A bispecific antibody and its application with T cells in the preparation of drugs for treating tumors

Abstract

The present invention provides a bispecific antibody and its use with T cells in the preparation of a drug for treating tumors, wherein the bispecific antibody comprises a first functional region targeting CD3 and a second functional region targeting PD-L1, wherein the first functional region is a scFv structure, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:2; the second functional region is a Fab structure, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:4. The bispecific antibody adopts an IgG type structure, which can prolong the half-life in vivo and reduce rejection. The T activation mode is optimized, and a combined stimulation strategy of TGF-β, IL-25 and IL-33 is used to maintain the activation state of T cells in vivo. The bispecific antibody provided by the present invention is used in combination with activated T cells to inhibit tumor cell growth, regulate the expression level of immune factors, and prolong the animal production cycle.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a bispecific antibody and its use with T cells in the preparation of a drug for treating tumors. Background Art

[0002] Tumor immunotherapy is an emerging tumor treatment method with the advantages of strong anti-tumor ability, long-lasting efficacy, and few side effects. It is widely used in tumor treatment, especially in refractory malignant tumors. Early tumor immunotherapy was mainly based on monoclonal antibodies, which developed from the initial mouse antibodies to humanized antibodies and then to fully human antibodies. Currently, many monoclonal antibody drugs have been approved for marketing, including blockbuster drugs such as rituximab, trastuzumab, bevacizumab, and cetuximab. However, due to factors such as off-target effects, short half-life, and difficulty adapting to the tumor microenvironment, their clinical performance is still unsatisfactory.

[0003] To this end, researchers have developed bispecific antibodies (BsAbs), which can target two target antigens at the same time, greatly reducing the risk of off-target effects, and have therefore attracted widespread attention in the industry. As of 2023, 14 BsAb drugs have been approved worldwide, of which 9 are used to treat hematological malignancies, including blinatumomab, mosunetuzumab, teclistamab, epcoritamab, and glofitamab, etc. (see Antonio TapiaGalisteo, et al. Bi-and trispecific immune cell engagers for immunotherapy of hematological malignancies. J Hematol Oncol. 2023; 16: 83).

[0004] Among many BsAbs, CD3 bispecific antibodies (CD3-BsAbs) have received increasing attention and have become an emerging treatment method in the field of cancer immunotherapy. CD3-BsAbs work by simultaneously binding to tumor-associated antigens (TAA) expressed on tumor cells and CD3 on T cells. CD3-BsAbs cross-link these two cell types to form an immune synapse, similar to the natural T cell receptor (TCR) / peptide-major histocompatibility complex (MHC) complex (see Xu H, et al. Retargeting T cells to GD2 pentasaccharide on human tumors using Bispecific humanized antibody. Cancer Immunol. Res. 2015; 3: 266–277). This synapse leads to T cell activation, secretion of inflammatory cytokines and cytolytic molecules, thereby killing tumor cells in the process. The advantage of CD3-BsAb is that any T cell can act as an effector cell, regardless of TCR specificity. For these BsAbs, TCR signaling does not require the participation of the antigen-binding domain of the TCR, but rather initiates the CD3 signal (see Wu Z, et al. T cell engaging bispecific antibody (T-BsAb): From technology to therapeutics. Pharmacol. Ther. 2018; 182: 161–175). Therefore, CD3-BsAbs can utilize all available T cells, not just tumor-specific T cells.

[0005] Currently, CD3-BsAb shows great potential in the treatment of blood cancers. FDA-approved blinatumomab (CD3xCD19) has been successfully used in the clinical treatment of some B-cell malignancies. Blinatumomab is a CD3xCD19 BsAb without an Fc tail. Compared with standard chemotherapy, more than 40% of adult patients treated with blinatumomab showed complete or partial remission, and the median overall survival was extended by several months (see Franquiz MJ, et al. Blinatumomab for the Treatment of Adult B-Cell Acute Lymphoblastic Leukemia: Toward a New Era of Targeted Immunotherapy. Biologics. 2020; 14: 23–34). In addition to blinatumomab, many other CD3-BsAbs are currently undergoing clinical trials targeting mature B cell markers such as CD19, CD20, CD38, and B cell maturation antigen (BCMA) as well as bone marrow markers such as CD33 and CD123. However, in contrast to the success of CD3-BsAb in hematological malignancies, the effects of these antibodies in solid tumors are still quite limited. This is due to the following obstacles in the treatment of solid tumors:

[0006] The first is off-target toxicity. In the case of blood cancer, temporary depletion of B cells or bone marrow subsets is reversible as long as hematopoietic stem cells are not targeted, so that the blood can be replenished. However, solid tumor targets are often expressed on tissues of healthy organs, which may lead to immunopathological phenomena, even organ failure and may be fatal, such as the results shown in preclinical mouse studies using CD3-BsAb targeting EGFR (see Lutterbuese R, et al. T cell-engaging BiTE antibodies specific for EGFR potently eliminate KRAS-and BRAF-mutated colorectal cancer cells. Proc. Natl. Acad. Sci. USA. 2010; 107: 12605–12610).

