal cysteine residue.
[0026] (2) Animal immunization: Two healthy adult alpacas (numbered A1 and A2) were immunized subcutaneously at multiple sites on days 0, 14, 28 and 42, respectively. Each time, 100 μg of the corresponding antigen was injected (first time with Freund's complete adjuvant, and thereafter mixed with Freund's incomplete adjuvant). 50 mL of peripheral blood was collected on day 56.
[0027] (3) Lymphocyte isolation and RNA extraction: Peripheral blood mononuclear cells were isolated using Ficoll-Paque PREMIUM density gradient centrifugation. Total RNA was extracted using a kit, and its concentration and purity (A260 / A280 ~ 2.0) were determined using NanoDrop.
[0028] (4) VHH gene amplification and library construction: cDNA was synthesized using a commercial kit (RevertAid First Strand cDNA Synthesis Kit). Nested PCR: First round reaction program: 95°C 5 min; 30 cycles of (95°C 30s, 55°C 30s, 72°C 45s); 72°C 10 min. 1 μL of the first round product was used for the second round of PCR, with the same program as the first round. The PCR product was verified by 1% agarose gel electrophoresis (expected ~700 bp) and purified using the QIAquick Gel Extraction Kit. Both the purified product and the pMECS phage display vector were digested with SfiI at 50°C for 3 hours. After purification, the digested product was ligated overnight at 16°C at a 1:3 (vector:insert) molar ratio. The ligation product was transformed into TG1 electrocompetent cells by electroporation (2.5 kV, 25 μF, 200 Ω). After resuscitation, the samples were plated on 2xYT-AG plates (containing 100 μg / mL ampicillin and 2% glucose) and incubated overnight at 37°C. Colonies were scraped off and the library size was determined.
[0029] (5) Phage panning
[0030] Anti-CD133 VHH (solid-phase panning): Immunotubes were coated with 10 μg / mL recombinant CD133 protein (dissolved in 0.1 M NaHCO3, pH 8.6) and incubated overnight at 4°C. After washing with PBS, the tubes were blocked with 2% skim milk / PBS for 2 hours. Approximately 10 μg / mL of the solution was added. 12 CFU phage particles (dissolved in 2% MPBS) were incubated at room temperature for 2 hours. The phages were then vigorously washed 10 times sequentially with PBST (0.1% Tween-20) and PBS. Eluting was performed with 1 mL of 100 mM triethylamine (pH 11.0) for 10 minutes, followed immediately by neutralization with 0.5 mL of 1 M Tris-HCl (pH 7.4). The eluted phages were used to infect logarithmic-phase TG1 bacteria, amplified, and used for the next round of panning. A total of 3 rounds were performed, with the washing intensity (Tween-20 concentration and number of washes) increasing in each round.
[0031] Anti-EGFRvIII VHH (competitive solution panning): Biotinylated EGFRvIII peptide (2 nM) was reacted with approximately 10 12CFU phages were mixed in a solution containing 2% MPBS, with 10 μM of wild-type EGFR-Fc added as a competitive agent, and incubated at room temperature for 1 hour. Then, 100 μL of pre-washed Dynabeads M-280 Streptavidin magnetic beads were added, and the mixture was incubated for 15 minutes. The mixture was then magnetically adsorbed, washed 10 times with PBST (containing 0.05% Tween-20), and then washed twice with PBS. The elution and amplification procedures were the same as for CD133 panning. Three rounds were performed, with the concentration of the wild-type EGFR-Fc competitive agent increased to 20 μM in the second and third rounds.
[0032] (6) Single-clone screening and sequencing: 96 single clones were randomly selected from the third round of panning and seeded into 96-well plates. Soluble VHH expression was induced for 16 hours at 30°C with 0.5 mM IPTG. Periplasmic extract was obtained by osmotic shock. ELISA detection: 96-well plates were coated with 1 μg / mL CD133 recombinant protein or EGFRvIII-BSA. Periplasmic extract was added and incubated, and detection was performed using HRP-labeled anti-HA tag antibody. Strong positive results were defined as OD450 > 1.0 (and more than 3 times higher than the negative control). Strongly positive clones were subjected to Sanger sequencing (Suzhou Genewiz), and the sequences were analyzed by IMGT / V-QUEST to remove duplicates and obtain unique VHH sequences.
[0033] (7) Results
[0034] Library size: The anti-CD133 immune library has a size of 3.2 × 10⁻⁶. 8 CFU; the anti-EGFRvIII immune library size was 2.8 × 10⁻⁶. 8 CFU.
[0035] Enrichment by panning: After the third round of panning, the phage production for CD133 recombinant protein was enriched by about 500 times compared to the first round of input; the production for EGFRvIII peptide was enriched by about 300 times, and in ELISA screening, about 85% of the clones were positive for EGFRvIII but negative for wild-type EGFR.
[0036] Sequence acquisition: Twelve unique anti-CD133 VHH sequences and nine unique anti-EGFRvIII VHH sequences were obtained. Based on sequence conservation, complementarity-determining region (CDR) length, and charge distribution, a representative clone of each was selected for in-depth study: anti-CD133 clone #7 (named VHH-CD133, its amino acid sequence is shown in SEQ ID NO:1) and anti-EGFRvIII clone #12 (named VHH-EGFRvIII, its amino acid sequence is shown in SEQ ID NO:2).
[0037] (8) Summary: This study successfully constructed an immune library with a large library capacity and enriched a large number of highly specific clones through a strict panning strategy, especially competitive panning for EGFRvIII, laying the foundation for obtaining high-quality VHH.
[0038] 1.2 VHH protein expression, purification and quality control analysis
[0039] (1) Expression vector construction: The gene fragments of VHH-CD133 and VHH-EGFRvIII were cloned into the NdeI and XhoI sites of the pET-22b(+) expression vector, respectively, so that the C-terminus of the expression product carries a 6×His tag. Sequencing confirmed that the expression was correct.
[0040] (2) Protein expression: The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells. Single colonies were picked and inoculated into 5 mL LB (containing 100 μg / mL ampicillin) and cultured overnight at 37°C and 220 rpm. The plasmid was then transferred to 1 L 2xYT medium at a 1:100 ratio and cultured at 37°C until OD600 ~0.8. IPTG was added to a final concentration of 0.5 mM and expression was induced at 30°C for 16 hours.
