Triple-effect T cell adapter for targeting T cell lymphoma TRBC1

Abstract

The invention relates to a triple-effect T cell adapter for targeting T cell lymphoma TRBC1. The invention discloses a TRBC1-targeted triple-effect T cell adapter which is composed of an Fc region, a hinge region, a first Fab arm and a second Fab arm, and a third targeting module is covalently connected to the C end of the Fc region to form a Y-shaped triple-head antibody. The first Fab arm and the second Fab arm are respectively combined with TRBC1 and CD3, and the third Fab arm is combined with CD28 or CD137, so that three functions of tumor recognition, T cell recruitment and co-stimulation amplification are integrated. The two polypeptide chains are heterodimerized through CH3 region button mutation, and correct assembly is ensured. The structure supports module interchange, programmable regulation and control of synergistic signals, significantly enhances TRBC1 positive T cell lymphoma killing, reduces toxicity to normal T cells, has high activity and a wide safety window, and is suitable for preparation of related drugs.

Description

Technical Field

[0001] This invention relates to a triple-effect T-cell binder targeting TRBC1 in T-cell lymphoma. Background Technology

[0002] Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of malignant tumors originating from mature T cells in the retrothymus, accounting for approximately 10%–15% of all non-Hodgkin lymphomas (NHL). PTCL includes more than 30 subtypes, with common types including nonspecific peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and anaplastic large cell lymphoma. This disease exhibits high heterogeneity in biological behavior, clinical presentation, and prognosis, typically characterized by high invasiveness and easy drug resistance. Most patients are diagnosed at an advanced stage, with rapid disease progression. The main challenges in PTCL treatment lie in its high relapse rate and short duration of remission. Even in patients achieving complete remission, the median duration of remission is usually only 6–7 months. Once relapse or refractory development occurs, treatment options are extremely limited, and patient survival is significantly shortened.

[0003] Currently, the standard treatment regimen for PTCL still largely follows the strategies used for aggressive B-cell lymphoma. In recent years, some targeted therapies have been approved for PTCL treatment, such as histone deacetylase inhibitors (e.g., romidadixin, chidamide, belistat), folate metabolism inhibitors (pralatrexate), and monoclonal antibodies (e.g., brentuximab vedotin). Although these drugs offer new treatment possibilities for relapsed / refractory patients, their monotherapy efficacy remains limited and they are often accompanied by adverse reactions such as hematologic toxicity, mucositis, and infections. Furthermore, current treatment decisions lack sufficient molecular subtyping support, making truly personalized precision treatment difficult to achieve.

[0004] In recent years, immunologically based treatment strategies, especially CAR-T cell therapy, have made breakthrough progress in B-cell malignancies. However, its application in T-cell lymphomas faces a series of unique challenges. Since T cells are the core executors of the immune system, treatment targeting widely expressed T-cell markers (such as CD3, CD5, and CD7) can lead to severe or even fatal immunosuppression. Specific challenges include: T-cell exhaustion (fratricide): CAR-T cells are T cells themselves; if they target pan-T-cell antigens, it can lead to self-killing, affecting the in vivo expansion and persistence of CAR-T cells; deep immunosuppression: the elimination of both malignant and normal T cells will cause severe T-cell deficiency, making patients susceptible to fatal infections; risk of EBV reactivation: loss of T-cell function may lead to uncontrolled EBV, thereby inducing post-transplant lymphoproliferative disorders or even lymphoma; manufacturing process challenges: T cells collected from patients may be contaminated with tumor cells, affecting the safety and quality of CAR-T products.

[0005] Bispecific antibodies (BsAbs), as another emerging class of immunotherapies, have shown potential in T-cell lymphomas, but key scientific questions and technological bottlenecks remain unresolved. BsAbs redirect T-cell killing of tumors by simultaneously binding to tumor antigens and CD3 molecules on the surface of T cells. However, their application in T-cell malignancies suffers from the following inherent limitations: Target selection and bidirectional killing issues: When targeting pan-T-cell antigens such as CD4, CD5, and CD7, BsAbs not only activate T cells to attack malignant cells but also lead to inter-T-cell killing, causing comprehensive T-cell exhaustion and severe immunosuppression. Unlike B-cell deficiency, there are currently no effective means of mitigating the risks associated with T-cell deficiency, making this type of immunosuppression a key toxicity factor limiting its clinical application. Insufficient treatment specificity: Theoretically, TRBC1 (T-cell receptor β-constant region 1) could be an ideal target because it can distinguish between malignant clones and normal polyclonal T-cell populations. In a normal T cell population, TRBC1 and TRBC2 expression exhibit a polyclonal distribution with a roughly balanced ratio; while malignant T cells such as PTCL typically express only TRBC1 or TRBC2. Therefore, targeting TRBC1 should theoretically eliminate only about half of the T cells (TRBC1+ population), preserving the other half, TRBC2+ T cells, to maintain immune function.

