Combination therapy of gosabezumab and TCR-T in solid tumors

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a combination therapy of gosabezumab and TCR-T in solid tumors. The invention relates to application of gosabezumab combined with TCR-T cells in preparation of drugs for treating solid tumors. According to the present invention, based on the drug action mechanism, in combination with clinical patient specimen data and single cell sequencing analysis, a variety of cytobiology and molecular biology means are adopted, and through a variety of in-vivo and in-vitro models, the macroscopic to microscopic deep research is performed; the application of TCR-T and SG in solid tumors is comprehensively and systematically researched, the research process has very strong technical comprehensiveness, and the synergistic effect of SG combined with TCR-T is found from the research process. At present, SG is already marketed and applied clinically, and TCR-T is proved to have good safety, so that the 'SG and TCR-T' duplex strategy has a good clinical transformation prospect, and the combination of precision medicine and transformation medicine is reflected.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a combined therapy of golatuzumab and TCR-T in solid tumors. BACKGROUND

[0002] TCR-T cell therapy (T-cell Receptor T-cell Therapy) is a kind of cell therapy that uses genetic engineering means to transfer TCR sequences that specifically bind to tumor target antigens into T cells derived from peripheral blood of patients, and then returns the modified T cells to the body, so that it can recognize and attack tumor antigen. TCR-T therapy can not only recognize cell membrane antigens, but also recognize intracellular tumor antigens presented by peptide-major histocompatibility complex (pMHC), and is not limited by the expression of target cell surface antigens, so it can more widely recognize target antigens, and therefore has greater potential in the treatment of solid tumors.

[0003] The applicant has developed more effective T cell sorting methods and personalized TCR-T cell therapy in previous TCR-T construction and related research work, which has significantly improved the success rate of TCR-T construction and the efficacy in solid tumors. TCR-T cell therapy has shown great potential in tumor treatment, but its efficacy needs a good "soil environment", but the immunosuppressive microenvironment in solid tumors often hinders TCR-T treatment, such as the presence of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and other immunosuppressive cells; high expression of immune checkpoint molecules such as PD-L1; metabolic disorders of the tumor microenvironment caused by hypoxic environment and lack of nutrients; abnormal structure of tumor blood vessels, physical barriers of tumor microenvironment composed of stromal cells and extracellular matrix. Therefore, appropriate methods are needed to reshape the tumor immune ecology to maintain the function of TCR-T.

[0004] At present, TCR-T therapy has made positive progress in clinical trials of melanoma, lung cancer, sarcoma and other solid tumors, but its objective response rate is only 15%-36%, and the clinical efficacy of TCR-T needs to be improved.

[0005] Previous studies have shown that poor efficacy of TCR-T is often closely related to insufficient T cell infiltration and activation, and the number and function of TCR-T cells in the tumor directly determine its anti-tumor effect. Considering that T cell activation first requires recognition of antigens presented by MHC on antigen-presenting cells (mainly dendritic cells, DCs) through TCR, therefore, the inhibition of dendritic cell (DC) function caused by the immunosuppressive microenvironment of solid tumors is an important reason for limiting the efficacy of TCR-T. Therefore, strategies to enhance DC cell activity, improve tumor immune microenvironment, and improve T cell activation are expected to significantly improve the anti-tumor efficacy of TCR-T.

[0006] DC is an important factor in mediating CD8+T cell activation. DCs can take up, process antigens and present antigen peptides through major histocompatibility complex class I molecules (MHC I). This antigen presentation is recognized by the T cell receptor (TCR) and CD8 co-receptor on CD8+T cells, leading to the activation and differentiation of CD8+T cells. And the mature activation of TCR-T cells also needs to receive the antigen presentation of DCs. In vitro and in vivo experimental results also show that the addition of SG-treated tumor cells can significantly induce the maturation of dendritic cells (DCs) and enhance their ability to cross-present CD8+T cells.