[0007] The second is the insufficient availability of effector cells in the immunosuppressive tumor microenvironment (TME). For hematological malignancies, cancer cells in the blood are surrounded by T cells, allowing CD3-BsAb to be drawn from an endless pool of effector cells, while solid tumors require T cell infiltration to exert therapeutic efficacy. In this case, "inflamed" tumors may appear, which are infiltrated by immune cells and often respond to immune checkpoint therapy (see Chen DS, et al. Elements of cancer immunity and the cancer-immune setpoint. Nature. 2017; 541: 321–330); or "immune desert" tumors, with reduced immune cells or lack of immunogenicity, resulting in few tumor-specific T cells homing to the tumor (see Anitis E, et al. Mechanisms regulating T-cell infiltration and activity in solid tumors. Ann. Oncol. 2017; 28: xii18–xii32).

[0008] The third is that the quality of infiltrating T cells is not high. TME can inhibit immune cells, including cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (T regs), producing factors such as TGF-β, IL-10, indoleamine 2,3-dioxygenase, and regulatory factors such as arginase, which hinder T cell metabolism and activation (see Tormoen G.W, et al. Role of the immunosuppressive microenvironment in immunotherapy. Adv. Radiat. Oncol. 2018; 3: 520–526). In addition, effector T cells show "exhaustion" characteristics due to chronic antigen stimulation, which is demonstrated in the expression of inhibitory immune checkpoints (see Grywalska E, et al. Immune-checkpoint inhibitors for combating T-cell dysfunction in cancer. Onco Targets Ther. 2018; 11: 6505–6524).

[0009] Based on the above research, the present invention designed and obtained a bispecific antibody targeting CD3 and PD-L1, and used it together with activated T cells for the treatment of solid tumors, thereby improving the anti-tumor effect and providing a new approach for the development of novel tumor immunotherapy. Summary of the invention

[0010] The first aspect of the present invention provides a bispecific antibody, characterized in that it comprises a first functional region targeting CD3 and a second functional region targeting PD-L1, the first functional region is a scFv structure, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; the second functional region is a Fab structure, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 4.

[0011] The target selection of CD3 BsAb is very important and directly related to tumor specificity and treatment safety. It has been reported that Her2, EGFR, and EpCAM have become qualified candidate targets. Immune checkpoint antibodies are a major breakthrough in the field of tumor immunotherapy in recent years, and have achieved amazing results in clinical practice. In addition to PD-1 / PD-L1, multiple inhibitory checkpoints have been identified and therapeutically verified, including CTLA-4, LAG-3, TIM-3, and TIGIT 9. There is evidence that LAG-3 and PD-L1 are both promising therapeutic targets. (See Reader CS, et al. Abstract 2874: The tetravalent structure of FS118, a bispecific antibody targeting LAG-3and PD-L1, is required for its novel mechanism of LAG-3shedding. Cancer Res. 2022; 82: 2874). Therefore, CD3 and PD-L1 are selected in this application to construct a bispecific antibody.

[0012] Furthermore, the first functional region is connected to the N-terminus of the second functional region.

[0013] Furthermore, the heavy chain of the bispecific antibody comprises, from N-terminus to C-terminus, the domains shown in scFv-VH-CH-hinge-Fc, wherein scFv targets CD3, VH is a heavy chain variable region targeting PD-L1, and CH is a heavy chain constant region; the light chain of the bispecific antibody comprises, from N-terminus to C-terminus, the domains shown in VL-CL, wherein VL is a heavy chain variable region targeting PD-L1, and CL is a light chain constant region.

[0014] Furthermore, the Fc region is a human IgG Fc region.

[0015] Furthermore, the Fc region is a human IgG4 Fc region.

