Bispecific T cell adapter targeting ICAM1 and CD3 and application thereof

Abstract

The invention discloses a bispecific T cell adapter targeting ICAM1 and CD3 and application of the bispecific T cell adapter, and belongs to the technical field of biological medicine. The bispecific T cell adapter HLE-BiIC does not depend on antigen presentation of MHC-I molecules, can overcome the problems of'first signal 'deficiency and immune escape of undifferentiated thyroid cancer caused by MHC-I down-regulation, and reconstructs efficient anti-tumor immune response in an'immune false fever' tumor model. The compound has good anti-tumor activity, shows remarkable anti-tumor activity in in-vivo and in-vitro experiments, can induce complete regression of tumors especially in a humanized mouse model, and has good biological safety, so that the compound has extremely high clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an immunotherapy drug and its preparation and application. More specifically, this invention relates to a bispecific antibody (T-cell connector) that simultaneously targets intercellular adhesion molecule 1 (ICAM1) and T-cell surface antigen CD3, and the application of this antibody in the preparation of drugs for treating thyroid cancer, particularly undifferentiated thyroid cancer. Background Technology

[0002] Anaplastic thyroid cancer (ATC) is a highly malignant solid tumor originating from the follicular epithelium of the thyroid gland. Although it accounts for only a small percentage of all thyroid cancers, it is extremely aggressive and deadly. Current standard treatment options include surgical resection, radiotherapy and chemotherapy, and small-molecule targeted therapies (such as inhibitors against BRAF mutations). However, due to the rapid progression of ATC, most patients are no longer candidates for surgery at diagnosis; and targeted therapy is limited by the low frequency of driver gene mutations and the rapid development of secondary resistance after treatment, resulting in a very limited clinical benefit population. Therefore, the development of new treatment methods is urgently needed.

[0003] In recent years, immunotherapy, represented by immune checkpoint inhibitors (ICIs, such as PD-1 / PD-L1 antibodies), has transformed the treatment landscape for various solid tumors. Pathological studies show that ATC tumor tissue often contains a large number of CD8+ cells. + T-cell infiltration presents characteristics of so-called "immune-hot tumors." Theoretically, these tumors should respond well to ICIs. However, clinical data show that ATC patients receiving ICIs have low objective response rates (ORR) and do not experience significant overall survival prolongation. This "pseudo-hot" phenomenon reveals a unique immune escape mechanism in ATC.

[0004] Existing research (including the research of the inventors' team) indicates that a key mechanism of immune escape in acute tumor cell (ATC) lies in the downregulation or absence of major histocompatibility complex class I (MHC-I) expression. In classic cellular immune responses, T cell receptors (TCRs) rely on the presentation of MHC-I molecules to recognize tumor antigens, serving as the "first signal" for T cell activation. Downregulation of MHC-I directly leads to a structural break in this first signaling pathway, rendering effector T cells "functionally blind" and unable to initiate their killing program even if they successfully infiltrate the tumor microenvironment due to their inability to recognize antigens. This mechanistic defect inherently limits conventional immunotherapies (such as ICIs and tumor vaccines) that rely on endogenous antigen presentation systems in ATC treatment. Therefore, developing a novel immunotherapy strategy that does not rely on MHC-I restriction and can directly reconstruct the recognition connection between T cells and tumor cells is crucial to overcoming the bottleneck in ATC treatment.

[0005] Intercellular adhesion molecule 1 (ICAM1 / CD54) is a transmembrane glycoprotein that has been shown to be abnormally highly expressed in various malignant tumors. The applicant's previous and current studies have demonstrated that ICAM1 is significantly highly expressed in ATC tissues, and this high expression is positively correlated with poor prognosis, while its expression is relatively low in normal thyroid tissues and other vital organs. This differential expression profile makes ICAM1 an ideal therapeutic target. Currently, investigational drugs targeting ICAM1 mainly include antibody-drug conjugates (ADCs) and chimeric antigen receptor T cells (CAR-T). However, ADC drugs face challenges related to endocytosis efficiency and systemic toxicity due to toxin shedding; CAR-T therapy in solid tumors faces problems such as difficulty in tissue invasion, long preparation cycles, high costs, and a high risk of T cell exhaustion.

[0006] T-cell engagers (TCEs), as a bispecific antibody technology, specifically bind to tumor surface antigens at one end and CD3 molecules on the surface of T cells at the other. The greatest advantage of TCEs is that their mechanism of action is completely independent of MHC-I antigen presentation. They can physically bring T cells closer to tumor cells and directly mimic the first signal to activate T cells, inducing them to release perforin and granzymes to lyse tumor cells. Given the prevalent pathological feature of MHC-I downregulation in acute tumor cell lesions (ATC), TCEs are theoretically one of the best strategies to address ATC immune evasion.