[0006] However, actual studies have found that BsAbs targeting TRBC1 can cause near-complete exhaustion of TRBC1+ and TRBC2+ T cells during treatment, leading to widespread immune damage. This unintended killing may be related to the mechanism of action of BsAbs—T cells are activated through the CD3 arm, independent of endogenous TCR recognition, which may cause broader T cell activation and killing effects, thereby weakening its targeting specificity.

[0007] Furthermore, although BsAbs have the advantage of being "off-the-shelf" and do not require individualized customization, developing BsAbs covering all subtypes based on the T cell receptor β chain variable region (TRBV) family would require constructing approximately 30 different antibodies, a massive undertaking. While 24 TRBV-targeting single-chain antibody fragments (scFvs) have been obtained, further developing them into highly effective and safe BsAbs still faces technical challenges and resource requirements. Summary of the Invention

[0008] Treatment of T-cell lymphoma (TCL) has long been limited by the lack of truly tumor-specific targets, making it difficult for existing therapies to balance efficacy and safety. Although targeting the T-cell receptor β chain constant region 1 (TRBC1) can theoretically selectively eliminate TRBC1+ malignant clones, traditional TRBC1-targeting T-cell adaptors (TCEs) still have serious limitations in practical applications: they cannot effectively distinguish between malignant and normal TRBC1+ T cells, resulting in broad-based non-tumor toxicity and limiting their therapeutic window and clinical application prospects.

[0009] To address the aforementioned technical bottlenecks, this invention breaks through the traditional design paradigm of bispecific or asymmetric trispecific antibodies, and innovatively constructs a novel TRBC1 / CD3 / CD28 trispecific antibody with a novel "1+1+1" symmetrical structure. Unlike the traditional "1+2" ​​structure (where two binding domains are located on one side of the antibody's Y-shaped structure, and the other binding domain is on the other side), the antibody of this invention independently places the three key binding domains—TRBC1, CD3, and CD28—at the three ends of the antibody's Y-shaped structure: the TRBC1 binding domain is on the left, the CD3 binding domain is on the right, and the CD28 binding domain is located below the Fc segment. This unique spatial conformation is the core innovation of this invention. By simultaneously targeting the TRBC1 tumor antigen and the CD3 and CD28 molecules on the surface of T cells, this molecule achieves dual synergistic regulation of T cell activation signals, thereby significantly reducing off-target toxicity to normal T cells while enhancing anti-tumor activity.

[0010] Therefore, the present invention discloses a triple-effect T cell conjugate targeting T-cell lymphoma TRBC1, wherein the antibody comprises an Fc region and a first Fab arm and a second Fab arm connected to the Fc region via a hinge region, and a third targeting module is further covalently linked to the C-terminus of the Fc region, thereby forming a Y-shaped three-headed antibody structure with three independent targeting modules.

[0011] In one feasible implementation, the three independent targeting modules specifically bind to three different antigens on T cells.

[0012] In one feasible implementation, the three different antigens are selected from T-cell receptor (TCR) complex components, co-stimulatory molecules, and combinations thereof.

[0013] In one feasible implementation scheme The first Fab arm contains a first variable domain that specifically binds to the first antigen; The second Fab arm contains a second variable domain that specifically binds to the second antigen; The third targeting module includes a third variable domain that specifically binds to a third antigen.

[0014] In one feasible implementation, the third targeting module is a single-chain variable fragment (scFv), a single-domain antibody (VHH), or a Fab fragment fused to the C-terminus of the Fc region heavy chain via a peptide chain.

