Study On The Anti-tumor Effect Of Radionuclide Coupled B7-H3 Humanized Antibody In Glioblastoma And Its Influence On Immune Microenvironment

Background:Glioblastoma（GBM）is the most common malignant tumor in the central nervous system.Due to the aggressive growth characteristics of glioma tumor cells,glioma has the characteristics of high incidence,high recurrence rate,high mortality and low cure rate.Despite the continuous development of treatment technology,the special microenvironment of glioma tumors cannot rely on the regulation of immune cell-mediated killing effect,resulting in little benefit from conventional immunotherapy（PD-1/PD-L1,CTLA-4inhibitors）.Radioimmunotherapy（RIT）is a valuable choice for clinical transformation.Antibodies to therapeutic targets are key elements of radioimmunotherapy.B7-H3 is known as a“tumor-associated antigen”,which is highly expressed in tumor cells and tumor blood vessels,but hardly expressed in normal tissues.Therefore,B7-H3 has attracted much attention in radioimmunotherapy for GBM RIT.The development of radionuclide coupled targeted B7-H3 antibodies is expected to bring clinical benefits to GBM patients.Objective:In this study,molecular imaging technology was adopted to screen out specific B7-H3targeted humanized antibodies from the self-developed anti-human B7-H3 humanized antibodies.And the characterization and antitumor efficacy of screening targeted B7-H3humanized antibodies were investigated in vitro.Furthermore,the antitumor effects of ¹³¹I-B7-H3 and its effects on immune microenvironment were performed in mouse GBM models,which provided a basis for the further development of radionuclide conjugate humanized antibody targeting B7-H3.Method:1.Construction,characterization and screening of B7-H3 targeted humanized antibody:（1）Antibody humanization:humanization was carried out by using http://www.abysis.org.3-D structures of the murine and humanized antibodies,including the variable domains of the heavy chain and light chain,were constructed.Fv fragments were modeled using computer-guided homology modeling.（2）Expression,purification and affinity determination of B7-H3 humanized antibody:The plasmids encoding human 4G4（hu4G4）as well as human 4H12（hu4H12）VH and VL were constructed and transfected into Expi CHO-S cells.The supernatant was collected and purified with Protein A.The purified antibody was measured by Bio Drop spectrophotometer and protein concentration assay kit,and the affinity of the humanized antibody was investigated.（3）¹²⁵I-SPECT imaging for antibody screening:（1）¹²⁵I labeling antibody and stability analysis in vitro:Antibody（hu4G4,hu4H12,Ig G）were labeled by ¹²⁵I with chloramine-T iodized method and the stability of ¹²⁵I labeled antibody in PBS and human serum was performed in vitro.（2）Biodistribution of ¹²⁵I-antibody in vivo:The nude mice were randomly divided into four groups（n=8）.Each group of mice were intravenously（i.v.）injected with Na¹²⁵I（11.1 MBq）,¹²⁵I-hu4G4（11.1 MBq,1.75 mg/kg bodyweight）,¹²⁵I-Ig G（11.1 MBq,1.75 mg/kg bodyweight）,¹²⁵I-hu4H12（11.1 MBq）,respectively.After treatment for 2 h,10-min static SPECT images were acquired at 2,24,48,and 72 h post-injection.Tumor-bearing mice were dissected at 72 h post-injection after SPECT imaging,and the major organs and tissues of each mouse,such as blood,heart,liver,spleen,kidney,bone,long bone joints,muscle,stomach,intestine,gonad,reticular tissue,and tumor samples,were collected,weighed and counted by using a gamma counter.（4）⁸⁹Zr-PET imaging for antibody screening:（1）⁸⁹Zr-labeling antibody and stability analysis in vitro:Antibodies（hu4G4,hu4H12,Ig G）were modified with DFO,and labeled by ⁸⁹Zr-oxalate.The stability of ⁸⁹Zr labeled antibody in acetate buffer and human serum was performed in vitro.