[0007] However, as of the date of this application, no prior art discloses a technical solution or application of bispecific antibodies targeting both ICAM1 and CD3 in the treatment of undifferentiated thyroid cancer; nor is there conclusive evidence regarding whether such bispecific drugs can effectively induce immune synapse formation and reconstruct anti-tumor immune responses in the ATC-specific MHC-I expression downregulation and immunosuppressive microenvironment. Therefore, there is an urgent need in the field to develop a novel T-cell connector drug that can specifically target ICAM1, effectively overcome the ATC immune escape mechanism (especially MHC-I restriction), and possess excellent safety profile to meet the pressing needs of clinical treatment. Summary of the Invention

[0008] This invention provides a method for preparing and applying a bispecific T-cell connector (HLE-BiIC) based on IgG Fc heterodimer with ICAM1 and CD3 as target antigens.

[0009] Specifically, this invention aims to achieve the following objectives: This invention provides a novel bispecific antibody entity: The present invention aims to provide a structurally stable bispecific T-cell connector with an extended half-life. This connector physically links the single-chain variable region of anti-ICAM1 and the single-chain variable region of anti-CD3, and utilizes Knob-into-Hole technology and LALA-PG mutation-optimized IgG Fc backbone to solve the problems of short half-life and poor safety of existing BiTE drugs.

[0010] Overcoming MHC-I-restricted immune escape: In response to the challenge of the failure of traditional immunotherapy due to the downregulation of MHC-I molecules in undifferentiated thyroid carcinoma (ATC), the connector of this invention aims to directly induce T cells to form artificial immune synapses with ICAM1-positive tumor cells in a manner that does not rely on MHC-I antigen presentation, thereby reconstructing the immune killing pathway.

[0011] The invention provides a method for preparing the aforementioned connector: The present invention aims to construct a recombinant vector expressing each polypeptide chain of the above-mentioned heterodimer through genetic engineering technology, and to efficiently prepare high-purity, conformally correct bispecific antibodies using a mammalian cell expression system.

[0012] Expanding the clinical application of the connector: This invention aims to verify and provide the application of the bispecific T-cell connector in the preparation of antitumor drugs, particularly for the treatment of refractory undifferentiated thyroid cancer that is ICAM1 positive and accompanied by MHC-I downregulation.

[0013] The specific technical solution of the present invention is as follows: This invention provides a bispecific T-cell connector targeting ICAM1 and CD3 (named HLE-BiIC). The connector is a fusion protein based on an IgG antibody constant region backbone, the backbone including an Fc domain comprising a hinge region, CH2, and CH3. The connector is a heterodimer formed by the binding of a first polypeptide chain and a second polypeptide chain. (1) The first polypeptide chain: from the N-terminus to the C-terminus, it includes: ICAM1-specific single-chain variable region (ICAM1-scFv), first flexible linker peptide, CD3-specific single-chain variable region (CD3-scFv), second flexible linker peptide, and first Fc domain subunit; (2) The second polypeptide chain: from the N-terminus to the C-terminus, it includes: second Fc domain subunit; the second polypeptide chain does not contain an antigen-binding domain.

[0014] Specifically, the ICAM1-scFv is composed of a heavy chain variable region (VH) and a light chain variable region (VL) derived from antibody R6.5 connected by a linker; the CD3-scFv is composed of a heavy chain variable region (VH) and a light chain variable region (VL) derived from antibody OKT3 connected by a linker. Both the first and second Fc domain subunits contain the hinge region, CH2 domain, and CH3 domain of the IgG antibody.

[0015] The first and second polypeptide chains form a stable "single-arm" bispecific antibody structure through heterodimerization in the Fc region. ICAM1-scFv and CD3-scFv are used to construct a bispecific recognition structure for simultaneously recognizing ICAM1 molecules on the tumor surface and CD3 molecules on the T cell surface. This bispecific T cell connector of the present invention can physically bridge T cells and tumor cells, inducing the formation of artificial immune synapses without relying on MHC-I class molecule antigen presentation, thereby activating T cells and mediating specific killing of ICAM1-positive tumor cells.

[0016] Preferably, the Fc domains of the first and second polypeptide chains are modified using a knob-into-Hole technique to form a stable hetero Fc dimer. Specifically, a mutation is introduced into the CH3 domain of one polypeptide chain to form a protrusion (knob), and a mutation is introduced into the CH3 domain of the other polypeptide chain to form a depression (holo), thereby promoting specific pairing of the two different polypeptide chains.

[0017] More specifically: a T366W mutation is introduced into the CH3 domain of the first polypeptide chain to form a Knob structure, and T366S, L368A and Y407V mutations are introduced into the CH3 domain of the second polypeptide chain to form a Hole structure. Alternatively, T366S, L368A, and Y407V mutations can be introduced into the CH3 domain of the first polypeptide chain to form a Hole structure, and T366W mutations can be introduced into the CH3 domain of the second polypeptide chain to form a Knob structure.

