| Cancer treatment has benefited from the accelerated development of modern biotechnology,and many immunotherapy strategies have sprung up,such as vaccines,immune checkpoint inhibitors,chimeric antigen receptor(CAR)cell therapy,monoclonal antibodies and bispecific antibodies.In tumor immunotherapy,T-cell redirected bispecific antibodies(Bs Ab)directly activate T-cells to kill cancer cells by binding to antigen proteins with tumor-specific expression and at the same time binding to CD3 molecules on the surface of T-cells.There is no need to rely on the classical T-cell activation pathway,providing a new therapeutic strategy especially for some patients with non-immunogenic tumors.For example,GPC3 protein is found to be specifically expressed in solid tumors such as liver cancer in recent years,which the clinical efficacy of the currently developed monoclonal monomer is not ideal,but it is a highly potential redirected T-Therapeutic targets of cellular antibodies.There are dozens of bispecific antibody structures in clinical practice,one of which is in the form of antibody fragments,such as the Bi TE configuration,which is formed by concatenating the variable regions(sc Fv)of two antibodies.The blood half-life is too short;the other type is Ig G type,which has the advantage of long half-life by Fc domain contribution,the bispecific antibody targeting malignant tumors is still in clinical trials.At present,for the research and development of bispecific antibodies,the balance between effectiveness,safety and druggability is more difficult than that of monoclonal antibodies.The design of antibody configuration in the early stage is particularly important.It is necessary to comprehensively consider stability,flexibility of antigen-antibody binding and factors such as compactness and ease of downstream process purification.In this study,a novel anti-GPC3/CD3 tetravalent bispecific antibody was designed in the Ig G-sc Fv configuration targeting hepatoma therapy.The main contents of this study are as follows:(1)On the basis of the constructed anti-GPC3/CD3 tetravalent bispecific antibody(Bs Ab-WT),in order to promote the pairing of the heavy and light chains of the antibody,seven new pairs were introduced between the CH1 and CL chains of the constant region of the human GPC3 antibody,respectively.disulfide bonds.It was secreted and expressed in the cell supernatant by HEK293 suspension cells.Quantitative analysis showed that the content of the target monomer increased after the introduction of seven pairs of mutation pairs.Among them,the content of the target monomer formed after the introduction of the CH1126/CL121(F126C/S121C)mutation pair Significantly improved by 20 %,anti-aggregation stability and thermal stability were also improved.The binding activity of the antibody to cell surface GPC3 and CD3 antigens was determined by flow cytometry(FCM),and the results showed that the affinity of the parental antibody was still maintained.(2)In order to further improve the stability of the tetravalent antibody introduced by the single disulfide bond,a disulfide bond(Q100C/G44C)was introduced in the conserved region of the VH/VL of the GPC3 antibody.The results showed that the anti-aggregation,degradation and thermal stability of the antibody were further improved,of which the thermal stability was increased,which was close to that of the parent antibody,and it was verified that the increase in stability benefited from the antibody VH-CH1/VL-CL domain(Fab)between the introduction of disulfide bonds;at the same time,the monomer content of the antibody was also increased by 28 %,and the yield was better than that of wild-type Bs Ab-WT.Formation of the engineered disulfide bonds introduced in the antibody was demonstrated by DTNB colorimetry.(3)By introducing double disulfide bonds,its affinity with the antigen was determined by FCM in the anti-GPC3/CD3 antibody Bs Ab-2SS,its affinity remained basically unchanged;the neonatal Ig G Fc receptor(Fc Rn)was in the Ig G blood circulation.The interaction between the antibody and Fc Rn was measured by biofilm interferometry(BLI),and it was found that the tetravalent antibodies Bs Ab-WT and Bs Ab-2SS dissociated normally from the Fc Rn receptor at p H=7.4.Compared with the Ig G control,the dissociation speed of both antibodies from Fc Rn was significantly reduced by nearly 26 times at p H=6.0,and the equilibrium dissociation constant KD was 3~7 times higher(Bs Ab-2SS was 7 times higher),which indicated that the antibody had binding activity to Fc Rn increase,potentially extending the half-life of the antibody in the body.(4)In order to evaluate the in vitro induction of T lymphocytes to kill cancer cells by tetravalent antibodies introduced by disulfide bonds,the half effective killing concentration was determined by CCK-8 method,and the antibody Bs Ab-2SS was 3~6 times better than the antibody Bs Ab-WT.The effect of Bs Ab-2SS on different liver cancer cells was further studied,and it was found that the killing effect of this antibody on cancer cells was concentration-dependent and GPC3 target antigen-dependent.Hep G2 with the highest abundance of GPC3 had the best killing effect,which was the same as the release of lymphocytes.In terms of inducing T-cell activation,BsAb-2SS can effectively activate T lymphocytes to proliferate and differentiate into CD4+ and CD8+ T lymphocytes.(5)The in vivo anti-tumor activity of anti-GPC3/CD3 antibodies was evaluated by using a liver cancer xenograft tumor model constructed in immunodeficient mice.The results showed that both tetravalent antibodies significantly inhibited tumor growth in mice at a dose of 1 mg/kg,and Bs Ab-2SS was slightly better than Bs Ab-WT.The content of T lymphocytes in tumor and blood of mice was further analyzed,and it was proved that after administration of Bs Ab-2SS,T lymphocytes could effectively infiltrate tumor tissue.(6)The antibody Fab domain was introduced through the same double disulfide bond mutation,and two Ig G-sc Fv tetravalent antibodies of different human antibodies were constructed: CEA-Bs Ab-2SS and h2E-Bs Ab-2SS.Compared with the wild type,the antibody target monomer The content was significantly increased by 40-60 %,and the purification yield was also significantly increased,among which CEA-Bs Ab-2SS increased nearly 38 times.The results of antibody affinity using BLI and FCM showed that the binding activity of antibody and antigen changed within 2 times after the introduction of disulfide bond,indicating that the introduction of engineered disulfide bond had no effect on the binding conformation of antibody and antigen.In conclusion,in this study,starting from the poor efficacy of anti-GPC3 monoclonal antibody and the structural instability of Ig G-sc Fv bispecific antibody,a novel anti-GPC3/CD3 tetravalent bispecific antibody was constructed.A series of biophysical and biological function studies were carried out on the introduction site of sulfur bond mutation,and the optimal combination of disulfide bond mutation pairs was obtained,which successfully promoted the correct pairing of the heavy and light chains of the tetravalent antibody and improved the stability of the antibody.Hence,it supplemented the application of engineered disulfide bond in Fab domain modification of antibodies.To verify the introduction of these two engineered disulfide bonds,two different Ig G-sc Fv configuration bispecific antibodies were designed that can significantly increase the monomer content and recovery yield without affecting the affinity activity of the antibody,which is the construction and optimization of bispecific antibodies provide new ideas.Based on the optimized anti-GPC3/CD3 bispecific antibody,the antibody can effectively recruit T lymphocytes to specifically kill GPC3 cancer cells through in vitro cancer cell killing experiments and in vivo mouse tumor growth inhibition experiments,which can be used as targeted therapy drugs for GPC3-positive tumors have potential clinical application value. |
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