[0007] Previous studies have found that the use of immune checkpoint inhibitors (ICIs) or IL-2 after TCR-T infusion can improve the tumor killing function of TCR-T to some extent, but these measures still have the problem of accumulation of adverse reactions and limited relief time. The current research focus on enhancing TCR-T often focuses on the activation of T cells after TCR-T infusion, but ignores the influence of pre-treatment before TCR-T infusion. SUMMARY

[0008] Applicants have found in previous studies that BRAF inhibitors can up-regulate the expression of tumor antigen MUC1, thereby improving the tumor killing effect of MUC-1 CAR-T cell therapy. This proves the effective effect of pre-treatment on adoptive T cell therapy. Based on this, the present application attempts to develop a combined therapy of TCR-T to improve its efficacy.

[0009] Applicants found that both chemotherapy and antibody targeted therapy can improve the immune microenvironment and promote anti-tumor immunity. Antibody-drug conjugate (ADC) is a conjugated drug of monoclonal antibody and cytotoxic load, which specifically targets tumor cells through antibody, releases potent cytotoxic drugs, and improves efficacy while reducing toxic side effects. Therefore, whether the antibody conjugated drug (ADC) combined with chemotherapy and antibody targeted therapy has stronger anti-tumor immune effect? Applicants collected tumor tissue specimens of patients before and after treatment with Sacituzumab govitecan (SG), and performed single cell RNA and TCR sequencing on these tumor specimens. The results showed that the number of CD8+ T cells infiltrating the tumor after treatment was more and the function was more activated, and the number of T cell clones also increased significantly. At the same time, immunofluorescence staining of tumor sections of patients also showed that the number of CD8+ T cell clones in tumor tissue after treatment was more and the activation degree was higher. In addition, applicants also found that the activated CD8+ T cell dependent anti-tumor immunity after SG treatment can produce systemic therapeutic benefit, and the immune effect of SG is indeed stronger than the anti-tumor immune effect of pure chemotherapy or antibody targeted drug.

[0010] Further in vivo experiments also showed that the SG treatment group not only had a significant reduction in tumor volume, but also had a significant activation of CD8+ T cells in the tumor microenvironment. This confirmed that SG can improve T cell infiltration and activation.

[0011] Further in vitro experiments co-cultured DC cells and 1G4 TCR-T cells stimulated by different drugs, and the activation level and expansion number of TCR-T cells in the SG group were significantly improved. Then the killing ability of TCR-T was measured, and the results showed that the killing ability of TCR-T cells in the SG stimulation group was significantly enhanced, which preliminarily proved that SG played a role in enhancing TCR-T by activating DC.

[0012] The present application studies the feasibility of SG combined with TCR-T therapy in solid tumors, and discloses the construction method of TCR-T and the synergistic effect of Sacituzumab govitecan (SG) on TCR-T.

[0013] Based on the above research results, in a first aspect, the present application provides the use of Sacituzumab govitecan in combination with TCR-T cells in the preparation of a medicament for treating solid tumors.

[0014] As a preferred embodiment of the first aspect, the tumor antigen targeted by the TCR-T cell is NY-ESO-1.

[0015] As a preferred embodiment of the first aspect, the solid tumor is breast cancer.

[0016] The application provides application of golatuzumab in preparation of a drug for enhancing the effect of TCR-T cell treatment on solid tumors.

[0017] In a second aspect, the application provides application of golatuzumab in preparation of a drug for enhancing the effect of TCR-T cell treatment on solid tumors.

[0018] The application finds through experiments that golatuzumab can enhance the killing ability of TCR-T cells, and therefore, the golatuzumab can be used as an enhancer of TCR-T cells.

[0019] In a third aspect, the application provides a pharmaceutical composition comprising: golatuzumab and TCR-T cells targeting NY-ESO-1 antigen.

[0020] As a preferred embodiment of the third aspect, the pharmaceutical composition further comprises an emulsifying agent, a disintegrating agent, a glidant, a preservative, a sustained-release material, a transdermal enhancer.

[0021] As a preferred embodiment of the third aspect, the sustained-release material is at least one of chitosan, hyaluronic acid, alginate, silk fibroin.