[0018] Preferably, the Fc CH2 domains of both the first and second polypeptide chains are mutated with LALA-PG mutations (i.e., L234A, L235A, and P329G mutations). This mutation silences the effector function of the antibody Fc fragment, significantly reducing or eliminating its binding ability to the Fcγ receptor and complement C1q, thereby inhibiting antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and avoiding systemic toxicity caused by nonspecific immune activation.

[0019] Preferably, the first flexible linker peptide and the second flexible linker peptide are (Gly4Ser)n structures, where n is 3 or 4 (i.e., 4GS linker peptide), to give the two antigen-binding domains sufficient spatial freedom.

[0020] Specifically, the amino acid sequence of the first polypeptide chain is shown as amino acid sequence 1-748 of SEQ ID NO.4, and the amino acid sequence of the second polypeptide chain is shown as amino acid sequence 1-231 of SEQ ID NO.5. The amino acid sequence of ICAM1-scFv is shown as SEQ ID NO.7, and the amino acid sequence of CD3-scFv is shown as SEQ ID NO.8.

[0021] The present invention also provides a nucleic acid molecule encoding the bispecific T-cell adaptor. As an example, the nucleotide sequence encoding the first polypeptide chain is a sequence of 1-2244 bp as shown in SEQ ID No. 1, and the nucleotide sequence encoding the second polypeptide chain is a sequence of 1-693 bp as shown in SEQ ID No. 2.

[0022] The present invention also provides a method for preparing the bispecific T cell connector, comprising the following steps: (1) constructing a recombinant expression vector: constructing a vector containing a nucleotide sequence encoding a first polypeptide chain and a vector containing a nucleotide sequence encoding a second polypeptide chain; (2) transfection and expression: co-transfecting the vector described in step (1) into mammalian host cells; (3) culture and purification: culturing the host cells under suitable conditions, collecting the culture supernatant, and purifying the protein using affinity chromatography to obtain the bispecific T cell connector. Preferably, the vector described in step (1) is pcDNA3.1; and the mammalian cells described in step (2) are HEK293F cells.

[0023] The present invention also provides the application of the bispecific T-cell connector in the preparation of antitumor drugs.

[0024] This invention also provides an antitumor drug, the active ingredient of which is the aforementioned bispecific T-cell connective or the aforementioned nucleic acid molecule. The tumor cell type is ICAM1-positive thyroid cancer cells, particularly undifferentiated thyroid cancer cells with downregulated or absent expression of MHC-I class molecules.

[0025] The beneficial effects of this invention are: Breakthrough in immune escape mechanism: The bispecific T cell connector HLE-BiIC of this invention does not rely on MHC-I molecules for antigen presentation, and can overcome the problem of "first signal" loss and immune escape caused by MHC-I downregulation in undifferentiated thyroid cancer, and reconstruct a highly efficient anti-tumor immune response in the "immune pseudo-heat" tumor model.

[0026] High specificity and safety: By introducing the LALA-PG silent mutation, the non-specific ADCC and CDC effects mediated by Fc are eliminated, avoiding off-target toxicity; at the same time, experiments have confirmed that this linker has a killing effect only on tumor cells with high ICAM1 expression, and has no significant effect on normal cells, thus having a good therapeutic window.

[0027] Pharmacokinetic advantages: Based on the design of the heterodimer Fc skeleton (Knob-into-Hole technology), not only is the stability of the molecular structure and the uniformity of the product guaranteed, but also the drug is given an extended in vivo half-life, which solves the clinical defect of short half-life of traditional BiTE molecules.

[0028] Significant antitumor activity: The connector of the present invention has shown significant antitumor activity in both in vivo and in vitro experiments. In particular, in humanized mouse models, it can induce complete tumor regression and has extremely high clinical translational value. Attached Figure Description

[0029] Figure 1 The images show the molecular form and purity identification of the bispecific T-cell connective HLE-BiIC and its control drug. (a) is a schematic diagram of the molecular structure; (b) is an SDS-PAGE electrophoresis analysis (under reduction conditions).

[0030] Figure 2 The graphs show the cell binding affinity analysis of HLE-BiIC and its control drug. (a) Graphs show the specific affinity analysis of HLE-BiIC and its control drug for KHM5M cells (ICAM1 highly expressed); (b) Graphs show the specific affinity analysis of HLE-BiIC and its control drug for Jurkat cells (CD3+-expressing cells). + Affinity analysis diagram of ).