[0015] In one feasible implementation, the triple-effect T cell connector comprises: The first polypeptide chain comprises, from the N-terminus to the C-terminus, the following components in sequence: a first light chain variable domain (VL1), a first constant domain (CL), a first heavy chain variable domain (VH1), a first heavy chain constant domain (CH1), a hinge region, a second heavy chain constant domain (CH2), a third heavy chain constant domain (CH3), and a fourth heavy chain variable domain (VH4). The second polypeptide chain comprises, from the N-terminus to the C-terminus, the following components in sequence: a second light chain variable domain (VL2), a second heavy chain constant domain (CH1'), a second heavy chain variable domain (VH2), a second light chain constant domain (CL'), a hinge region, a fourth heavy chain constant domain (CH2'), a fifth heavy chain constant domain (CH3'), and a third light chain variable domain (VL3). The first polypeptide chain and the second polypeptide chain form a dimer through the interaction between the CH3 and CH3' domains, thereby constituting the Fc region of the antibody; and Among them, VL1 and VH1 together form the first antigen binding site that specifically binds to TRBC1, VL2 and VH2 together form the second antigen binding site that specifically binds to CD3, and VH4 and VL3 together form the third antigen binding site that specifically binds to CD28.

[0016] In one feasible implementation, the first constant structural domain (CL) is CL kappa or CL lambda, and the second light chain constant structural domain (CL') is CL kappa or CL lambda.

[0017] In one feasible implementation, the CH3 and CH3' domains contain buttonhole mutation pairs to facilitate proper pairing of the first polypeptide chain with the second polypeptide chain.

[0018] In one feasible implementation, the triple-effect T cell connector comprises: The first polypeptide chain, from the N-terminus to the C-terminus, comprises: αTRBC1 VL, CL, αTRBC1 VH, CH1, hinge region, CH2, CH3, and αCD28 VH; The second polypeptide chain, from the N-terminus to the C-terminus, comprises: αCD3 VL, CH1, αCD3 VH, CL, hinge region, CH2, CH3, and αCD28 VL.

[0019] The technical solution provided in this application has at least the following beneficial effects: 1. Spatial Optimization of Signal Coordination: Traditional "1+2" ​​structures may experience spatial steric hindrance or signal interference due to the two binding domains being located on the same side. The "1+1+1" structure of this invention provides independent and optimal spatial binding orientations for TRBC1, CD3, and CD28, ensuring that all three molecules can bind to their respective targets simultaneously and efficiently. This maximizes the synergistic activation of the CD3 primary signal and the CD28 co-stimulatory signal, avoiding mutual inhibition between signals.

[0020] 2. Enhanced antigen binding efficiency and stability: The independent localization of each binding domain reduces intramolecular interference, enabling antibodies to bind to both tumor cells (via TRBC1) and T cells (via CD3 and CD28) with better affinity and kinetic properties. This structure improves the overall efficiency and stability of immune synapse formation, creating a superior microenvironment for full T cell activation.

[0021] 3. Mechanism verification of functional advantages: Highly Efficient and Low-Toxicity Killing: In vitro experiments have confirmed that, thanks to the aforementioned structural advantages, the TRBC1 / CD3 / CD28 trispecific antibody of this invention can efficiently mediate the clearance of various TRBC1+ tumor cell lines by T cells at extremely low concentrations (0.1–10000 pM), with significantly superior efficacy compared to traditional control antibodies. More importantly, at concentrations as high as 10–1000 nM, the non-specific killing effect of this invention on healthy T cells is significantly lower, demonstrating a significantly expanded safety window brought about by its unique structure. Synergistic Activation and Delayed Exhaustion: Mechanistic studies have shown that this "1+1+1" structure can more effectively promote T cell activation, proliferation, and cytokine release. Compared to bispecific antibodies (TRBC1 / CD3) that only activate CD3, this invention can induce stronger phosphorylation of downstream signaling molecules, while significantly reducing the expression of T cell exhaustion markers (such as PD-1 and LAG-3) and promoting T cell differentiation towards the memory phenotype. This is directly attributed to the independent and efficient spatial access of the CD28 co-stimulatory signal. Excellent in vivo efficacy: In a tumor-bearing mouse model, the trispecific antibody (mouse-derived alternative version) of this invention exhibited strong in vivo anti-tumor activity, significantly inhibiting tumor growth. Its effect far exceeded that of the control group, fully verifying the in vivo effectiveness of this structural design.