[0022] As a preferred embodiment of the third aspect, the transdermal enhancer is at least one of polyethylene glycol, oleic acid, isopropyl myristate, sodium lauryl sulfate.

[0023] As a preferred embodiment of the third aspect, the emulsifying agent is at least one of polysorbate, poloxamer, sucrose fatty acid ester, sodium dodecyl sulfate.

[0024] In a fourth aspect, the application provides application of the pharmaceutical composition of the third aspect in preparation of a drug for treating solid tumors.

[0025] Compared with the prior art, the application has the following beneficial effects: (1) The application first proposes the theory of antibody conjugate drugs (SG) remodeling the immune microenvironment to enhance TCR-T therapy. The existing treatment methods for solid tumors are limited, resulting in poor prognosis. TCR-T cell therapy shows great potential in tumor treatment, but its anti-tumor effect needs a good “soil environment”, but the immunosuppressive microenvironment in solid tumors often hinders TCR-T treatment. The immunogenic death and DC activation induced by SG not only release a large amount of tumor antigens (including neoantigens), but also may provide a functionally enhanced “soil” for TCR-T by remodeling the immune microenvironment, improve the anti-tumor effect of TCR-T, and have strong clinical application value.

[0026] (2) The research plan is comprehensive and highly feasible for clinical translation. This invention starts from the drug's mechanism of action, combining clinical patient specimen data and single-cell sequencing analysis, employing various cell biology and molecular biology techniques, and utilizing multiple in vivo and in vitro models to conduct in-depth research from macroscopic to microscopic levels. A comprehensive and systematic study of the application of TCR-T and SG in solid tumors was conducted. The research process exhibits strong technical integration, revealing the synergistic effect of SG combined with TCR-T. Currently, SG is already on the market and used clinically, while TCR-T has been proven to have good safety. Therefore, the "SG combined with TCR-T" dual-strategy has excellent prospects for clinical translation, reflecting the integration of precision medicine and translational medicine. Attached Figure Description

[0027] Figure 1 This is a diagram of 1G4 TCR-T cells targeting NY-ESO-1, as verified using pentamers in Example 1.

[0028] Figure 2 The diagram shows the in vitro activation of T cells using a combination of SG and TCR-T in Example 2, where A represents the activation level of TCR T cells; B represents the proliferation level of TCR T cells; and C represents the killing ability of TCR T cells.

[0029] Figure 3 This is a TCR-T that targets NV-ESO-1 in vivo with SG in Example 3. A is an experimental flowchart; B is a graph showing the changes in tumor volume in mice in the two treatment groups; C is a graph showing the survival curves of mice in the two treatment groups; and D is a graph showing the body weight of mice in the two treatment groups. Detailed Implementation

[0030] Example 1: Construction of TCR-T(1G4) targeting NY-ESO-1 TOP1i-ADC in vitro activation of TCR-T Based on previous research findings that TOP1i-ADC (topoisomerase 1 inhibitor-antibody conjugate) can enhance the ability of dendritic cells (DCs) to activate CD8+ T cells, it is hypothesized that TOP1i-ADC may enhance the efficacy of adoptive T-cell therapy (ACT) for tumors. Given that ADC therapy relies on the high expression of cell membrane antigens, using TCRT (thin-cell retrieval therapy) that recognizes intracellular antigens, rather than CAR-T (carbohydrate artery retrieval therapy) that recognizes membrane antigens, in combination with ADCs prevents conflicts between the two therapies.

[0031] Step 1: Construct an expression vector targeting the human NY-ESO-1 antigen peptide Based on the pLVX-IRES-Puro vector, the DNA fragment expressing NY-ESO-1 1G4-LYTCR was inserted by digestion with EcoRI-BamHI (codon optimization based on human, synthesized by the company's whole genome, amino acid sequence is shown in SEQ ID NO.1) to construct the final vector pLVX-IRES-Puro-NY-ESO-1-1G4-LY.

[0032] The amino acid sequence of NY-ESO-1 1G4-LY is shown in SEQ ID NO.1: MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQD PGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNT GELFFGEGSRLTVLDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEND EWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRGSGRAKRSGSGATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSS KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRL NASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVY QLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*(SEQ ID NO. 1).