[0041] (3) Periplasmic Extraction and Purification: Collect bacterial cells by centrifugation (4,000 g, 20 min, 4°C), resuspend in 30 mL of pre-chilled TES buffer (0.2 M Tris-HCl, 0.5 mM EDTA, 0.5 M sucrose, pH 8.0), and gently stir on ice for 1 hour. Add an equal volume of pre-chilled 0.5×TES buffer and continue stirring on ice for 1 hour. Centrifuge (12,000 g, 30 min, 4°C) to collect the supernatant containing soluble VHH protein (periplasmic extract). Load the periplasmic extract onto a HisTrap HP 5 mL pre-packed column equilibrated with binding buffer (20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0). Elute with a linear gradient using elution buffer containing 250 mM imidazole and collect the protein peak.
[0042] (4) Protein purification and concentration: To further remove impurities and aggregates, the nickel column elution peak collection solution was further purified using a Superdex 75 Increase 10 / 300 GL gel filtration chromatography column in PBS buffer (pH 7.4). The major monomer peak was collected. The purified protein was concentrated to approximately 1 mg / mL using an Amicon Ultra-15 centrifugal ultrafiltration tube (3 kDa molecular weight cutoff). The protein concentration was determined using NanoDrop One (calculated using the theoretical extinction coefficient at 280 nm).
[0043] (5) Quality control
[0044] Protein yield: The final yield of VHH-CD133 was 18.5 mg of purified protein per liter of culture; the yield of VHH-EGFRvIII was 21.2 mg per liter of culture.
[0045] SDS-PAGE: 5 μg of purified protein was subjected to 12% SDS-PAGE electrophoresis under reducing conditions and stained with Coomassie Brilliant Blue. Results are as follows: Figure 1 As shown, both VHH-CD133 and VHH-EGFRvIII exhibited a single main band at approximately 15 kDa, consistent with the expected molecular weight, and had a purity >95%.
[0046] 1.3 Surface Plasmon Resonance (SPR) Kinetic Analysis and Affinity Measurement
[0047] (1) Chip activation and antigen fixation: A standard amine conjugation kit was used. The chip surface was activated for 7 minutes by injecting an EDC / NHS mixture (1:1) at a flow rate of 10 μL / min. Channel 1 (reference channel): 1 M ethanolamine (pH 8.5) was injected for 7 minutes to block. Channel 2 (CD133 test channel): Human CD133 recombinant protein diluted to 20 μg / mL with 10 mM sodium acetate (pH 5.0) was injected, with a fixation level of approximately 6000 RU. Channel 3 (EGFRvIII test channel): EGFRvIII-BSA conjugate diluted to 30 μg / mL with 10 mM sodium acetate (pH 4.5) (peptide conjugated to BSA via SMCC) was injected, with a fixation level of approximately 5500 RU. After fixation, 1 M ethanolamine was injected to block all channels.
[0048] (2) Kinetic analysis: The purified VHH protein was serially diluted in HBS-EP+ buffer to eight concentrations (VHH-CD133: 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 nM; VHH-EGFRvIII: 0.19, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25 nM). The program was set as follows: sample injection time 120 seconds, dissociation time 300 seconds, flow rate 30 μL / min. After each analysis, the chip surface was regenerated with 10 mM Glycine-HCl (pH 2.0) for 20 seconds to ensure baseline stability. Each concentration was injected twice. All data were subtracted from the reference channel signal and the blank buffer injection signal.
[0049] (3) Data fitting: Using Biacore T200 Evaluation Software 3.2, the sensorgram data was globally fitted using a 1:1 Langmuir binding model, and the binding rate constant (ka, M) was calculated. -1 s -1 ), dissociation rate constant (kd, s) -1 ) and equilibrium dissociation constant (KD = kd / ka, M).
[0050] (4) Results
[0051] VHH-CD133: Global fitting yields ka = (3.85 ± 0.21) × 10 5 M -1 s -1 kd = (1.62 ± 0.09) × 10 -3 s -1 The calculated KD = 4.21 ± 0.32 nM (mean ± standard deviation, n = 3 independent experiments).
[0052] VHH-EGFRvIII: Its binding and dissociation kinetics are similar to those of VHH-CD133. The fitting yields ka = (5.12 ± 0.28) × 10⁻⁶. 5 M -1 s -1 kd = (1.43 ± 0.08) × 10 -3 s -1 , KD = 2.79 ± 0.23 nM (n=3).
[0053] Cross-reactivity validation: A high concentration (10 μM) of wild-type EGFR recombinant protein (e.g., ab155639) was used as the analyte and flowed through a chip channel immobilized with VHH-EGFRvIII. The maximum detected change in response value (ΔRU) was only 0.8, far below the positive binding signal (>100 RU), and there was no retention during the dissociation phase, confirming that VHH-EGFRvIII did not bind to wild-type EGFR in any detectable way.
[0054] 1.4 Performance Comparison Analysis with Existing Antibodies
[0055] (1) Selection of control antibodies and data sources:
[0056] Anti-CD133 control: A commercially available anti-human CD133 monoclonal antibody (ab222782) that is widely accepted and commonly used for enriching tumor stem cells was selected. Its affinity data (KD ~ 5-10 nM).
[0057] Anti-EGFRvIII control: A widely accepted commercial anti-human EGFRvIII monoclonal antibody (ab313646) was selected.
[0058] Its affinity data (KD ~ 5-10 nM).
[0059] (2) Comparative analysis is shown in Table 1.
[0060] Table 1. Comparison of key performance characteristics of the VHH antibody of this invention with existing technologies.
[0061]
[0062] (3) Analysis and summary of experimental results:
[0063] Comparable or superior affinity: The VHH screened by this invention has the same order of magnitude (low nanomolar level) affinity as the best targeting antibody in the prior art, proving its effectiveness as an antigen binding module.
[0064] Breakthrough in Specificity: Targeting EGFRvIII, the VHH-EGFRvIII of this invention achieves virtually zero cross-reactivity with wild-type EGFR (wtEGFR), an unexpected and significant technological advantage. While existing antibodies exhibit high selectivity, they still show detectable weak binding to wtEGFR. This difference is crucial for the safety of CAR-T therapy because wtEGFR is expressed in various normal tissues, such as skin and the gastrointestinal tract. The VHH of this invention fundamentally reduces the risk of on-target off-tumor toxicity.
[0065] Molecular properties offer potential advantages: The molecular weight (~15 kDa) of VHH in this invention is significantly smaller than that of traditional scFv (~25 kDa) and full-length antibodies (~150 kDa). This characteristic may directly translate into superior tumor tissue penetration in CAR-T cells constructed based on it. Simultaneously, the high yield and high stability of VHH in E. coli facilitate its large-scale, low-cost production, demonstrating clear advantages for industrial applications.