[0022] In summary, the "1+1+1" type TRBC1 / CD3 / CD28 trispecific antibody provided by this invention, through its original spatial structure design, solves key problems such as low signal synergy efficiency and high toxicity of traditional TCEs, and achieves simultaneous improvement in anti-tumor efficacy and safety, providing an unprecedentedly efficient and low-toxicity solution for TCL immunotherapy. Attached Figure Description

[0023] Figure 1 The different forms of trispecific antibodies constructed in this invention; Figure 2 The killing effects of different forms of trispecific antibodies on target cells (A) and effector cells (B) at different concentrations were investigated. Figure 3 The killing effect of TRBC1 / CD3 / CD28 on different TRBC1+ tumor cells; Figure 4 The killing effect of different human PBMCs on TRBC1+ tumor cells mediated by TRBC1 / CD3 / CD28; Figure 5 The activation effect of TRBC1 / CD3 / CD28-mediated T cells; Figure 6 The effects of TRBC1 / CD3 / CD28-mediated release of cytotoxic proteins and cytokines from T cells; Figure 7 It affects the proliferation and differentiation of TRBC1 / CD3 / CD28-mediated T cells; Figure 8 The effects of TRBC1 / CD3 / CD28-mediated T cell exhaustion; Figure 9 Pathway enrichment analysis (A) and differential gene analysis (B) for TRBC1 / CD3 / CD28-mediated T cells; Figure 10 This refers to the expression of pathway-related proteins in T cells mediated by TRBC1 / CD3 / CD28. Figure 11 The therapeutic effect of TRBC1 / mCD3 / mCD28 on TRBC1-MC38 tumor-bearing mice. Detailed Implementation

[0024] Unless otherwise specified, the following terms used in this specification and claims have the broadest possible technical meaning and are not limited to the specific molecules, sequences, or configurations listed in the embodiments: TRBC1 is given the broadest definition in this invention, including not only the full-length wild-type human TRBC1 protein and any fragments, point mutations, glycoforms or splice variants, but also homologous sequences from other mammals that can be recognized by the same binding molecule, and even artificially modified or simulated epitopes, as long as they retain the constant region characteristics that can be specifically bound, they fall within the scope of protection.

[0025] CD3 binding modules refer to any structure that can trigger or amplify the TCR signal cascade. They can be antibodies against CD3ε, CD3δ, or CD3γ, as well as their Fab, scFv, VHH, biantibodies, etc. They can also be non-antibody scaffolds such as DARPin, anticalin, affibody, nucleic acid aptamers, and peptide ligands. They also include variants that have undergone humanization, affinity maturation, deimmunization, or extended half-life, as well as monovalent, bivalent, or multivalent arrangements. The sequence source can be human, mouse, chimeric, or total synthetic.

[0026] Co-stimulatory binding modules refer to any ligand-receptor binding unit capable of providing a second activation signal, encompassing antibodies, natural ligands, and soluble receptor fragments of CD28, CD137, and other TNFR superfamily members such as OX40, CD27, GITR, or Ig superfamily members such as ICOS, CD2, and also including any form that is agonist or antagonist, monovalent or multivalent, linear or cyclized, chemically synthesized, or biologically expressed.

[0027] Triple-headed antibodies or Y-shaped symmetrical trispecific antibodies are not limited to the traditional IgG backbone. They include structures with three functional ends formed based on Fc heterodimerization technologies such as Knob into Hole, CrossMab, SEEDbody, AZEPbody, DuoBody, or orthogonal Fab. They can also be minimal Y configurations lacking the Fc region, Y-extended configurations with added fourth and fifth functional domains, and variants that have undergone glycosylation, polyethylene glycolation, albumin fusion, or Fc silencing mutations.

[0028] Button mutation refers to any engineering modification that can promote heterodimerization of two different heavy chains. This includes the classic Knob into Hole pairing of T366W and T366S L368A Y407V, as well as charge pair guidance, disulfide bond strengthening, chain exchange engineering, or future alternative heterodimerization strategies, all of which are considered equivalent technical means.

[0029] Module interchangeability or positional programmability refers to any operation that, without altering the overall framework, rearranges the relative spatial positions of CD3 and costimulatory binding modules at the gene level, including arm-end interchange, N-terminal / C-terminal inversion, or connector length adjustment, thereby quantitatively regulating signal intensity, cytokine profiles, or memory formation tendencies. It also encompasses computer-aided design, construction, testing, and learning loops.