[0033] Step 2: Knockout of hTRBC1 / 2 and hTRAC1 domains to prevent mismatch between endogenous and recombinant TCR chains: Two sgRNAs were designed by knocking out the hTRBC1 / 2 and hTRAC1 domains (KO) via CRISPR / Cas9-mediated knockout; sgTRAC1 (SEQ ID NO.2): TCTTCTCAGCTGGTACACGGC; sgTRBC1 / 2 (SEQ ID NO.3): CAAACACAGCGACCTCGGGT. The sgRNAs were delivered using RNP-electroporation to remove endogenous TCRα and β from recipient T cells, thus preventing mismatch between endogenous and recombinant TCR chains.

[0034] Step 3: Construct the complete TCR sequence structure: The complete TCR sequence structure is: TCRβV-TCRβC-autohydrolytic linker protein-TCRαV-TCRαC.

[0035] TCRβV or TCRαV, including FR1-CDR1-FR2-CDR2-FR3-CDR3.

[0036] The self-hydrolyzing linker protein selected was furin-SGSG-P2A.

[0037] The following method, based on CRISPR-Cas9, was used for construction: 1. Cut the TRAC1 exon 1 in the genome and insert T2A-TCRβV-TCRβC-P2A-TCRαV-TCRαC (to fill in the part that was cut off earlier); 2. Cut the TRBC1 exon 1 in the genome and insert T2A-TCRαV-C-P2A-TCRβV-TCRβC (to fill in the part that was cut off earlier); 3. Cut the TRAC1 exon 1 in the genome and insert T2A-TCRαV-C (to fill in the previously cut-out part). Then cut the TRBC1 exon 1 and insert T2A-TCRβV-C (to fill in the previously cut-out part).

[0038] T2A is a "self-cleaving" peptide used to distinguish the portion before the cleavage site in exon 1 of TRAC1 from the newly inserted sequence.

[0039] The original complete TCR sequences were all humanized, with the stable regions (TCRβC, TCRαC) replaced with murine-derived sequences (Uniprot: P01849, P01851, P01852) for detection using anti-mouse TCR.

[0040] Step 4: Amplify and transfect the recombinant plasmid. TCR-T cells were further amplified (50 IU / ml IL-2 and 10 ng / ml IL-7), and lentivirus was packaged in 293T cells using psPAX2 and pMD2.G vectors and then used to infect CD8+ T cells. The results are as follows: Figure 1 As shown, significant TCRs were transferred into T cells.

[0041] The specific methods for packaging lentiviruses and infecting CD8+ T cells are as follows: 293T cells were cultured in complete medium. One day later, the medium was changed to 1 mL complete medium for 2 wells and transfected. The transfection reagents were: 1*{(1)A: 50 μL opti-MEM + 2 μL PEI 40000; (2)B: 50 μL opti-MEM + 0.3 μg psPAX2 + 0.15 μg pMD2.G + 0.55 μg pLVX-IRES-Puro NY-ESO-11G4-LY / PLVX-Puro}; the medium was changed with complete medium on the second day, and the cell culture supernatant was collected on the third day, which was rich in virus; 1 / 3 volume of lentivirus concentrate (4X LentiV-X Lentivirus Concentration Solution (ES-7011)) was added to the supernatant and shaken overnight at 4℃ (40 rpm); then centrifuged at 3000 rpm to obtain concentrated virus.

[0042] Peripheral blood mononuclear cells (PBMCs) were isolated from the patient's peripheral blood using density gradient centrifugation, followed by magnetic bead sorting of CD8+ T cells. The cells were resuspended in 2 mL of complete culture medium, cultured in 24-well plates for 1 day, and then treated with 10 μL / mL ImmunoCult. TM HumanCD3 / CD28 / CD2 T Cell Activator (Catalog#10970 / 10971) amplification, followed by viral concentration on day 4. Two viral solutions, one with knockout hTRBC1 / 2 / hTRAC1 and the other with inserted TCR sequences, were centrifuged at 3000 rpm for 30 min at 4°C to precipitate the cells. The cells were resuspended in 0.1 mL of T cell culture medium (complete medium containing 25 IU / mL interleukin-2), and polybrene 1:1000 was added. T cells were evenly distributed into 24-well plates, with 0.1 mL of viral solution added to each well. The plates were centrifuged at 400 g for 60 min at room temperature. 1 mL of culture medium was added to obtain TCR-T (1G4).