[0066] Example 2: Construction, validation, and lentivirus production of different chimeric antigen receptor (CAR) expression vectors
[0067] 2.1 Precise design, codon optimization, and gene synthesis of CAR molecules
[0068] (1) Domain composition: All CARs adopt the same basic architecture, from N-terminus to C-terminus: human CD8α signal peptide (its sequence is based on UniProt accession number P01732); extracellular antigen binding domain; human CD8α hinge region and transmembrane region (its sequence is based on UniProt accession number P01732); intracellular co-stimulatory domain of human 4-1BB (CD137) (its sequence is based on UniProt accession number Q07011); and intracellular signaling domain of human CD3ζ chain (its sequence is based on UniProt accession number P20963).
[0069] (2) Precise design of extracellular antigen-binding domains
[0070] Group A (core building block of this invention): [SEQ ID NO:1: VHH-CD133] - [SEQ ID NO:3:(GGGGS)3linker] - [SEQ ID NO:2: VHH-EGFRvIII]. This sequence is an optimized cascade of "CD133-EGFRvIII".
[0071] Group B (Key Sequence Comparison): [SEQ ID NO:2] - [SEQ ID NO:3] - [SEQ ID NO:1]. This is a reversed “EGFRvIII-CD133” cascade, used to directly demonstrate the criticality of the cascade sequence.
[0072] Group C (single target control 1): [SEQ ID NO:1].
[0073] Group D (single target control 2): [SEQ ID NO:2].
[0074] Group E (Traditional Technology Control): The VH and VL sequences of the anti-human CD133 monoclonal antibody and the anti-human EGFRvIII monoclonal antibody were used to construct scFv (VH-linker-VL format) and then linked together with (G4S)3 linker peptides to form the structure "scFv-CD133-linker-scFv-EGFRvIII".
[0075] (3) C-terminal tag: To facilitate high-sensitivity detection of CAR expression on the cell membrane surface by flow cytometry, a His tag was introduced at the C-terminus of each CAR (after the CD3ζ domain).
[0076] (4) Codon optimization and gene synthesis: The amino acid sequences of the above five CARs were submitted to GenScript Biotech Co., Ltd., which was entrusted to perform human codon optimization. The optimized DNA sequences were chemically synthesized by GenScript and cloned into its standard cloning vector pUC57, providing pUC57-CAR-A to pUC57-CAR-E plasmids. Sequencing results showed that the sequencing results of all five synthesized genes were 100% consistent with the designed sequences, with no frameshifts or unexpected mutations.
[0077] 2.2 Construction and Strict Quality Control of Lentiviral Expression Vectors
[0078] (1) Vector backbone: Select commercially available pCDH-EF1-MCS-T2A-copGFP (such as Shanghai Zeye Biotechnology Co., Ltd.). This vector contains the EF1α promoter (driving CAR expression), 2A peptide sequence and copGFP reporter gene (used for initial transduction evaluation; in subsequent experiments, GFP+ cells can be removed by flow cytometry or a reporter gene-free vector can be used).
[0079] (2) Enzyme digestion and ligation: The pCDH vector and the pUC57 plasmid containing the CAR gene were double-digested with restriction endonucleases XbaI and NotI at 37°C for 3 hours. The CAR fragment and the linearized pCDH vector backbone were purified by agarose gel electrophoresis using the Monarch DNA Gel Extraction Kit. Ligation was performed overnight at 16°C using T4 DNA ligase at a vector:insert molar ratio of 1:3.
[0080] (3) Transformation and screening: The ligation product was transformed into Stbl3 chemocompetent cells and plated on LB agar plates containing 100 μg / mL ampicillin. At least 10 single colonies were picked, inoculated into 5 mL of LB medium and cultured overnight for preliminary colony PCR identification.
[0081] (4) Plasmid extraction and verification: For PCR-positive clones, high-quality, low-endotoxin plasmids were extracted using the EndoFree Plasmid Maxi Kit. The final constructs were named pCDH-EF1-CAR-A to pCDH-EF1-CAR-E, respectively.
[0082] (5) Sequencing verification: To ensure complete sequence accuracy, each final plasmid was sent to a sequencing company for Sanger sequencing using a set of primers covering the entire CAR expression cassette. The sequencing maps of all five constructs were completely consistent with the expected optimized sequences, with no nucleotide errors, deletions, or insertions.
[0083] (6) The final concentration of the extracted plasmids was between 800-1200 ng / μL (NanoDrop determination), the A260 / A280 ratio was between 1.8-1.9, and the A260 / A230 ratio was >2.0, indicating that the purity was qualified.
[0084] 2.3 Packaging, Concentration, and Quantification of Lentiviral Particles
[0085] (1) Cells and reagents: HEK293T cells were cultured in DMEM high glucose medium + 10% FBS. A three-plasmid packaging system was used: transfer plasmid (pCDH-CAR), packaging plasmid (psPAX2), and envelope plasmid (pMD2.G). Polyethyleneimine (PEIMax) was used as the transfection reagent.
[0086] (2) Virus packaging: Inoculate approximately 1.2 × 10⁻⁶ virus cells into a 15 cm culture dish. 7 HEK293T cells were transfected to a density of 70-80%. Transfection complex preparation: 30 μg of transfer plasmid, 22.5 μg of psPAX2, and 7.5 μg of pMD2.G were mixed in 1.5 mL of Opti-MEM (tube A). 120 μL of PEI Max (1 mg / mL) was diluted in 1.5 mL of Opti-MEM (tube B). After incubating at room temperature for 5 minutes, the solution from tube B was added dropwise to tube A, vortexed, and allowed to stand at room temperature for 20 minutes. The complex was then added dropwise and evenly to a culture dish containing 15 mL of fresh culture medium. After 6-8 hours, the medium was replaced with 20 mL of fresh complete culture medium.
[0087] (3) Virus collection and preliminary treatment: Cell supernatant was collected 48 hours and 72 hours after transfection and immediately filtered through a 0.45 μm PES membrane to remove cell debris. The filtrates collected in the two batches were mixed.
[0088] (4) Virus concentration by ultracentrifugation: Transfer the mixed virus supernatant to an ultracentrifuge tube and centrifuge at 25,000 rpm for 2 hours at 4°C. Carefully discard the supernatant and resuspend the virus pellet in 1 / 100 of the original volume of pre-cooled PBS (containing 1% BSA) and gently shake overnight at 4°C. Aliquot and store at -80°C.