[0030] In addition to TRBC1, the tumor targeting module also includes any alternative antigens selectively present on the surface of malignant T cells, such as CD30, CD5, CD7, CD4, CD8, CD2, CD52, CXCR4, CCR4, CD99, TCR, Vβ family members LAG3, TIM, 3TIGIT, or any combination thereof. The binding form can be antibody, ligand, peptide, nucleic acid aptamer, small molecule, or the aforementioned non-antibody scaffold, while maintaining high selectivity for malignant T cells.

[0031] Effector T cells refer to all T cell subsets capable of performing cytotoxic or helper functions, including CD8-positive αβ T cells, CD4-positive αβ T cells, γδ T cells, NKT cells, tissue-resident memory T cells, stem cell-like memory T cells, central memory T cells, effector memory T cells, and the aforementioned cells modified by CAR or TCR genes. All of these belong to the effector population that can be recruited and activated.

[0032] Pharmaceutically acceptable carriers refer to any excipients, diluents, stabilizers, buffers, preservatives, lyophilization protectants, solubilizers, sustained-release matrices, or delivery systems that do not interfere with the biological activity of trispecific antibodies and comply with pharmaceutical regulations. Specifically, they may include water, physiological saline, sucrose, trehalose, histidine buffer, polysorbate 80, PLGA microspheres, liposomes, nanoparticles, or in vivo biodegradable hydrogels, as well as novel pharmaceutical excipients to be developed in the future.

[0033] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Simple modifications made to the present invention based on its essence are all within the scope of protection claimed by the present invention.

[0034] Example 1: Expression and purification of different forms of bispecific and trispecific antibodies targeting TRBC1 To systematically evaluate the impact of different antibody conformations on function, we designed and constructed a variety of bispecific and trispecific antibodies targeting TRBC1. These multispecific antibodies can specifically bind to TRBC1, CD3, CD28, or CD137 antigens on different cells, respectively. Their structural diagrams are shown below. Figure 1 As shown.

[0035] 1. Traditional "1+2" ​​type trispecific antibodies ( Figure 1 a) This antibody employs a classic asymmetric design: the left arm displays the TRBC1 binding unit, specifically, its light chain variable region (αTRBC1 VL) is directly fused to the constant light chain (CL) via its N-terminus, while the heavy chain variable region (αTRBC1 VH) is fused to the heavy chain constant region (CH1-CH2-CH3) via its N-terminus. The right arm sequentially connects the CD3 and CD28 binding units via flexible / rigid linkers—at the N-terminus of the light chain, αCD3 VL and αCD28 VL are tandemly linked to the CL domain via a flexible linker; at the N-terminus of the heavy chain, αCD28 VH and αCD3 VH are tandemly linked to the CH1 domain via a rigid linker. To promote correct chain pairing and reduce heterodimer formation, a "knob-into-hole" structure is introduced in the Fc region. The specific amino acid sequences of each element (including VH / VL of αTRBC1, αCD3, and αCD28, as well as CH1, CL, CH2, and CH3) are listed in Table 1.

[0036] 2. The innovative "1+1+1" type trispecific antibody of this invention ( Figure 1 be) The core of this invention lies in proposing a novel symmetrical "tri-headed antibody" structure (taking TRBC1 / CD3 / CD28 as an example). This design independently places three targeting modules (TRBC1, CD3, and CD28) at the three ends of the antibody's Y-shaped structure. Compared to traditional antibodies, its characteristic is the introduction of a new antibody at the lower end, as detailed below: The left arm is dedicated to TRBC1 recognition. Its light chain is a fusion of αTRBC1 VL and CL, and its heavy chain is a fusion of αTRBC1VH and CH1-CH2-CH3, with αCD28 VH further fused to the C-terminus of the heavy chain. The right arm is dedicated to CD3 recognition. Its light chain is a fusion of αCD3 VL and CH1, and its heavy chain is a fusion of αCD3 VH and CL-CH2-CH3, with αCD28 VL further fused to the C-terminus of the heavy chain. This structure also introduces a "button" mutation in the Fc region to ensure correct assembly. Based on the same platform design concept, by interchanging the positions of the CD3 and CD28 modules (e.g., TRBC1 / CD28 / CD3), the co-stimulatory signaling synergistic mode can be systematically regulated.