（2）Biodistribution of ⁸⁹Zr-DFO-antibody in vivo:U87-xenografted mice randomized into five groups of six mice were intravenously（i.v.）injected with ⁸⁹Zr-DFO-B7-H3（hu4G4,hu4H12）（1.94 MBq,1.75mg/kg bodyweight）,⁸⁹Zr-DFO-Ig G（1.94 MBq,1.75 mg/kg bodyweight）,⁸⁹Zr-DFO（1.94MBq）,⁸⁹Zr-oxalate（1.94 MBq）.Then,10-min static PET images were acquired at 2,12,24,48,72,96,120,144 and 168 h post-injection.Tumor-bearing mice were dissected at 72 h and 168 h post-injection after PET imaging,and the major organs and tissues of each mouse,such as blood,brain,heart,liver,spleen,kidney,bone,long bone joints,muscle,stomach,intestine,gonad,and tumor samples,were collected and counted by using a gamma counter.（3）For the pharmacokinetics study,blood samples were collected from the tail at 5 min,10min,15 min,30 min,and 1,2,6,24,48,72,96,120,144 and 168 h after injection,weighed immediately and detected with a gamma counter.Pharmacokinetic parameters were analyzed by Phoenix Win Non Lin.Through comparison of biological properties in vivo and in vitro,the most suitable target B7-H3 humanized antibody for subsequent radioimmunotherapy research was selected.2.Characterization of ¹³¹I labeled B7-H3 targeted humanized antibody（hu4G4）in vitro:（1）The process exploration of ¹³¹I labeling hu4G4:The ratio of ¹³¹I activity（GBq）to B7-H3 antibody（μmol）was set for radiolabeling reaction and the most suitable labeling technology was selected for ¹³¹I labeling hu4G4 by studying the stability in vitro.（2）The performance of ¹³¹I-hu4G4 antibody in vitro:The uptake,internalization and competitive binding of ¹³¹I-hu4G4 in U87 glioma cells were preliminarily evaluated and the optimal concentration was analyzed to provide a basis for the evaluation of efficacy in vitro.（3）Pharmacodynamic analysis of ¹³¹I-hu4G4 in vitro:The inhibitory effect of ¹³¹I-hu4G4antibody was evaluated by CCK8,cell cloning,cell migration,cell immunogenic death,cell apoptosis and cell cycle detection.3.The anti-tumor efficacy of ¹³¹I-hu4G4 in U87-xenografted model:（1）Antibody dose escalation:（1）A dose escalation design with 5 different m Abs doses of ¹³¹I-hu4G4（0.05 mg/kg,0.50 mg/kg,1.75 mg/kg,7 mg/kg,28 mg/kg bodyweight,respectively）.Biological distribution analysis was performed at 2 h,24 h,72 h and 120 h after administration and counted by using a gamma counter.The correlation between tumor tissue-specific uptake rate and antibody concentration was analyzed.The IC50 value of tumor tissue-specific binding ¹³¹I-hu4G4 was calculated by the nonlinear fitting curve,and combined with the efficacy evaluation of different antibody doses,to find the most appropriate antibody amount and radioactive dose for RIT treatment.（2）Efficacy and safety evaluation at 1.5 mg/kg 11.1 MBq ¹³¹I-hu4G4 dose:（1）U87-xenografted mice with established tumors were randomly divided into four groups（n=14 per group）and injected with ¹³¹I-hu4G4,¹³¹I-Ig G,hu4G4 or PBS（negative control）for 2 cycles on a two-weekly basis with 3 weeks follow-up in total.The tumor volume was measured with a digital caliper during treatment and relative tumor proliferation rate（T/C%）was analyzed.¹⁸F-FDG was used to evaluate the early efficacy（D7 after administration）.（2）Further,the safety was evaluated by H&E staining,body weight and blood routine.4.The antitumor effect of ¹³¹I-hu4G4 by intracranial injection and its effect on immune microenvironment in situ GBM:（1）The construction of GL261-flag-CD276cells GL261-Red-FLuc cells were infected with lentivirus by expressing human B7-H3（hu B7-H3）antigen.The expression of CD276 in GL261-flag-CD276 cells was detected by Western blot.（2）Antitumor efficacy evaluation of ¹³¹I-hu4G4 in situ GBM model:GL261-flag-CD276 cells were injected in situ injection in C57 BL/6 mice（n=30）.These mice were randomly divided into four groups,which were PBS group,hu4G4 group（1.5 mg/kg）,Na¹³¹I group（1.11 MBq）and ¹³¹I-hu4G4 group（1.11 MBq,1.5 mg/kg）,respectively.Five mice in each group were injected intracranially on Day 0 and Day 3.Then,on Day 1 before administration and Day 2 and Day 6 after administration,the small animal in vivo fluorescence imaging system was used to develop the optical signal value by delineating the part of the interest of the brain,and the changes of brain tumor burden in each group were analyzed.