[0031] Figure 3The in vitro activity of HLE-BiIC in PBMCs killing tumor cells is shown. The dose-response curves of HLE-BiIC and its control drug at different concentrations inducing KHM5M cell lysis are presented, demonstrating the dose-dependent cytotoxicity of HLE-BiIC.

[0032] Figure 4 The following figures validate the targeting dependence and structure-activity relationship of HLE-BiIC. (a) Validation of the construction of a stable CHO-K1-ICAM1 cell line; (b): The upper figure shows the fluorescence activation detection after co-culturing HLE-BiIC with Jurkat-NFAT reporter cells and different target cells (CHO-K1 or CHO-K1-ICAM1); the lower figure shows the fluorescence intensity statistical analysis, which shows that the activation signal is strictly dependent on ICAM1 expression; *** p<0.001, and the scatter points represent different samples.

[0033] Figure 5 The diagram shows the specific activation of T cells stimulated by HLE-BiIC; (a) shows CD3. + (a) Expression ratio of CD69, an early activation marker on the surface of T cells; (b) Expression ratio of CD3. + Expression ratio of CD25, a marker of late activation on the surface of T cells; *** p<0.001, where the scatter plots represent different samples.

[0034] Figure 6 A diagram showing the expansion of T cells stimulated by HLE-BiIC; displaying CFSE-labeled (a)CD3. + (b) CD8 + The fluorescence intensity attenuation histogram of T cells after drug treatment indicates that HLE-BiIC induces T cell proliferation.

[0035] Figure 7 The graph shows the release of interferon-gamma (IFN-γ) from T cells stimulated by HLE-BiIC; it also shows the trend of IFN-γ concentration in the co-culture supernatant as a function of HLE-BiIC concentration; ** p < 0.01, *** p < 0.001, where the scatter plots represent different samples.

[0036] Figure 8The following graphs are used to evaluate the in vivo specific antitumor activity and safety of HLE-BiIC in the NSG humanized mouse model: (a) Flowchart of animal experiments; (b) Tumor growth curves (tumor volume changes) of the control group and different doses of HLE-BiIC groups; (c) Tumor weight statistics of the control group and different doses of HLE-BiIC groups at the experimental endpoint; (d) Curves of body weight changes of mice in each experimental group during the treatment period; (e) H&E stained pathological sections of major organs (heart, liver, spleen, lung, kidney) of mice in each experimental group at the experimental endpoint; *** p<0.001, ns p>0.05, where the scatter plots represent different samples. Detailed Implementation

[0037] Example 1: Preparation of a bispecific T-cell adaptor targeting ICAM1 and CD3 (HLE-BiIC) The anti-ICAM1 antibody chosen was R6.5 (targeting the extracellular domains D1-D2 of human ICAM1), sequence derived from patent: US5821337A. The anti-CD3 antibody chosen was OKT3 (targeting CD3ε), sequence derived from patent: US4361549A. A schematic diagram of the bispecific T-cell connector is shown below. Figure 1 As shown in (a), three expression plasmids for human kidney epithelial cells (293F) were constructed.

[0038] HLE-BiIC (targeted drug): First polypeptide chain (functional chain): R6.5 scFv-(G4S)3-OKT3 scFv-(G4S)3-Hinge-CH2(LALA-PG)-CH3(knob)-His6 (gene sequence as shown in SEQ ID No. 1, amino acid sequence as shown in SEQ ID No. 4); Second polypeptide chain (stabilizing chain): Hinge-CH2(LALA-PG)-CH3(hole)-Flag (gene sequence as shown in SEQ ID No. 2, amino acid sequence as shown in SEQ ID No. 5).

[0039] Ctrl-CD3 (control drug): First polypeptide chain (control chain): Ctrl-scFv-(G4S)3-OKT3 scFv-(G4S)3-Hinge-CH2(LALA-PG)-CH3(knob)-His6 (gene sequence as shown in SEQ ID No. 3, amino acid sequence as shown in SEQ ID No. 6); Second polypeptide chain (stable chain): Hinge-CH2(LALA-PG)-CH3(hole)-Flag (gene sequence as shown in SEQ ID No. 2, amino acid sequence as shown in SEQ ID No. 5).

[0040] R6.5scFv and OKT3scFv are the variable regions of single-chain antibodies against ICAM1 and CD3, respectively, with amino acid sequences shown in SEQ ID NO.7 and SEQ ID NO.8. Ctrl-scFv is the variable region of a single-chain antibody recognizing an irrelevant antigen (KRAS-G12V). (G4S)3 is a flexible linker of three GGGGS lengths. Hinge, CH2, and CH3 are constant region structures of human IgG1. Knob and hole structures were designed between the two CH3 structures using the knock-in-hole technique to promote heterodimerization. Simultaneously, LALA-PG mutations (L234A, L235A, P329G) were introduced into CH2 to silence the Fc effector function. His6 is a 6-histidine tag for protein purification, and the Flag tag is an 8-amino acid tag (DYKDDDDK) for protein purification.