[0037] 3. Replacement and construction of the co-stimulatory signal domain To further investigate the effects of different co-stimulatory signals on antibody function, we replaced the CD28 binding module with a CD137 (4-1BB) binding module based on the above "1+1+1" structure, and constructed two novel antibodies, TRBC1 / CD137 / CD3 and TRBC1 / CD3 / CD137, respectively, to compare the differences between CD28 and CD137 as co-activating signals in T cell activation and anti-tumor effects.

[0038] Based on the above description, the heavy and light chains of TRBC1, CD3, CD28, and CD137 antibodies were cloned into mammalian expression vectors, respectively. The expression system vectors included fusion DNA sequences linked with suitable transcriptional and translational regulatory sequences. Different plasmids were transfected into mammalian cells according to the target protein to express and purify the fusion protein, yielding trispecific antibodies. Transfection methods could be chemical transfection or electroporation transfection, and the mammalian cells could be HEK293 cells or CHO cells. After expression, the proteins were purified using NiBeads and molecular sieves.

[0039] Table 1: Amino acid sequences of the proteins involved .

[0040] Example 2: To verify the killing effect of different forms of antibodies on TRBC1-positive tumor cells, activated T cells and CFSE-labeled target cells (Jurkat cells) were co-incubated at an effector-target ratio of 1:10. The test antibodies were added to final concentrations ranging from 0.1 to 10000 pM. After culturing in 96-well plates for 24 hours, the apoptosis of target cells was detected using the Annexin V / 7-AAD apoptosis detection kit. The results are as follows: Figure 2As shown in Figure A, all constructed antibodies exhibited cytotoxic effects on Jurkat cells, with TRBC1 / CD3_CD28 and TRBC1 / CD3 / CD28 showing the most significant cytotoxic effects.

[0041] Given previous studies reporting that TRBC1-targeting T-cell adaptors may cause off-target toxicity to healthy T cells, we further evaluated the safety of various antibodies at high concentrations (10-1000 nM). The results are as follows: Figure 2 As shown in Figure B, TRBC1 / CD3 / CD28 exhibits the lowest non-specific killing effect on healthy T cells, indicating that it has the optimal safety window. Therefore, TRBC1 / CD3 / CD28 was selected as the focus of subsequent research and named the "triple-effect T cell binder".

[0042] Example 3: Killing effect of TRBC1-targeting triple-effect T cell conjugates on tumor cells with different TRBC1 groups. To investigate the cytotoxic effect of the triple-effect T cell conjugate TRBC1 / CD3 / CD28 on different TRBC1-positive tumor cells, in addition to the Jurkat and H9 cell lines, we also constructed SKW3-TRBC1 and CEM-TRBC1 cell lines overexpressing TRBC1. Under an effector-to-target ratio of 1:10, the cytotoxic effects of different concentrations of the triple-effect T cell conjugate and its control antibody on each cell line were evaluated. The results are as follows: Figure 3 As shown in A-3D, the triple-effect T cell agonist TRBC1 / CD3 / CD28 exhibited superior killing efficacy against all tested TRBC1-positive tumor cells compared to the control group.

[0043] Example 4: Killing effect of PBMCs from different human sources on TRBC1 tumor cells To evaluate the universality of the triple-effect T cell conjugate TRBC1 / CD3 / CD28 in immune cells from different individuals, we collected peripheral blood mononuclear cells from multiple healthy donors, activated them, and co-cultured them with tumor cells at a 1:10 effector-to-target ratio. We then examined the cytotoxic effects of different concentrations of the triple-effect T cell conjugate. The results are as follows: Figure 4 As shown, the triple-effect T cell binders TRBC1 / CD3 / CD28 can effectively mediate the killing of TRBC1-positive tumor cells by PBMCs from different donors, indicating that their effects are independent of the immune background of a specific individual.