（3）Biodistribution experiment in situ glioma model in vitro:major organs and tissues of 3 mice in each group were collected,gamma-counter counting was performed on Day 6,and%ID/g of each tissue sample was calculated;（4）Blood pharmacokinetics study of in situ glioma model:blood samples collected from the tail at 1 h,12 h,36 h,48 h and 72h after the first administration was weighed immediately,and%ID/g in the blood sample was calculated.（5）Morphological analysis,immunogenic death and changes of immune cells in the immune microenvironment of mouse brain glioma were investigated by flow cytometry and multiple immunofluorescences.Results:1.Construction,characterization and screening of B7-H3 targeted humanized antibodies:（1）Flow cytometric analysis and binding curve showed that 4G4 and 4H12specifically bind to the B7-H3 protein rather than the other proteins of the B7 family（B7-H1,B7-H2,B7-H4,B7-H5,B7-H6,B7-H7）.（2）After humanizing 4G4 and 4H12,unbinding forces between hu4G4/hu4H12 and h B7-H3 were mainly on the order of 10^-10 M K_d,2.625E^-10 for hu4G4 and 7.543E^-10 for hu4H12,indicating that the humanized antibody retained the specificity and affinity of the original antibody.（3）Antibody was radiolabeled with ¹²⁵I and⁸⁹Zr at a radiolabeling yield of more than 95%,and the radiochemical purities（RCPs）of all were>99%.The stability of ¹²⁵I/⁸⁹Zr-（hu4G4,hu4H12,Ig G）in solvent buffer at room temperature and in human serum at 37°C were greater than 90%for 168 h in vitro.（4）¹²⁵I-SPECT and ⁸⁹Zr-PET imaging results were basically consistent,both showed that hu4G4had higher tumor targeting and lower liver uptake in GBM mice model compared with hu4H12 and Ig G.It is suggested that hu4G4 has a better advantage as a candidate antibody for the development of tumor radioimmunotherapy drugs targeted on B7-H3.2.Characterization of ¹³¹I-hu4G4 humanized antibody in vitro:（1）When the ratio of ¹³¹I activity（GBq）to B7-H3 antibody（μmol）was 55.5 GBq/μmol,the stability of ¹³¹I-hu4G4 in PBS and human serum was best in vitro.（2）Performance of ¹³¹I-hu4G4 antibody in vitro:（1）The radioactive uptake of ¹³¹I-hu4G4 in U87 cells at different time points showed that cell uptake increased gradually with the incubation time increased.When the incubation time exceeded 4 h,the uptake value of U87 cells was basically unchanged,about11.52±0.12%.（2）After the same radioactive dose of ¹³¹I-hu4G4 antibody was incubated with the U87 cells for 4 h,the cell radioactive uptake rate had a strong positive correlation with the number of incubated U87 cells,and the linear relationship was Y=5.209E^-5X+0.2397,R² was 0.9900.（3）With the increase of radioactive dose of ¹³¹I-hu4G4,the cell uptake decreased gradually and reached a plateau after incubation（>370 KBq）.（4）The¹³¹I-hu4G4 antibody could be internalized by U87 cells after incubation,and the cell internalization rate reached the maximum 22.99±1.76%after incubation for 1 h.（5）The competitive binding experiment showed that the K_d value of ¹³¹I-hu4G4 was 0.99±0.07 n M,the B_max was 741.26 pmol/mg,and the saturation concentration of binding receptor and ligand was about 143 ng with 10⁵ cells.（4）Pharmacodynamic evaluation of ¹³¹I-hu4G4antibody showed in vitro that:（1）¹³¹I-hu4G4 significantly inhibited the proliferation of U87cells（1×10⁴ cells/m L）,IC50=18.75 KBq/m L;（2）Cell cloning and cell migration experiments showed that ¹³¹I-hu4G4 could significantly inhibit the formation of cell cloning and the migration of U87 cells compared with other groups.（3）Analysis of cell cycle analysis results:¹³¹I-hu4G4 blocked the G2/M phase of U87 cells to inhibit cell proliferation.（4）Hoechst staining showed that the ¹³¹I-hu4G4 group could further lead to apoptosis of U87 cells by inducing nuclear shrinkage,thus inhibiting cell proliferation and reducing cell viability.