[0043] Example 2: SG enhances TCR-T(1G4) targeting NY-ESO-1 in vitro. Co-culture of breast cancer cell lines MDA-MB-468, which highly express NY-ESO-1 and TROP2, with DCs: MDA-MB-468 cells were cultured using RPMI-1640 + 10% FBS; mononuclear cells were seeded in 24-well plates and DC induction medium (DMEM, 50 ng / mL GM-CSF, 20 ng / mL IL-4, 10% FBS, 5 x 10⁶ cells per well) was added. 5 Immature DCs were obtained by culturing DCs for 5 days. After counting, DCs and MDA-MB-468 were co-cultured at a 1:1 cell ratio for 24 hours. After one day of treatment with SG (1 μM), DCs were used to activate TCR-T (1G4) cells. It was found that this significantly activated TCR-T cells showed increased expression of CD69 and IFNγ. Figure 2 A), its proliferation is significantly enhanced ( Figure 2 B); and its ability to kill cancer cells is also significantly enhanced. Figure 2 C), whose effect was significantly stronger than the control ADC drug (sacituzumab, S).

[0044] Example 3: SG in vivo enhancement of TCR-T(1G4) targeting NY-ESO-1 To further clarify the efficacy of this combination therapy in human tumor models, tumor PDX from TROP2+HLA-A2+ breast cancer patients was transplanted into the fat pads of NCG mice: the tumor tissue was cut into 2-4 mm pieces using sterile scissors. 3 In NCG-treated mice, after anesthetizing the fourth mammary gland region, the skin was incised 5-10 mm to locate the fat pad. The tumor mass was then embedded into the fat pad using forceps and adhered with bio-adhesive. Finally, 1×10⁻⁶ CD8+ T cells from the same individual, transfected with 1G4 TCR, were introduced. 6 Mice were inoculated with cells / mouse and divided into groups and given maintenance treatment with control antibody S and SG (dosage 10 mg / kg / week). Figure 3 A). The results showed that administering one injection of SG (50 mg / kg) before and after TCR T treatment significantly inhibited tumor progression. Figure 3 B), to prolong survival time ( Figure 3 C), while there was no significant difference in body weight among the groups of mice. Figure 3 D). This demonstrates that ADC is an effective induction protocol before TCRT treatment.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of goxatocilizumab combined with TCR-T cells in the preparation of drugs for treating solid tumors.

2. The application according to claim 1, characterized in that, The tumor antigen targeted by the TCR-T cells is NY-ESO-1.

3. The application according to claim 1, characterized in that, The solid tumor was breast cancer.

4. Application of goxatocilizumab in the preparation of drugs that enhance the efficacy of TCR-T cell therapy for solid tumors.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: goxatocilizumab and TCR-T cells targeting the NY-ESO-1 antigen.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also includes emulsifiers, disintegrants, flow aids, preservatives, sustained-release materials, and transdermal penetration enhancers.

7. The pharmaceutical composition according to claim 6, characterized in that, The sustained-release material is at least one of chitosan, hyaluronic acid, alginate, and silk fibroin.

8. The pharmaceutical composition according to claim 6, characterized in that, The transdermal penetration enhancer is at least one of polyethylene glycol, oleic acid, isopropyl myristate, and sodium lauryl sulfate.

9. The pharmaceutical composition according to claim 6, characterized in that, The emulsifier is at least one of polysorbate, poloxamer, sucrose fatty acid ester, and sodium lauryl sulfate.

10. Use of the pharmaceutical composition of claim 5 in the preparation of a medicament for treating solid tumors.

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