[0044] Example 5: The effect of a TRBC1-targeting triple-effect T cell conjugate on T cell activation To investigate the effect of the triple-effect T cell conjugate TRBC1 / CD3 / CD28 on T cell activation, activated T cells were co-cultured with different CFSE-labeled TRBC1-positive tumor cells at a 1:1 ratio for 12 hours, and different concentrations of the triple-effect T cell conjugate were added. The trispecific antibodies EGFP / CD3 / CD28, bispecific antibodies TRBC1 / CD3, and TRBC1 / CD28 were used as controls. After culture, the expression levels of CD25 and CD69 on the surface of CD4+ and CD8+ T cells were measured. The results are as follows: Figure 5 As shown in AB, for all tested TRBC1-positive tumor cells, the proportion of CD25+ and CD69+ cells in CD4+ and CD8+ T cells treated with the triple-effect T cell binder TRBC1 / CD3 / CD28 was the highest, indicating that it can more effectively promote T cell activation.

[0045] Furthermore, using Jurkat cells as an example, the expression changes of CD137 (4-1BB) and CD107a during T cell co-culture with target cells were further evaluated. The results are as follows: Figure 5 As shown in the CE diagram, the treatment group with the triple-effect T cell binder TRBC1 / CD3 / CD28 had the highest proportion of CD137+, NTB-NA+, and CD107a+ T cells, further confirming its ability to enhance T cell activation.

[0046] Example 6: Effects of TRBC1-targeting triple-effect T cell conjugates on T cell cytokine secretion To investigate the effects of the triple-effect T cell binder TRBC1 / CD3 / CD28 on T cell cytokine secretion, T cells and Jurkat cells were co-cultured at a 1:1 ratio at a concentration of 1000 pM, and the release of T cell cytotoxic proteins and cytokines was detected. The results are as follows: Figure 6 As shown in AF, compared with the control groups, the triple-effect T cell agonist TRBC1 / CD3 / CD28 can more effectively promote the release of CD8+ T cell cytotoxic proteins and key cytokines.

[0047] Example 7: Effects of TRBC1-targeting triple-effect T cell conjugates on T cell proliferation and differentiation To evaluate the effects of the triple-effect T cell conjugate TRBC1 / CD3 / CD28 on T cell proliferation and differentiation, CFSE-labeled PBMCs and Jurkat cells were co-cultured at a 1:1 ratio for 3 days with an antibody concentration of 1000 pM. After culture, the proliferation of CD4+ and CD8+ T cells and Ki67 expression were analyzed. The results are as follows: Figure 7As shown in Figures AB, the triple-effect T cell conjugate TRBC1 / CD3 / CD28 more effectively promotes CD8+ T cell proliferation, with the highest proportion of Ki67+ cells. Cell differentiation analysis results show that this antibody mainly promotes T cell differentiation into memory effector T cells.

[0048] Example 8: The effect of a TRBC1-targeting triple-effect T cell conjugate on T cell exhaustion To investigate the effect of the triple-effect T cell binder TRBC1 / CD3 / CD28 on T cell exhaustion, PBMCs and CFSE-labeled Jurkat cells were co-cultured at a 1:1 ratio for 3 days with an antibody concentration of 1000 pM. After culture, the expression of PD-1 and LAG-3 in CD4+ and CD8+ T cells was analyzed. The results are as follows: Figure 8 As shown, compared with TRBC1 / CD3, the T cell exhaustion level in the TRBC1 / CD3 / CD28 triple-effect T cell binder treatment group was significantly reduced.

[0049] Example 9: Transcriptome analysis of CD8+ T cells by a TRBC1-targeting triple-effect T cell conjugate PBMCs were activated and co-cultured with TRBC1-positive tumor cells for one day at an antibody concentration of 1000 pM. CD8+ T cells were then sorted using magnetic beads, and RNA was extracted for transcriptome sequencing analysis. Results are as follows: Figure 9 As shown in Figures AB, the triple-effect T cell binder TRBC1 / CD3 / CD28 is mainly enriched in the TCR signaling pathway, the PI3K-AKT signaling pathway, and the cytokine-receptor interaction pathway. Volcano plot analysis showed that this antibody significantly promoted the transcription of interferon-γ-related genes in CD8+ T cells.

[0050] Example 10: The effect of a TRBC1-targeting triple-effect T cell conjugate on the TCR signaling pathway To investigate the effects of the triple-effect T cell binder TRBC1 / CD3 / CD28 on the TCR signaling pathway, we analyzed the phosphorylation levels of key signaling molecules during T cell co-culture with Jurkat cells. The results are as follows: Figure 10 As shown, the triple-effect T cell binder TRBC1 / CD3 / CD28 can significantly promote the phosphorylation of PLCγ1, p38, Erk1 / 2 and JUN.