[0089] (5) Virus titer determination
[0090] Physical titer (genomic titer): using Lenti-X TM qRT-PCR Titration Kit. Viral RNA is extracted, reverse transcribed, and then the viral genome copy number is determined by quantitative PCR (qPCR). The standard curve is generated using known copy number standards provided with the kit.
[0091] Functional titer (transduction unit, TU / mL): 5 × 10⁶ cells / mL seeded in a 24-well plate. 4 HEK293T cells / well. The next day, the concentrated virus solution was serially diluted with complete culture medium (10⁻⁶ cells / well). -2 Up to 10 -5 Add 500 μL of diluted virus solution and a final concentration of 8 μg / mL polybrene to each well. After 72 hours, observe and count the proportion of GFP-positive cells under a fluorescence microscope (because the vector carries copGFP). Calculate the titer (TU / mL) using the formula: (cell number × GFP positivity rate % × dilution factor) / virus solution volume (mL).
[0092] (6) Results
[0093] ① Packaging process observation: 48 hours after transfection, fluorescence microscopy ( Figure 2 Approximately 70% of HEK293T cells expressed GFP (from the pCDH vector), indicating high transfection efficiency.
[0094] ② Physical titer (qPCR assay):
[0095] CAR-A virus: average Ct value 18.5 (10 -3 (Dilution), titer 2.3 × 10⁻⁶ 9 Virus particles (vp) / mL.
[0096] CAR-B virus: average Ct value 18.7 (10 -3 (Dilution), titer 2.0 × 10⁻⁶ 9 vp / mL.
[0097] CAR-C virus: Average Ct value 19.0 (10 -3 (Dilution), titer 1.8 × 10⁻⁶ 9 vp / mL.
[0098] CAR-D virus: average Ct value 18.8 (10 -3 (Dilution), titer 2.1 × 10⁻⁶ 9 vp / mL.
[0099] CAR-E virus: average Ct value 19.2 (10 -3 (Dilution), titer 1.6 × 10⁻⁶ 9 vp / mL.
[0100] ③ Functional titer: at 10 -4 At the dilution, the GFP positivity rate is approximately 15%-20%. Calculations show that:
[0101] CAR-A virus: Functional titer = (5 × 10) 4 (× 0.18) / (0.5 × 10) -4 ) = 1.8 × 10 8 TU / mL.
[0102] The calculated CAR-B is 1.5 × 10⁻⁶. 8 TU / mL; CAR-C: 1.7 × 10⁻⁶ 8 TU / mL; CAR-D: 1.9 × 10⁻⁶ 8 TU / mL; CAR-E: 1.4 × 10⁻⁶ 8 TU / mL.
[0103] ④ Titer Standardization: To eliminate the influence of titer differences on subsequent T cell transduction experiments, all viral stock solutions were diluted with PBS containing 1% BSA to uniformly adjust their functional titers to (1.5 ± 0.2) × 10⁻⁶. 8 TU / mL.
[0104] (7) Quality control
[0105] Sterility test: A small amount of the original virus solution was spread on LB agar plates and thioglycolate fluid medium and incubated at 37°C for 7 days. No microbial growth was observed in either case.
[0106] Mycoplasma test: The result was negative using the MycoAlert PLUS test kit (Lonza).
[0107] Example 3: Preparation, Expression Validation and Analysis of CAR-T Cells
[0108] 3.1 Isolation, activation, and lentiviral transduction of primary T cells
[0109] (1) Materials and reagents
[0110] Peripheral blood from healthy volunteers; Lymphoprep TM Density gradient centrifuge solution; DPBS, calcium and magnesium-free; CD3 / CD28T Cell Activator (Human), i.e., anti-CD3 / CD28 magnetic beads; Recombinant Human IL-2, formulated to 10 5 IU / mL stock solution; X-VIVO TM 15 Serum-free medium, supplemented with 5% Human AB Serum and 1% Penicillin-Streptomycin, is labeled "Complete Medium + 5% HABS".
[0111] The CAR-A to CAR-E viruses prepared in Example 2 have had their functional titers standardized to (1.5 ± 0.2) × 10⁻⁶. 8 TU / mL; Polybrene, prepared as an 8 mg / mL stock solution.
[0112] (2) Steps
[0113] PBMC isolation: Dilute approximately 30 mL of peripheral blood with an equal volume of DPBS and gently add it to the surface of 15 mL of Lymphoprep solution. Centrifuge at 800 g for 30 minutes at room temperature (without brake). Carefully aspirate the white membrane layer (peripheral blood mononuclear cells, PBMCs) and wash twice with DPBS (300 g, 10 minutes).
[0114] T cell activation: PBMCs were counted and resuspended in complete culture medium + 5% HABS to a final volume of 1×10⁻⁶. 6 cells / mL. Following the supplier's instructions, CD3 / CD28 T cell activation beads were added at a ratio of magnetic beads to cells of 1:2 (number ratio). Recombinant human IL-2 was also added to a final concentration of 100 IU / mL. Cells were seeded into 24-well plates, 1 mL per well (i.e., 1 × 10⁻⁶ cells / mL). 6 Cells). Incubate at 37°C in a 5% CO2 incubator.
[0115] Lentiviral transduction (centrifugation infection method): 24 hours after activation, confirm under a microscope that T cells have formed clones and are activated. Prepare viral infection mixture: per 1×10 6 Cells require 100 μL of viral stock solution (MOI=5) and 8 μL of Polybrene stock solution (final concentration 8 μg / mL), brought to a final volume of 500 μL with complete culture medium + 5% HABS. Aspirate most of the culture medium from the original wells, reserving approximately 200 μL to prevent cell drying. Add 500 μL of viral infection mixture. Seal the 24-well plate with sealing film and place it in a centrifuge rotor pre-cooled to 4°C. Centrifuge at 1200 ×g for 90 minutes at 32°C. Immediately after centrifugation, transfer the cells back to a 37°C, 5% CO2 incubator.
[0116] Medium change and culture: 6-8 hours after transduction, carefully add 1 mL of fresh complete culture medium + 5% HABS (containing 100 IU / mL IL-2) to each well. Thereafter, perform a half-volume medium change or passage every 2-3 days to maintain a cell density of 0.5-2.0 × 10⁶ cells / well. 6 cells / mL, and IL-2 is continuously supplemented.
[0117] 3.2 CAR Expression Detection and Immunophenotypic Analysis
[0118] (1) Materials and reagents: Flow cytometry staining buffer DPBS + 2% FBS. Fixable Viability DyeeFluor TM 780, used to remove dead cells. APC anti-human CD279 (PD-1) Antibody (used to non-specifically block Fc receptors);
[0119] PE anti-His Tag Antibody, used to detect CAR (CAR with His tag at the C-terminus); BV510 anti-human CD45RA Antibody; APC anti-human CCR7 Antibody; and corresponding isotype control antibodies.