[0016] BsAbs have many structural variations and can be roughly divided into IgG-like BsAbs and non-IgG-like BsAbs. IgG-like BsAbs are Y-shaped, and the Fc fragment has many advantages in production and clinical treatment. The complete antibody structure is not only conducive to the purification process, but also increases the stability of the product. Compared with non-IgG-like BsAbs, IgG-like BsAbs have a longer in vivo half-life. More importantly, the Fc fragment mediates innate and adaptive immune responses and plays a vital role in anti-tumor activity; however, due to linkage associations, more stringent quality control processes are required (see Ma J, et al. Bispecific antibodies: from research to clinical application. Front Immunol. 2021; 12: 626616). Non-IgG-like BsAbs do not contain Fc fragments, which have unique advantages. This type of BsAb has a simple structure and can be easily produced in eukaryotic and prokaryotic expression systems. In addition, the number of antigen binding sites is more flexible, and the valence of the two targets can be customized (see Johnson S, et al. Effector cell recruitment with novel Fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo B-cell depletion. J Mol Biol. 2010; 399: 436–49). In the present invention, in order to reduce the graft-versus-host reaction of the antibody during use and improve the safety and effectiveness of treatment, an IgG-like BsAb structure was selected.

[0017] The second aspect of the present invention provides an anti-tumor composition comprising the bispecific antibody and T cells.

[0018] Furthermore, the method for preparing T cells includes: extracting mononuclear cells from peripheral blood, and obtaining T cells by CD3 / CD28 antibody magnetic bead sorting; and the T cells are incubated with TGF-β, IL-25 and IL-33.

[0019] Furthermore, the method for preparing T cells includes: using TGF-β with a final concentration of 1.5 ng / mL, IL-25 with a final concentration of 50 ng / mL, and IL-33 with a final concentration of 80 ng / mL, and incubating at 37° C. for 24 hours.

[0020] As mentioned above, a major obstacle in the treatment of solid tumors is the lack of sufficient and fully activated T cells in the tumor microenvironment. Therefore, the present invention attempts to supplement T cells by in vitro injection, thereby exerting a synergistic effect with the bispecific antibody targeting CD3 and PD-L1, and in order to increase the activity of T cells, cytokines are also used to activate them in vitro. The activation and differentiation of T cells are related to a variety of cytokines, including the interleukin family, the interferon family, the tumor necrosis factor family, the transforming growth factor family, and the like. Among them, the mechanism of action of TGF-β is relatively complex. It is generally believed that it is not conducive to T cell activation and anti-tumor therapy. However, it can produce synergistic effects when used in combination with certain cytokines to promote T cell proliferation and differentiation. For example, TGFβ synergizes with IL-6, which is beneficial to the differentiation of Th17 cells that express retinoic acid-related orphan receptor γ (RORγ) and produce high levels of IL-17 (see Park H, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 2005, 6: 1133–41); and synergizes with IL-4 to induce the differentiation of Th9 cells that secrete IL-9 (Veldhoen M, et al. TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 2006, 24: 179–89). The present invention creatively combines TGFβ with IL-25 and IL-33 and adjusts their dosage, which can well promote T cell activation and produce a strong anti-tumor effect.

[0021] The third aspect of the present invention provides a use of the bispecific antibody and / or the anti-tumor composition in the preparation of an anti-tumor drug.

[0022] Furthermore, the tumor is lung cancer and liver cancer.

[0023] Beneficial Effects

[0024] The present invention provides a bispecific antibody and its use with T cells in the preparation of a drug for treating tumors, with the following specific beneficial effects:

[0025] (1) Screening obtained antigen-binding domains targeting CD3 and PD-L1 that can bind to target antigens with high specificity;

[0026] (2) A bispecific antibody targeting both CD3 and PD-L1 was constructed. The antibody has an IgG structure, which can prolong the half-life in vivo and reduce rejection reactions;

[0027] (3) Optimized T cell activation methods, using a combined stimulation strategy of TGF-β, IL-25, and IL-33 to maintain the activated state of T cells in vivo;

[0028] (4) The bispecific antibody provided by the present invention, when used in combination with activated T cells, can inhibit tumor cell growth, regulate the expression level of immune factors, and prolong the animal production cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Schematic diagram of the bispecific antibody structure;

[0030] Figure 2 : In vitro tumor inhibition assay;

[0031] Figure 3 : Animal survival curve;

[0032] Figure 4 : TNF-α expression level in serum;

[0033] Figure 5 : IL-2 expression level in serum;

[0034] Figure 6 : Expression level of IFN-γ in serum. DETAILED DESCRIPTION

[0035] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0036] Example 1 Design and preparation of bispecific antibodies

[0037] The present invention designs a bispecific antibody with an Fc fragment. This bispecific antibody not only significantly prolongs the half-life in vivo due to the addition of the Fc fragment, but also uses the human IgG4 Fc region to reduce immune rejection and avoid serious toxic side effects.