[0051] Example 11: Effect of TRBC1-targeting triple-effect T cell conjugates on MC38-TRBC1 growth in mice To evaluate the in vivo antitumor activity of the triple-effect T cell conjugate TRBC1 / CD3 / CD28, we constructed an MC38 cell line overexpressing TRBC1 and replaced the CD3 and CD28 antibody fragments in the triple-effect T cell conjugate with corresponding mouse fragments, obtaining TRBC1 / mCD3 / mCD28. We also constructed corresponding control antibodies. After subcutaneous inoculation of MC38-TRBC1 cells into mice, mice were treated with different antibodies, and mouse body weight and tumor volume were monitored regularly. Results are as follows: Figure 11 As shown, TRBC1 / mCD3 / mCD28 can significantly inhibit the growth of MC38-TRBC1 tumors, and their tumor volume and weight are significantly lower than those of the control groups.

[0052] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A triple-effect T-cell binder targeting TRBC1 in T-cell lymphoma, characterized in that, The antibody comprises an Fc region and a first Fab arm and a second Fab arm connected to the Fc region via a hinge region, and the C-terminus of the Fc region is further covalently linked to a third targeting module, thereby forming a Y-shaped trihead antibody structure with three independent targeting modules.

2. The triple-effect T-cell connector as described in claim 1, characterized in that, The three independent targeting modules specifically bind to three different antigens on T cells, respectively.

3. The triple-effect T-cell connector as described in claim 2, characterized in that, The three different antigens are selected from T-cell receptor (TCR) complex components, co-stimulatory molecules, and combinations thereof.

4. The triple-effect T cell connector as described in claim 1, characterized in that: The first Fab arm contains a first variable domain that specifically binds to the first antigen; The second Fab arm contains a second variable domain that specifically binds to the second antigen; The third targeting module includes a third variable domain that specifically binds to a third antigen.

5. The triple-effect T-cell connector as described in claim 4, characterized in that, The third targeting module is a single-chain variable fragment (scFv), a single-domain antibody (VHH), or a Fab fragment fused to the C-terminus of the Fc region heavy chain via a peptide chain.

6. The triple-effect T-cell connector as described in claim 1, characterized in that, Include: The first polypeptide chain comprises, from the N-terminus to the C-terminus, the following components in sequence: a first light chain variable domain (VL1), a first constant domain (CL), a first heavy chain variable domain (VH1), a first heavy chain constant domain (CH1), a hinge region, a second heavy chain constant domain (CH2), a third heavy chain constant domain (CH3), and a fourth heavy chain variable domain (VH4). The second polypeptide chain comprises, from the N-terminus to the C-terminus, the following components in sequence: a second light chain variable domain (VL2), a second heavy chain constant domain (CH1'), a second heavy chain variable domain (VH2), a second light chain constant domain (CL'), a hinge region, a fourth heavy chain constant domain (CH2'), a fifth heavy chain constant domain (CH3'), and a third light chain variable domain (VL3). The first polypeptide chain and the second polypeptide chain form a dimer through the interaction between the CH3 and CH3' domains, thereby constituting the Fc region of the antibody; and Among them, VL1 and VH1 together form the first antigen binding site that specifically binds to TRBC1, VL2 and VH2 together form the second antigen binding site that specifically binds to CD3, and VH4 and VL3 together form the third antigen binding site that specifically binds to CD28.

7. The triple-effect T-cell connector as described in claim 6, characterized in that, The first constant structural domain (CL) is CL kappa or CL lambda, and the second light chain constant structural domain (CL') is CL kappa or CL lambda.

8. The triple-effect T-cell connector as described in claim 6 or 7, characterized in that, The CH3 and CH3' domains contain buttonhole mutation pairs to facilitate proper pairing of the first polypeptide chain with the second polypeptide chain.

9. The triple-effect T-cell connector as described in claim 6, characterized in that, Include: The first polypeptide chain, from the N-terminus to the C-terminus, comprises: αTRBC1 VL, CL, αTRBC1 VH, CH1, hinge region, CH2, CH3, and αCD28 VH; The second polypeptide chain, from the N-terminus to the C-terminus, comprises: αCD3 VL, CH1, αCD3 VH, CL, hinge region, CH2, CH3, and αCD28 VL.

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