[0120] (2) Steps:
[0121] ① Sample preparation: On day 7 after transduction (mid-amplification), at least 1×10⁻⁶ cells were collected from each experimental group (AE group) and the untransduced T cell control group. 5 Each cell.
[0122] ② Cell staining
[0123] a. Live / dead staining: Wash cells once with pre-chilled flow cytometry buffer, resuspend in 100 μL buffer, add 1 μL Fixable Viability Dye, and incubate at 4°C in the dark for 15 minutes. Wash with 2 mL buffer.
[0124] b. Surface staining: Resuspend the cells in 50 μL of staining buffer containing Fc receptor blocker (optional), add the pre-titrated detection antibody mixture (anti-His-PE, anti-CD45RA-BV510, anti-CCR7-APC), and incubate at 4°C in the dark for 30 minutes.
[0125] c. Washing and fixation: Wash cells twice with 2 mL staining buffer, and finally resuspend in 200 μL staining buffer. Immediately perform instrumental analysis or fix with 1% paraformaldehyde and store at 4°C in the dark (analyze within 24 hours).
[0126] ③ Flow cytometry data acquisition: At least 10,000 live cells were collected using a flow cytometer (lymphocyte populations were delineated based on forward scattering FSC-A / side scattering SSC-A, adhesions were excluded based on FSC-H / FSC-A, and live cells were finally delineated using live / dead staining negative).
[0127] ④ Data Analysis
[0128] CAR expression rate: The fluorescence intensity of the His-PE channel was analyzed within the viable cell phylum. Threshold setting: The percentage of His-PE positive cells in each CAR-T group was calculated based on the proportion of PE-positive cells in the untransduced T cell group (or isotype control staining group) being <1%.
[0129] T cell subset analysis: Within the CAR-positive cell (His-PE+) phylum, four subsets were defined based on the expression of CD45RA and CCR7: Naïve T cells (T_N): CD45RA+ CCR7+; Central memory T cells (T_CM): CD45RA- CCR7+; Effector memory T cells (T_EM): CD45RA- CCR7-; Terminally differentiated effector memory T cells (T_EMRA): CD45RA+ CCR7-.
[0130] 3.3 Analysis and Summary of Experimental Results
[0131] (1) The results of the CAR expression efficiency experiment are shown in Table 2: One-way ANOVA showed that there was no statistically significant difference in the transduction efficiency and CAR surface expression rate among the five groups of CAR-T cells (F(4, 10) = 2.15, P = 0.148). This indicates that different CAR structures (including the specific sequence of this invention) can be effectively integrated and expressed on the T cell membrane, excluding the possibility of bias in subsequent functional comparisons due to differences in expression levels.
[0132] Table 2 Summary of T cell preparation results from three independent healthy donors (%, mean ± SD)
[0133]
[0134] (2) The results of the T-cell memory subset analysis are shown in Table 3: Intergroup comparisons were made regarding the proportion of T_CM. Unpaired t-tests showed that the proportion of T_CM in group A (this invention) was significantly higher than that in group E (scFv tandem) (t=6.12, df=4, P=0.0036). Meanwhile, the proportion of T_CM in group A was also higher than that in groups B, C, and D, but the differences between group A and groups B, C, and D did not reach statistical significance in this analysis (P values were 0.07, 0.25, and 0.11, respectively). Compared with the untransduced T-cell group, the proportion of T_CM in group A was significantly increased (P<0.001).
[0135] Table 3. Summary of results from n=3 independent preparations using CAR+ cells from donor HD01 as an example.
[0136]
[0137] (3) Summary
[0138] This study established a stable and reliable process for primary T cell activation, lentiviral transduction and amplification, and successfully prepared five groups of CAR-T cells with comparable CAR expression levels, laying the foundation for subsequent fair functional comparisons.
[0139] The specific “VHH-CD133-linker peptide-VHH-EGFRvIII” tandem sequence of this invention (Group A) confers an unexpected phenotypic advantage to CAR-T cells: inducing a higher proportion of central memory T cells (T_CM) in CAR+ cells. This difference is statistically significant compared to dual-target CAR-T cells constructed based on conventional scFv (Group E).
[0140] In CAR-T therapy, the T_CM subset is considered to possess stronger in vivo persistence, self-renewal capacity, and anti-tumor efficacy. Therefore, the CAR structure of this invention not only overcomes antigenic heterogeneity through dual-target design, but its unique tandem sequence can also potentially enhance the long-term efficacy and durability of therapeutic products at the level of cell fate regulation. This discovery highlights the ingenuity of VHH tandem sequence optimization and its resulting multi-level synergistic effects.
[0141] Example 4: In vitro functional verification
[0142] 4.1 Preparation of target cell lines and validation of antigen expression
[0143] (1) Cell lines and culture
[0144] U87-MG: Cultured in DMEM high-glucose medium (Gibco) + 10% FBS. Known to express CD133 at low levels and not EGFRvIII.
[0145] U87-EGFRvIII: In our laboratory, the human EGFRvIII mutant was stably expressed in U87-MG cells via lentiviral transduction, and maintained and screened using a medium containing 1 μg / mL puromycin. Flow cytometry validation showed that the EGFRvIII expression rate was >98%.
[0146] GBM-SC01: Primary patient-derived glioma stem cell-like cells, stored in the laboratory. Cultured in neural stem cell culture medium in ultra-low adsorption dishes to form spheroids.
[0147] (2) Flow cytometry verification of antigen expression
[0148] U87-MG, U87-EGFRvIII, and GBM-SC01 cells in the logarithmic growth phase were collected. Surface staining was performed using anti-human CD133-APC antibody and anti-human EGFRvIII-Alexa Fluor 488 antibody, with isotype controls included.
[0149] Flow cytometry analysis confirmed that U87-MG was CD133-EGFRvIII-; U87-EGFRvIII was CD133-EGFRvIII+; and GBM-SC01 was CD133+EGFRvIII-.
[0150] (3) Reporter gene markers: The three types of cells were infected with lentiviruses expressing NucLight Red and a population of monoclonal cells with >99% red fluorescence positive was obtained by flow cytometry for real-time killing analysis.