[0041] The heavy chain variable regions and light chain variable regions of R6.5 and OKT3, the constant region of the IgG1 antibody heavy chain, and the sequences of each linker peptide were synthesized by Shanghai Sangon Biotech Co., Ltd. The gene segments were combined according to the requirements of recombinant plasmids using PCR and overlap PCR, and introduced upstream of the recombinant gene. Nhe I restriction enzyme site (GCTAGC), Kozak sequence (ACCACC), and a stop codon (TGA) are introduced downstream. Hind The AAACTT restriction site is ligated to the pCDNA3.1 plasmid by T4 ligase (the plasmid is also ligated via...). Nhe I and Hind Recombinant plasmids expressing the first, second, and control polypeptide chains were constructed using a double enzyme digestion (IIII), and their correctness was verified by sequencing. Subsequently, protein expression was performed using transient transfection technology. The constructed first-peptide recombinant plasmid and the second-peptide recombinant plasmid were mixed at a 1:1 mass ratio and co-transfected into 293F cells in suspension culture. After 5-7 days of culture, the supernatant was collected and purified using a tandem affinity chromatography strategy: First, the HisTrap HP affinity column (GE Healthcare, catalog number: 17-5248-02) was used to capture the His-tagged first polypeptide chain and its assembly. The elution buffer was then passed through an anti-FLAG... ® Secondary purification was performed using M1 affinity gel (Sigma-Aldrich, catalog number: A4596) to specifically enrich the second polypeptide chain containing the Flag tag. This dual-screening strategy effectively removed homodimeric impurities, yielding high-purity heterodimeric proteins.

[0042] Finally, the purified product was analyzed by SDS-PAGE electrophoresis (see [link]). Figure 1(b) shows that both HLE-BiIC and its control drug exhibit two clear bands under reducing conditions, corresponding to the theoretical molecular weights of the first polypeptide chain (approximately 81 kDa) and the second polypeptide chain (approximately 27 kDa), respectively. This result indicates that the Fc heterodimer modified using the Knob-into-Hole technology has high assembly efficiency in eukaryotic cells, and the purified product has a correct structure and few impurities, meeting the requirements for subsequent biological activity evaluation.

[0043] Example 2: Verification of the affinity and binding specificity of HLE-BiIC This embodiment utilizes flow cytometry to evaluate the binding affinity and specificity of HLE-BiIC for the tumor target ICAM1 and the T cell target CD3. The specific experimental procedure is as follows: Jurkat cells (CD3-positive T cells) and KHM5M cells (ICAM1-highly expressing tumor cells, undifferentiated human thyroid cancer cells) in logarithmic growth phase were collected and centrifuged at 400 ×g for 5 minutes, then the supernatant was discarded. The cell pellet was washed with PBS buffer, and 500 μL of HLE-BiIC or the control antibody Ctrl-CD3 at different concentration gradients (with an equal volume of PBS as a blank control) was added. The cells were incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with PBS buffer to remove unbound primary antibody. The cells were then resuspended in 500 μL of PBS, and 1 μL of FITC-labeled goat anti-human IgG (H+L) secondary antibody (purchased from Beyotime Biotechnology Co., Ltd., catalog number: A0556) was added. After mixing, the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS to remove unbound secondary antibody, and then resuspended in 500 μL PBS. Signal acquisition and data analysis were performed using a BD flow cytometer.

[0044] Experimental results show (see) Figure 2 HLE-BiIC exhibited good antigen-binding specificity. Specifically, in binding assays against ICAM1-positive KHM5M cells (see [link to assay]), [the specific antigen-binding specificity was observed]. Figure 2 In (a) of the study, HLE-BiIC stably binds to the ICAM1 antigen on the cell surface, and the mean fluorescence intensity (MFI) increases significantly in a dose-dependent manner with increasing drug concentration. In contrast, the control antibody Ctrl-CD3 (whose ICAM1 recognition module is replaced by an irrelevant TCR sequence) did not show any detectable binding signal. This result clarifies that the recognition of tumor cells by HLE-BiIC is strictly dependent on the presence of the ICAM1 antigen. Further evaluation of the binding ability of the two antibodies on CD3-positive Jurkat cells (see [reference]) was conducted. Figure 2(b) shows that HLE-BiIC and Ctrl-CD3 exhibit highly overlapping affinity curves, suggesting that their CD3 recognition structures maintain consistent spatial conformation and biological function. In summary, the functional difference of HLE-BiIC mainly stems from the conformational specificity of the tumor-targeting end, rather than the non-specific difference of the CD3-binding end, confirming the correctness of the molecular design.