[0038] In the early use of the present invention, a monoclonal antibody targeting PD-L1 has been obtained, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 4. It was identified that the affinity equilibrium constant KD with the target antigen was 5.51E-10M. Subsequently, a scFv structure targeting human CD3 was screened and obtained by phage display technology, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2. It was identified that the affinity equilibrium constant KD with the target antigen was 3.07E-10M. Figure 1 As shown, based on the above foundation, the present invention uses the monoclonal antibody of PD-L1 as the second functional region, which has a complete antibody structure, and the Fc region is a human IgG4 Fc region, which can prolong the half-life in the body and reduce the immune rejection reaction caused by heterologous proteins; its N-terminus is connected to the scFv targeting human CD3 to form the first functional region, thereby forming a bispecific antibody. The specific steps include:

[0039] It comprises a first functional region targeting CD3 and a second functional region targeting PD-L1, wherein the first functional region is a scFv structure, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:2; the second functional region is a Fab structure, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:4. The nucleotide sequence encoding the anti-CD3 scFv is connected to the N-terminus of the heavy chain nucleotide of the anti-PD-L1 antibody, and the light chain portion still uses the light chain nucleotide of the anti-PD-L1 antibody. The nucleotide is transferred into the pGEX-4T-1 expression vector, and the recombinant plasmid is named pGEX-CD3-PD-L1, which is transfected into Escherichia coli DH5α infected cells. It is spread on LB solid medium containing ampicillin resistance, positive clones are screened, plasmids are extracted for preliminary enzyme digestion identification, and the positive clones are sequenced to identify the correct nucleotide sequence.

[0040] Take 1 μL of recombinant plasmid pGEX-CD3-PD-L1, transform it into E. coli BL21 competent cells, select positive clones, inoculate them into 100 mL LB liquid medium, and culture at 37°C and 200 r / min with shaking until OD 600About 0.8, add IPTG with a final concentration of 0.2mmol / L for induction culture, and shake culture at 20℃ and 200r / min for 24h. After the induction expression is completed, the bacteria are collected by centrifugation at 12000r / min for 5min, and after ultrasonic disruption, the antibody is separated and purified using a GST chromatography column. SDS-PAGE and Coomassie Brilliant Blue staining are used to identify the purity of the antibody. The purity is tested to be more than 90%, which meets the experimental requirements.

[0041] Example 2 Extraction and activation of T cells

[0042] 12 mL of peripheral venous blood from healthy volunteers was collected into a heparin anticoagulation tube, 6 mL of lymphocyte separation solution was added to a 15 mL centrifuge tube, and then 6 mL of blood was added to the wall, and centrifuged at 4°C and 1000 rpm for 30 min; the buffy coat layer, i.e., the mononuclear cell layer (PBMCs) was aspirated into a 50 mL centrifuge tube, X-VIVO medium was added for washing, centrifuged at 500 rpm for 10 min, and the supernatant was discarded; 15 mL of red blood cell lysis buffer was added, incubated at room temperature for 10 min, centrifuged at 500 rpm for 10 min, and the supernatant was discarded; X-VIVO medium was added for washing once, and X-VIVO complete medium containing anti-human CD3 / CD28 antibody magnetic beads was used for activation and screening, and the obtained cells were subcultured for 3-5 generations and recorded as T cells.

[0043] Take T cells in the logarithmic growth phase, discard the original culture medium, wash three times with sterile PBS, add TGF-β containing a final concentration of 1.5 ng / mL, 50 ng / mL IL-25 and 80 ng / mL IL-33, and incubate at 37°C for 24 hours; discard the culture medium, digest the cells with 0.5% trypsin, wash three times with sterile PBS, collect the cells by centrifugation at 500 rpm for 10 minutes, and record them as AT cells.

[0044] Example 3 In vitro anti-tumor experiment of bispecific antibodies and T cells

[0045] In the present invention, human liver cancer cells HepG2 and human non-small cell lung cancer cells A549 were used as objects to investigate the tumor suppression ability of T cells. The tumor cells were revived and cultured until the logarithmic growth phase, and T cells and tumor cells were inoculated in a 96-well plate at an effect-target ratio of 5:1, and the cells were grouped, with 3 duplicate wells in each group, namely: BsAb+T cells, T cells extracted by traditional methods and 100ng / mL of BsAb targeting CD3 and PD-L1 provided in the present invention were added; BsAb+AT cells, activated T cells and 100ng / mL of BsAb targeting CD3 and PD-L1 provided in the present invention were added, and tumor cells without T cells and antibodies were used as negative control groups, and wells containing only culture medium were used as blank control groups. After 24h of co-culture, 10mL of MTT solution was added to each well, incubated at 37°C for 4h, and the absorbance (OD value) at 450nm was measured with an enzyme marker, and each group of experiments was repeated 3 times. The inhibition rate was calculated according to the following formula: inhibition rate = (OD value of negative control group - OD value of experimental group) / (OD value of negative control group - OD value of blank control group) × 100%.