[0151] 4.2 Real-time dynamic cell killing analysis
[0152] Target cell plating: NucLight Red-labeled target cells (U87-EGFRvIII, GBM-SC01, or a 1:1 mixture thereof) were plated at 5.0 × 10⁶ cells per well. 3 Cells were precisely seeded at a density of 100 μL into 96-well plates. "Target Cells Only" wells (background control) and "Culture Blank" wells were included. The plates were incubated overnight at 37°C in a 5% CO2 incubator.
[0153] Effector cell preparation: On the day of co-culture, collect all CAR-T cells prepared and expanded to day 8-10 in Example 3, as well as untransduced T cells. Wash twice with complete medium without IL-2, count and adjust cell density.
[0154] Co-culture setup and data acquisition: Remove the 96-well plate from the incubator and aspirate the old culture medium. Add 100 μL of culture medium containing a specific number of effector cells to the corresponding well according to the preset effector-to-target ratio (E:T = 0.5:1, 1:1, 2:1, 5:1). Set up three biological replicates (CAR-T from different donors) for each group, and three technical replicates for each biological replicate. Place the culture plate into the Incucyte® S3 instrument chamber.
[0155] Scanning settings: The instrument is set to automatically scan the entire plate every 2 hours (using a 10× objective lens and the red fluorescence channel). Continuous monitoring lasts for a total of 72 hours.
[0156] Data Analysis: Using Incucyte® Basic Analyzer software, the number of NucLight Red+ target cells in each well was quantified through the "Red Fluorescent Object Count" module. Specific killing rate (%) was calculated using the formula: [1 - (Number of target cells in experimental wells at time T / Average number of target cells in "target cells only" control wells at time T)] × 100%.
[0157] 4.3 Multiplex detection of cytokines and chemokines
[0158] Co-culture supernatant collection: CAR-T cells and mixed target cells (U87-EGFRvIII:GBM-SC01 = 1:1) were co-cultured in parallel 96-well plates at an E:T ratio of 2:1. After 24 hours of co-culture, all supernatant from each well was collected into labeled centrifuge tubes, centrifuged at 500 g for 5 minutes to remove cell debris, and the supernatant was transferred to new tubes and stored at -80°C for analysis. Commercially available kits, such as LEGENDplex, were used. TM Human CD8 / NK Panel (13-plex), operate according to the product instructions. Use LEGENDplex. TM The Data Analysis Software Suite performs concentration calculations. The software automatically converts the fluorescence signal into the concentration (pg / mL) of each factor based on the standard curve.
[0159] 4.4 Experimental Results, Statistical Analysis and Summary
[0160] (1) Experimental results
[0161] Real-time killing kinetics (against mixed target cells): Summary of 24-hour targeted killing rates (Table 4), data from CAR-T cells derived from three independent healthy donors (HD01, HD02, HD03). All data points were derived from Incucyte raw counts. At multiple E:T ratios, especially the lower E:T ratios (0.5:1, 1:1) which are closer to physiological conditions, the killing rate of group A was significantly higher than that of all control groups, demonstrating its strong synergistic killing ability.
[0162] Table 4 Summary of 24-hour specific killing rate results for mixed target cells (CD133+ / EGFRvIII+)
[0163] (%, mean ± SEM, n=3 independent donors)
[0164]
[0165] Conclusion: At various E:T ratios, especially the low E:T ratios (0.5:1, 1:1) which are closer to physiological conditions, the killing efficiency of single-positive target cells and cytokine release is shown in Table 5. Under the condition of E:T=2:1, the killing rate of cells expressing only CD133 (GBM-SC01) or only EGFRvIII (U87-EGFRvIII) in group A was not statistically different from that of the corresponding single-target CAR-T (group C or group D) (P > 0.05), indicating that the dual-target CAR-T of the present invention completely retains the full killing efficacy against a single target without mutual interference.
[0166] Table 5. Kill rate of single-positive target cells (E:T=2:1, 24h, %) and release results of key cytokines
[0167]
[0168] (2) Experiment Summary
[0169] The synergistic effect has been clearly demonstrated: the specific “VHH-CD133-EGFRvIII” tandem sequence of this invention (Group A) exhibits significantly superior killing ability against heterogeneous tumor cells expressing dual antigens compared to single-target CAR-T (Groups C and D) and reverse-sequence CAR-T (Group B). This advantage is particularly pronounced at low target-to-cell ratios and has significant clinical implications.
[0170] Confirming structural advantages: Compared with dual-target CAR-T (Group E) constructed based on traditional scFv technology, the optimized structure based on VHH in this invention (Group A) still has statistically significant advantages in killing activity and cytokine release, proving that the VHH tandem structure is not a simple replacement, but brings functional improvement.
[0171] Perfectly addressing antigen heterogeneity: The CAR-T of this invention has a killing effect on single-positive target cells comparable to that of single-target CAR-T, indicating that its dual-target design is effective and free from internal interference, and can provide a reliable guarantee for dealing with the loss or downregulation of tumor antigens during treatment.
[0172] Example 5: Three-dimensional tumor spheroid penetration experiment
[0173] 5.1 Spheroid formation and co-cultivation
[0174] (1) Materials and reagents: target cells GBM-SC01 (CD133+, EGFRvIII-).
[0175] Effector cells, group A (VHH-CAR-T of this invention) and group E (scFv-CAR-T) cells prepared in Example 3, were cultured to a high viability state on days 9-11. Spheroidal culture medium, NeuroCult... TM NS-A Proliferation Kit, supplemented with 20 ng / mL EGF, 10 ng / mL bFGF, and 2 μg / mL heparin. Cell tracking dye: CellTracker. TM Green CMFDA (5-Chloromethylfluorescein Diacetate), prepared as a 10 mM stock solution with DMSO, stored at -20°C protected from light. Co-culture medium: RPMI 1640 + 10% FBS.
[0176] (2) Preparation of tumor spheroids: GBM-SC01 cells were digested into single-cell suspensions using Accutase and counted. Cells were resuspended in 150 μL of spheroid culture medium at a density of 5,000 cells per well and seeded into each well of a 96-well ultra-low adsorption round-bottom plate. The culture plate was incubated statically at 37°C in a 5% CO2 incubator, without moving or shaking. After 5-7 days of culture, spheroid formation was observed daily under an inverted microscope. The goal was to form single spheroids with a compact structure, smooth edges, and a diameter between 280-320 μm (measured using a microscope scale). Irregularly shaped or fused spheroids were discarded.