[0045] Example 3: Validation of in vitro specific antitumor activity and targeting mechanism This embodiment first evaluates the direct killing ability of HLE-BiIC against ICAM1-overexpressing tumor cells using an in vitro co-culture system. The specific experimental procedure is as follows: KHM5M cells and PBMCs were seeded in cell culture plates at an effector-to-target ratio (E:T) of 1:10. Then, a series of gradient concentrations of HLE-BiIC or the control antibody Ctrl-CD3 were added to the system (each concentration was set in triplicate, n=3). After gently mixing the culture plates, they were incubated at 37°C in a 5% CO2 incubator. After 48 hours of co-incubation, the experimental results were detected and analyzed. The results show (see...). Figure 3 HLE-BiIC exhibited typical dose-dependent cytotoxic effects in the KHM5M cell line, with an effective concentration (EC50) of approximately 64.95 nM. 50 HLE-BiIC significantly induced T cell aggregation with target cells and mediated target cell lysis. In contrast, the control antibody Ctrl-CD3 did not induce a significant killing response under the same experimental conditions. This result effectively ruled out non-specific cross-linking or background activity caused by the CD3 binding domain, indicating that the antitumor activity of HLE-BiIC depends on the simultaneous recognition of both ICAM1 and CD3 targets.

[0046] To further verify at the molecular mechanism level whether ICAM1 expression is a necessary and sufficient condition for conferring sensitivity to target cells, the applicant constructed a "target conferral" verification model. Specifically, human ICAM1 was exogenously and stably overexpressed in CHO-K1 cells that do not naturally express ICAM1 using genetic engineering techniques to construct the CHO-K1-ICAM1 cell line (see [link to model]). Figure 4 (a)). Subsequently, it was co-cultured with Jurkat-8xNFAT-ZsGreen reporter cells under the conditions described above. The experimental results showed (see [reference]). Figure 4As shown in (b), HLE-BiIC effectively induced the activation of the NFAT signaling pathway in T cells only in the presence of CHO-K1 cells expressing ICAM1, manifested as a strong upregulation of ZsGreen fluorescence intensity in reporter cells. In contrast, no activation signal was detected in wild-type CHO-K1 cells not transfected with ICAM1, or in the group treated with the control antibody Ctrl-CD3. These results further confirm that the structure-activity relationship of HLE-BiIC is strictly dependent on the spatial localization and physical bridging mechanism mediated by ICAM1, demonstrating its high specificity.

[0047] Example 4: Detection of HLE-BiIC-induced T cell-specific activation This embodiment aims to evaluate the ability and specificity of HLE-BiIC in inducing the expression of T cell surface activation markers in an in vitro co-culture system. The specific procedure is as follows: KHM5M cells (target cells) in logarithmic growth phase and healthy human PBMCs (effective cells) were mixed at an effector-to-target ratio (E:T) of 1:10 and seeded in cell culture plates. A series of gradient concentrations of HLE-BiIC or control antibody were added (three replicates per group, n=3). After mixing, the plates were incubated at 37°C in a 5% CO2 incubator. After 72 hours of culture, the cell suspension was collected, centrifuged at 400×g for 5 minutes, and the supernatant was discarded. Cell pellets were washed once with 1×PBS buffer, resuspended in 1×PBS, and stained with the following fluorescently labeled antibodies: 5 μL APC-labeled mouse anti-human CD3 antibody (BioLegend, catalog number 317318), 5 μL FITC-labeled mouse anti-human CD69 antibody (BioLegend, catalog number 310904), and 5 μL PE-labeled mouse anti-human CD25 antibody (BioLegend, catalog number 302606). After mixing, the mixture was incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with 1×PBS to remove unbound antibodies. Finally, the cells were resuspended in 500 μL 1×PBS, and data were acquired using a BD flow cytometer to analyze the expression ratios of CD69 and CD25 in the CD3-positive T cell population.

[0048] CD69 and CD25 are early and late marker proteins in the T cell activation process, respectively. Experimental results are as follows: Figure 5 As shown, in the co-culture system of PBMC and KHM5M cells, HLE-BiIC can significantly induce CD3+. +The expression of CD69, an early activation marker, and CD25, a late activation marker, on the T cell surface was upregulated, and this activation effect showed a clear dose-dependent relationship. In contrast, the control antibody Ctrl-CD3 failed to induce a significant T cell activation response (activation level remained at baseline) due to its lack of targeting ability for ICAM1 on the tumor cell surface. These results strongly confirm that HLE-BiIC-induced T cell activation is highly specific and strictly depends on the dual bridging effect of the drug molecule on the tumor target ICAM1 and the T cell target CD3.