[0046] The results are as follows Figure 2 As shown, the bispecific antibodies provided in the present invention can effectively activate T cells to exert their anti-tumor effects, and the activated T cells can produce a stronger tumor killing effect. After the T cells are dually activated by the bispecific antibodies and the combined cytokines, the tumor killing ability is greatly improved.

[0047] Example 4 In vivo anti-tumor experiment of bispecific antibodies and T cells

[0048] 4.1 Animal model preparation and treatment

[0049] A549 cells were revived and cultured until the logarithmic growth phase, and then digested with 0.25% trypsin to adjust the cell concentration to about 2 × 10 6 C57BL / 6 mice were used as the research subjects. Each mouse was subcutaneously inoculated with 0.2 mL of cell suspension in the right armpit, and the tumor growth was observed. 30 mice with successful modeling were selected and randomly divided into 3 groups, namely the BsAb+AT group, which was injected with 50 mg / kg of bispecific antibody targeting CD3 and PD-L1 every 3 days and 1×10 6 The activated T cells were administered for 4 weeks; the BsAb+T group was injected with 50 mg / kg of bispecific antibody targeting CD3 and PD-L1 every 3 days, and 1×10 6 T cells extracted by traditional methods were administered for 4 weeks; the control group was injected with the same amount of saline for 4 weeks. Ten normal mice were selected as the normal control group.

[0050] 4.2 Animal survival observation

[0051] Observe the survival status of animals daily, record and draw a survival curve. Figure 3 As shown, the use of BsAb and T cell therapy provided by the present invention can significantly prolong the survival of experimental animals, and the activated T cells seem to have a better therapeutic effect, which shows that due to the influence of the tumor microenvironment in the experimental animals, the T cells in the body are difficult to meet the needs of anti-tumor effects, while the external supplementation of T cells, especially the activated T cells, significantly enhances the anti-tumor effect.

[0052] 4.3 Detection of inflammatory factors

[0053] Blood was collected from the eye sockets and placed in anticoagulant tubes containing heparin, and the supernatant was collected by centrifugation to collect plasma. The levels of serum cytokines TNF-α, IL-2, and IFN-γ in tumor-bearing mice were detected using an enzyme-linked immunosorbent assay kit (purchased from Suzhou Calvin Biotechnology Co., Ltd.), and the specific steps were carried out according to the kit instructions.

[0054] The results are as follows Figure 4-6 As shown in the figure, the expression of TNF-α, IL-2, and IFN-γ in the tumor model group decreased significantly compared with the normal group. After BsAb and T cell treatment, the expression levels of various immune factors were restored, indicating that this method can effectively activate the body's immune system, stimulate the secretion of immune factors, and mobilize the body's own immune mechanism to fight tumor cells. Considering that the body's own immune system has been fully mobilized by BsAb, the use of activated T cells does not have a significant advantage in stimulating the secretion of immune factors. There was no significant difference in TNF-α and IL-2 compared with the BsAb+T group, but the expression of IFN-γ was significantly increased, showing a strong immunomodulatory effect.

Claims

1. A bispecific antibody, characterized in that: It comprises a first functional region targeting CD3 and a second functional region targeting PD-L1, wherein the first functional region is a scFv structure, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:2; the second functional region is a Fab structure, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

4.

2. The bispecific antibody according to claim 1, characterized in that The first functional region is connected to the N-terminus of the second functional region.

3. An anti-tumor composition comprising the bispecific antibody as claimed in any one of claims 1 to 2 and T cells.

4. The antitumor composition according to claim 3, characterized in that: The method for preparing T cells comprises: extracting mononuclear cells from peripheral blood, and obtaining T cells by CD3 / CD28 antibody magnetic bead sorting; and the T cells are incubated with TGF-β, IL-25 and IL-33.

5. The antitumor composition according to claim 4, characterized in that: The method for preparing T cells comprises: using TGF-β with a final concentration of 1.5 ng / mL, IL-25 with a final concentration of 50 ng / mL, and IL-33 with a final concentration of 80 ng / mL, and incubating at 37° C. for 24 hours.

6. Use of the bispecific antibody according to any one of claims 1 to 2 and / or the anti-tumor composition according to any one of claims 3 to 5 in the preparation of anti-tumor drugs, wherein the tumor is lung cancer or liver cancer.

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