[0177] (3) CAR-T cell fluorescent labeling: CAR-T cells from groups A and E were collected and washed once with pre-warmed PBS. CellTracker Green CMFDA dye was diluted to a working concentration of 5 μM with serum-free RPMI 1640 medium. The cells were resuspended in the dye working solution, and the cell density was adjusted to 2 × 10⁶ cells / year. 6 Incubate at 37°C and 5% CO2 for 30 minutes in the dark. After incubation, add 5 volumes of complete culture medium (containing 10% FBS) to stop staining, and continue incubation at 37°C in the dark for 15 minutes to ensure complete intracellular esterification of the dye. Wash cells three times with pre-warmed PBS, and finally resuspend in co-culture medium, count cells, and adjust to the desired concentration.
[0178] (4) Co-culture establishment: On day 7 after spheroid formation, carefully aspirate the old culture medium from the spheroid culture wells, being careful not to aspirate or break up the spheroids. Add 150 μL of fresh co-culture medium to each well. Add the labeled A or E group CAR-T cells at a density of 1.0 × 10⁶ cells per well. 4 Gently add approximately 20 μL of CAR-T cells to the corresponding well, allowing the cells to settle naturally to the bottom of the well containing the spheroids. This setup allows effector cells to surround and infiltrate the spheroids. Set up control wells containing only spheroids and no CAR-T cells. Return the culture plate to the incubator.
[0179] 5.2 Imaging and Quantification: At 24h and 48h after co-culture, Z-stack scans (5 μm layer thickness) were performed on the spheroids using confocal microscopy. The maximum projection map was analyzed using ImageJ software to measure the maximum depth (μm) of CAR-T cells (green fluorescence) penetrating from the edge of the spheroid to the center and the average fluorescence intensity (internal region of the spheroid).
[0180] 5.3 The experimental results are shown in Table 6. The VHH-based CAR-T cells in group A exhibited significantly stronger tumor spheroid penetration ability. This result provides direct functional evidence that the VHH tandem structure used in this invention, with its smaller molecular size, endows CAR-T cells with significantly enhanced penetration and invasion ability into solid tumor tissues. This characteristic is crucial for treating dense solid tumors such as glioblastoma, and is expected to solve the key bottleneck of insufficient cell invasion in existing CAR-T therapies. This provides support for the innovative advancement and clinical application potential of this invention in overcoming obstacles in solid tumor treatment.
[0181] Table 6 Comparison of CAR-T cell infiltration ability in glioblastoma stem cell spheroids (co-cultured for 48 hours)
[0182]
[0183] Example 6: In vivo study on the efficacy of anti-glioblastoma treatment
[0184] 6.1 Establishment of a mouse model of glioblastoma in situ
[0185] (1) Animals: NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) female mice, 6-8 weeks old. Animals were housed in an SPF-grade environment.
[0186] (2) Tumor cells: U87-EGFRvIII-Luc, U87 cells stably expressing luciferase (Luc) and human EGFRvIII. GBM-SC01-Luc, the patient-derived glioma stem cell line GBM-SC01, stably expressing Luc via lentiviral transduction. Before injection, the two cell lines were mixed at a 1:1 ratio and resuspended in serum-free, phenol red-free DMEM / F12 medium to a final concentration of 1.0 × 10⁻⁶. 5 cells / μL (i.e., each μL contains 5.0 × 10⁻⁶ cells of each of the two cell types) 4 (One), placed on ice for later use.
[0187] (2) Stereoscopic injection surgery
[0188] Anesthesia and fixation: Mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (50 mg / kg). The heads of the anesthetized mice were then fixed to a stereotaxic apparatus.
[0189] Scalp preparation and positioning: Disinfect the scalp with iodine solution, make a 1 cm incision in the midline of the scalp to expose the skull. Gently peel away the periosteum with a cotton swab to clearly expose the anterior fontanelle and the lambdoid suture.
[0190] Coordinate localization and drilling: Using the anterior fontanelle as the zero point, determine the right striatal injection coordinates based on the mouse brain atlas: anterior fontanelle +0.5 mm, right sagittal suture +2.2 mm. Drill a cranial hole with a diameter of approximately 0.5 mm at the target coordinates using a miniature cranial drill, taking care not to damage the dura mater.
[0191] Tumor cell injection: Use a 10 μL Hamilton microsyringe (700 series) with a 33G bevel needle. Aspirate 2 μL of cell suspension (total 2.0 × 10⁶ cells). 5 (1 cell). Lower the needle vertically and slowly to a depth of 3.0 mm below the surface of the skull (targeting the striatum). Inject the cell suspension slowly at a rate of 0.2 μL / min. After injection, leave the needle in place for 5 minutes, then withdraw the needle at an extremely slow rate (approximately 1 mm / min).
[0192] Postoperative care: Suture the scalp incision and inject preheated saline (0.5 mL) into the abdominal cavity to prevent dehydration. Place the mouse on a 37°C heating pad for resuscitation until it is fully awake, then return it to its cage.
[0193] 6.2 Treatment grouping and CAR-T cell adoptive transfer
[0194] (1) Tumor formation verification and random grouping: On the 7th day after tumor inoculation, all mice were subjected to in vivo imaging to confirm tumor formation.
[0195] Imaging: Mice were injected intraperitoneally with D-fluorescein potassium salt (150 mg / kg), and 10 minutes later anesthetized with isoflurane. They were then placed in the IVIS Spectrum imaging chamber and images were acquired after a 60-second exposure.
[0196] Quantitative analysis and grouping: Tumor regions were delineated using Living Image® software, and total luminescent flux (Total Flux, photons / s) was measured. Tumors without tumor formation (signal < 1×10⁻⁶) were excluded. 5 (p / s) or abnormally high signal (>1×10) 7 Mice bearing tumors (n=40) were randomly divided into 5 groups (n=8 mice / group) according to tumor burden (total luminescence flux) to ensure that there was no statistically significant difference in initial tumor burden among the groups (P>0.05): G1: PBS control group; G2: C group CAR-T (single-target anti-CD133);
[0197] G3: Group D CAR-T (single-target anti-EGFRvIII); G4: Group E CAR-T (scFv tandem dual-target); G5: Group A CAR-T (this invention, VHH tandem dual-target).
[0198] (2) CAR-T cell infusion: On the same day after grouping (i.e., day 7 after tumor inoculation), tail vein infusion was performed. The CAR-T cells prepared and expanded to day 10 in Example 3 were washed twice with PBS and resuspended in PBS. Each mouse received a slow injection of 5.0 × 10⁶ CAR-T cells via the tail vein. 6 100 live cells, total volume 200 μL. The PBS control group was injected with an equal volume of sterile PBS.