[0049] Example 5: Detection of HLE-BiIC-induced T cell proliferation ability This embodiment utilizes the CFSE dilution method to evaluate the ability of HLE-BiIC to drive T cell clonal expansion. The specific procedures are as follows: First, a suspension of healthy human PBMCs was prepared, and CFSE cell proliferation tracking fluorescent dye (purchased from Thermo Fisher Scientific, catalog number: 65-0850-84) was added to a final concentration of 1 μM. The suspension was then incubated at 37°C in the dark for 15 minutes for labeling. Subsequently, the cells were washed with 1×PBS buffer and centrifuged to completely remove unbound free dye. KHM5M cells (target cells) and labeled PBMCs (effective cells) were mixed at an effector-to-target ratio (E:T) of 1:10 and seeded into cell culture plates. Gradual concentrations of HLE-BiIC were added, and the mixture was incubated at 37°C in a 5% CO2 incubator. After 96 hours of culture, cell samples were collected, and CD3+ was detected using flow cytometry. + and CD8 + CFSE fluorescence intensity of T cell subsets. CFSE is a cell membrane-permeable fluorescent dye that nonspecifically covalently binds to intracellular proteins to produce green fluorescence; as cells undergo mitosis, the fluorescent dye is equally distributed to the two daughter cells, resulting in a gradual decrease in fluorescence intensity per cell. Experimental results show (see...) Figure 6 CD3 of the HLE-BiIC processing group + and CD8 + The fluorescence intensity of CFSE in T cells was significantly reduced (manifested as a leftward shift of the fluorescence peak in the flow cytometry histogram), and this proliferative effect was dose-dependent. These results indicate that HLE-BiIC can significantly drive functional and sustained proliferation of T cells, providing a cellular basis for achieving a durable in vivo anti-tumor immune response.

[0050] Example 6: Detection of HLE-BiIC-induced release of cytokines (IFN-γ) from T cells This embodiment aims to evaluate the release of key effector cytokines after HLE-BiIC activates T cells. The specific procedure is as follows: KHM5M cells (target cells) in logarithmic growth phase and human PBMCs (effector cells) were seeded in cell culture plates at an effector-to-target ratio (E:T) of 1:10. A gradient concentration of HLE-BiIC or control antibody was added to each well (three replicates per group, n=3), and the mixture was incubated at 37°C in a 5% CO2 incubator. After 72 hours of culture, the cell culture supernatant was aspirated and centrifuged at 400 ×g for 5 minutes to remove cell debris. The supernatant was diluted appropriately and analyzed strictly according to the instructions of the Human Interferon-gamma (IFN-γ) ELISA kit (Thermo Fisher Scientific, catalog number: 88-7316-88) to determine the concentration of IFN-γ in the supernatant of each experimental group.

[0051] Experimental results show (see) Figure 7 HLE-BiIC significantly promoted the secretion of the key pro-inflammatory cytokine IFN-γ by T cells, exhibiting a clear dose-dependent trend. High levels of IFN-γ secretion suggest that T cells were effectively activated, which can further amplify the immune-killing effect and inhibit tumor proliferation by enhancing the antigen-presenting capacity of tumor cells and recruiting other immune cells. In contrast, the cytokine levels in the control antibody treatment group remained at a very low baseline, indicating that IFN-γ release strictly depends on the dual recognition and synergistic effect of HLE-BiIC on ICAM1 on the surface of tumor cells and CD3 on the surface of T cells, further confirming the highly specific immune activation capacity of HLE-BiIC.

[0052] Example 7: Evaluation of the in vivo dose-dependent antitumor activity and safety of HLE-BiIC in NSG humanized mouse model In this embodiment, healthy female NSG mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., SPF grade) weighing 20-22 grams and aged 6-8 weeks were used to establish a humanized subcutaneous xenograft model of undifferentiated thyroid cancer to evaluate the in vivo efficacy and systemic safety of HLE-BiIC. The specific procedures were as follows: On day -1 of the experiment, each mouse received 1×10⁻⁶ HLE-BiIC via tail vein infusion. 7 Peripheral blood mononuclear cells (PBMCs) from healthy individuals were used to reconstruct the human immune system; the following day (day 0), 1 × 10⁻⁶ cells were subcutaneously injected into the right axilla of mice. 7 KHM5M cells were collected. On day 9 post-inoculation, orbital venous blood was collected from mice, and human CD3+ was detected by flow cytometry. +After confirming successful immune reconstitution, mice were randomly divided into three groups (n=8) on day 10 based on tumor volume and body weight: a solvent control group (administered an equal volume of physiological saline), a low-dose group (administered 50 μg / kg HLE-BiIC), and a high-dose group (administered 200 μg / kg HLE-BiIC). All groups received the medication via tail vein injection once a week for a total of two administrations.

[0053] During the experiment, the long and short diameters of the tumor were measured periodically and the tumor volume was calculated. The results showed that HLE-BiIC exhibited significant and dose-dependent in vivo antitumor activity (see [link to study]). Figure 8 (b) and Figure 8 (c)). Compared with the solvent control group, tumor growth in mice in the 50 μg / kg low-dose group was significantly inhibited, with a tumor growth inhibition rate (TGI) of approximately 50.8%; while in the 200 μg / kg high-dose group, HLE-BiIC showed excellent therapeutic response, with 87.5% (7 out of 8 mice) achieving complete tumor regression, and no recurrence was observed after drug withdrawal. Regarding safety evaluation, the general condition and weight changes of the mice were monitored throughout the experiment, and histopathological examination was performed at the experimental endpoint (Day 17). The results showed that the weight of mice in each treatment group remained stable throughout the experiment (see [reference]). Figure 8 In (d), no symptoms of graft-versus-host disease or acute toxicity reactions such as erect hairs and arched backs were observed.

[0054] Mice were euthanized after the experiment, and tissues from major organs such as the heart, liver, spleen, lungs, and kidneys were collected. After fixation, embedding, and sectioning, hematoxylin-eosin staining was performed. Microscopic examination results (see...) Figure 8 In (e) of the study, the major organ structures of mice in each treatment group were intact, myocardial fibers were neatly arranged, liver lobule structure was clear, glomeruli and renal tubules were normal in morphology, and alveolar structure was clear. No obvious pathological changes such as inflammatory cell infiltration, tissue necrosis, edema, or fibrosis were observed. In summary, this example demonstrates that HLE-BiIC can induce complete tumor regression at a dose of 200 μg / kg, and has no obvious toxic side effects on major organs at effective doses, exhibiting good biocompatibility.

Claims

1. A bispecific T-cell connector targeting ICAM1 and CD3, comprising a backbone containing a constant region of IgG antibody, said backbone including an Fc domain, the Fc domain including a hinge region, CH2, and CH3, characterized in that, The bispecific T cell connector is a heterodimer, specifically composed of a first polypeptide chain and a second polypeptide chain: The first polypeptide chain comprises, from the N-terminus to the C-terminus, the following components in sequence: an ICAM1-specific single-chain variable region, a first flexible linker peptide, a CD3-specific single-chain variable region, a second flexible linker peptide, and one chain with an Fc domain. The second polypeptide chain, from the N-terminus to the C-terminus, includes: another chain with an Fc domain; The first polypeptide chain and the second polypeptide chain form a stable heterodimer through the Knob-into-Hole structure in the Fc domain; The ICAM1-specific single-chain variable region in the first polypeptide chain is composed of a heavy chain variable region and a light chain variable region derived from the R6.5 clone, and the CD3-specific single-chain variable region is composed of a heavy chain variable region and a light chain variable region derived from the OKT3 clone.

2. The bispecific T-cell connector as described in claim 1, characterized in that, The IgG antibody backbone is a human IgG1 antibody backbone; The CD3-specific single-stranded variable region specifically binds to the CD3ε strand; The amino acid sequence of the ICAM1-specific single-stranded variable region is shown in SEQ ID NO.7, and the amino acid sequence of the CD3-specific single-stranded variable region is shown in SEQ ID NO.

8. The first and second flexible linker peptides have a (Gly4Ser)n structure, where n is 3 or 4.

3. The bispecific T-cell connector as described in claim 1, characterized in that, The first polypeptide chain and the second polypeptide chain are designed with a knot structure and a hole structure respectively using the knot-into-hole technique between their CH3 domains. The CH2 domains of the first and second polypeptide chains introduce the LALA-PG mutation.

4. The bispecific T-cell connector as described in any one of claims 1 to 3, characterized in that, The amino acid sequence of the first polypeptide chain is as shown in SEQ ID No. 4, amino acid sequence 1-748, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID No. 5, amino acid sequence 1-231.

5. An isolated nucleic acid molecule encoding the bispecific T-cell connective as described in any one of claims 1 to 3.

6. The nucleic acid molecule as described in claim 5, characterized in that, The nucleotide sequence encoding the first polypeptide chain is as shown in SEQ ID No. 1, which is a sequence of 1-2244 bp, and the nucleotide sequence encoding the second polypeptide chain is as shown in SEQ ID No. 2, which is a sequence of 1-693 bp.

7. A recombinant expression vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the nucleic acid molecule of claim 6 or transformed with the recombinant expression vector of claim 7.

9. The use of the bispecific T-cell connector as described in any one of claims 1 to 3 or the nucleic acid molecule as described in claim 5 or 6 in the preparation of antitumor drugs.

10. An antitumor drug, characterized in that, The active ingredient is the bispecific T-cell connector as described in any one of claims 1 to 3 or the nucleic acid molecule as described in claim 5 or 6, and a pharmaceutically acceptable carrier.

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