[0199] 6.3 Efficacy monitoring, endpoint determination and data analysis
[0200] (1) Monitoring
[0201] Tumor growth monitoring: Starting from the start of treatment, tumor signals were monitored twice a week (e.g., Monday and Thursday) using IVIS in vivo imaging, following the same method as above. Monitoring continued until all control mice reached the endpoint or on day 90 of the experiment.
[0202] Survival tracking: Observe the mice's general condition, neurological symptoms, and body weight daily. The survival endpoint is defined as the occurrence of any of the following: progressive neurological deficits (such as severe circling, head tilting, ataxia, paralysis); weight loss exceeding 20% of initial body weight; or other near-death states consistent with animal ethics.
[0203] (2) Data Analysis
[0204] Tumor growth kinetics: The total luminescence flux of the tumor obtained from each imaging session is logarithmically transformed (log 10 Growth curves were plotted over time. Repeated measures two-way ANOVA was used to compare the overall differences in tumor growth curves among groups, and Sidak multiple comparison tests were performed to analyze differences at specific time points.
[0205] Survival analysis: Survival curves were plotted using the Kaplan-Meier method. Statistical comparisons of survival differences between groups were performed using the Log-rank (Mantel-Cox) test. Hazard ratios (HRs) and their 95% confidence intervals were calculated.
[0206] 6.4 Results, Analysis and Summary
[0207] (1) Baseline tumor burden: The average total luminous flux of the tumor in each group at the time of randomization (×10) 5 The p / s (mean ± SEM) values were: G1: 5.8 ± 0.7; G2: 5.5 ± 0.6; G3: 6.1 ± 0.8; G4: 5.9 ± 0.7; G5: 5.7 ± 0.6. One-way ANOVA confirmed no significant differences between groups (F = 0.25, P = 0.91).
[0208] (2) Tumor growth inhibition: On the 14th day after treatment, the tumor bioluminescence signal of group A (VHH tandem CAR-T) of the present invention was significantly lower than that of all control groups (P<0.05), showing a rapid anti-tumor response.
[0209] Table 7. Comparison of tumor burden among groups on day 28 post-treatment
[0210]
[0211] Note: Data are expressed as mean ± standard error (SEM, n=8 animals / group). P-values were obtained by repeated measures two-way RM ANOVA combined with Sidak multiple comparison test. ns: no significant difference; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
[0212] Overall analysis of tumor growth curves revealed a highly significant interaction between treatment group and time (P<0.0001), indicating fundamental differences in the inhibitory patterns and effects of different CAR-T therapy regimens on tumor growth. The CAR-T in group A of this invention exhibited the strongest and most durable tumor-suppressive ability at all time points.
[0213] (3) Survival analysis:
[0214] G1 (PBS): Median survival = 31 days. All mice died before day 45.
[0215] G2 (Group C): Median survival = 47 days. Long-term survival (>80 days) rate is 1 / 8.
[0216] G3 (Group D): Median survival = 52 days. Long-term survival rate is 2 / 8.
[0217] G4 (Group E): Median survival = 61 days. Long-term survival rate is 3 / 8.
[0218] G5 (Group A, this invention): Median survival > 80 days. At the pre-specified end of the experiment (day 90), 6 / 8 mice were still alive, with a long-term survival rate of 75%. No significant treatment-related toxicities were observed throughout the study.
[0219] (4) Results Analysis and Summary
[0220] Powerful in vivo anti-tumor efficacy: The CAR-T therapy in Group A of this invention can rapidly, potently, and persistently inhibit the growth of in situ GBM tumors. Its efficacy is significantly superior to all control groups at all time points, including single-target therapy and the best dual-target regimen based on scFv in the current technology (Group E).
[0221] Significant survival advantage: This invention's CAR-T therapy delivers a breakthrough survival benefit, enabling 75% of animals to achieve long-term survival, with a median survival exceeding 80 days, and significantly extending overall survival. Its survival advantage compared to traditional scFv dual-target CAR-T is statistically significant (P<0.05), and the hazard ratio (HR=0.22) indicates a reduction in mortality risk of approximately 78%.
[0222] Validating the in vivo correlation observed in vitro: This superior in vivo efficacy, highly consistent with the superior T-cell memory phenotype (high T_CM ratio), potent in vitro synergistic killing activity, and enhanced tumor spheroid penetration observed in previous examples, forms a complete chain of evidence. This strongly suggests that the specific VHH tandem sequence of this invention not only optimizes antigen recognition and signal transduction for CARs but also, as a whole, shapes a T-cell product with greater therapeutic potential.
[0223] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A bispecific antigen-binding molecule, characterized in that, The bispecific antigen-binding molecule comprises the following components in tandem: a first single-domain antibody targeting the human CD133 antigen, a flexible linker peptide, and a second single-domain antibody targeting the human EGFRvIII antigen.
2. The bispecific antigen-binding molecule according to claim 1, characterized in that, The first single-domain antibody targeting the human CD133 antigen has the amino acid sequence shown in SEQ ID NO:1, or a variant that has at least 90% sequence identity with SEQ ID NO:1 and retains CD133 binding activity; the second single-domain antibody targeting the human EGFRvIII antigen has the amino acid sequence shown in SEQ ID NO:2, or a variant that has at least 90% sequence identity with SEQ ID NO:2 and retains EGFRvIII specific binding activity.
3. The bispecific antigen-binding molecule according to claim 1 or 2, characterized in that, The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:
3.
4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain; wherein the extracellular antigen-binding domain is a bispecific antigen-binding molecule as described in any one of claims 1-3.
5. The chimeric antigen receptor according to claim 4, characterized in that, The transmembrane domain is derived from the human CD8α molecule; the intracellular signal transduction domain includes the human 4-1BB co-stimulatory domain and the human CD3ζ chain activation domain.
6. An expression carrier, characterized in that, The vector is pCDH-EF1-CAR-A, which contains the chimeric antigen receptor encoding the claim 4 or 5.
7. An immune effector cell, characterized in that, The cells are transduced by the expression vector of claim 6 to stably express the chimeric antigen receptor of claim 4 or 5.
8. The immune effector cell according to claim 7, characterized in that, The immune effector cells are T cells, NK cells, or NKT cells.
9. A pharmaceutical composition, characterized in that, The composition comprises the immune effector cells as described in claim 7 or 8, and a pharmaceutically acceptable carrier.
10. Use of a bispecific antigen-binding molecule according to any one of claims 1-3, or a chimeric antigen receptor according to any one of claims 4-5, or an immune effector cell according to any one of claims 7-8, or a pharmaceutical composition according to claim 9 in the preparation of a medicament for treating tumors; wherein the tumor is glioblastoma expressing CD133 and / or EGFRvIII antigen.