（5）Analysis of immunogenic death:¹³¹I-hu4G4 group treated U87 cells showed significantly increased CRT expression,and at the same time,HMGB1 was released the most.In conclusion,in vitro experiments confirmed that the novel humanized antibody hu4G4 targeting B7-H3 labeled by ¹³¹I has good internalization performance,tumor targeting and specificity.At the same time,the results showed that ¹³¹I-hu4G4 could significantly inhibit the proliferation and migration of U87 cells,block the G2/M phase of U87 cells,and promote cell apoptosis and cell immunogenic death.3.The anti-tumor efficacy of ¹³¹I-hu4G4 in U87-xenografted model:（1）Antibody dose escalation:（1）The antibody dose escalation experiment:（1）The biodistribution of¹³¹I-hu4G4 in different total antibody levels（0.05 mg/kg,0.50 mg/kg,1.75 mg/kg,7 mg/kg,28 mg/kg）showed that the radioactive uptake of ¹³¹I-hu4G4 in each group was mainly concentrated in the tissues with rich blood supply after administration for 2 h,such as heart,liver,spleen,lung and kidney,followed by tumors,thyroid.Subsequently,with the extension of time,the radioactive uptake in tumor tissues gradually increased,while that in other tissues showed a trend of gradual decline.At the same time,with the increase of hu4G4 dose,the tissues uptake decreased and remained unchanged in vitro.For example,120 h after administration,the radioactivity uptake of tumor tissues in each dose group was 18.27±4.75%ID/g,11.74±1.88%ID/g,9.87±1.93%ID/g,6.60±0.97%ID/g and 6.60±1.00%ID/g,respectively.（2）According to the tumor tissue uptake values corresponding to the five doses at 120 h,the competitive binding curve between the tumor specific uptake and dose was fitted by the non-linear fitting method,and the IC50 was 0.56 mg/kg.（3）The pharmacokinetic results showed that T_（1/2）of the five dose groups in mice was basically the same with a significant difference.The AUC_（0-180h）of 0.05 mg/kg ¹³¹I-hu4G4（1335.00±142.70%ID/g*h）was significantly higher than that in the other four groups,p<0.001.（4）PD results showed that with the increase of the radioactive dose（0.37 MBq,3.7 MBq,11.1MBq）of ¹³¹I-hu4G4 antibody levels（0.05 mg/kg,0.50 mg/kg,1.75 mg/kg）,tumor inhibition was also increased.For example,T/C%in the three groups was 116.09±63.85,126.59±65.96,80.56±21.23,respectively,after 15 days of administration.For ¹³¹I-hu4G4 groups of1.75 mg/kg,7 mg/kg,and 28 mg/kg,with the increase of hu4G4 dose,although the conjugate radioactivity dose was basically the same.However,T/C%in the 1.75mg/kg ¹³¹I-hu4G4group was lower than that in the other two groups after 15 days administration,showing better antitumor efficacy,indicating that the same radioactivity dose of ¹³¹I-hu4G4 has more obvious anti-tumor effect.（5）The efficacy and safety evaluation of 1.5 mg/kg 11.1 MBq showed that the T/C%of ¹³¹I-hu4G4 group was 40%,and lower than other groups after 21days of administration.H&E staining results showed that there was no abnormal tissue morphology in the heart,liver,spleen,lung and kidney before and after treatment,and there was no significant change in body weight during treatment,indicating that ¹³¹I-hu4G4 had good therapeutic effect,high safety and no obvious toxicity.4.The antitumor effects of ¹³¹I-hu4G4 on intracranial injection of in situ GBM models and its effects on immune microenvironment:（1）GL261-flag-CD276 cells were constructed and detected by Western Blot.The results showed that GL261-flag-CD276 cells were highly expressed CD276 protein,while GL261-flag-vector and GL261-wt were not expressed.This indicated that GL261-flag-CD276 cells were successfully constructed.（2）Biodistribution experiment in situ glioma model:¹³¹I-hu4G4 group mainly concentrated in the brain,and the total radioactive uptake of brain（including tumor）was 9.23±5.24%ID/g,followed by liver（1.57±0.19%ID/g）,spleen（1.41±0.43%ID/g）,kidney（0.92±0.28%ID/g）,thyroid（0.54±0.68%ID/g）and lung（0.26±0.22%ID/g）.The Na¹³¹I group had virtually no radioactive uptake in all tissues.（3）Blood pharmacokinetics in situ glioma model showed that compared with Na¹³¹I group,¹³¹I-hu4G4 group had rarely entered blood after administration.At 1 h after the first administration,the blood uptake values of ¹³¹I-hu4G4 group were 5.21±2.82%ID/g,respectively,significantly lower than those of Na¹³¹I group（24.82±7.91%ID/g）（p<0.001）,indicating that the ¹³¹I-hu4G4 group was less likely to enter the blood after administration,and probably concentrated mainly in the brain tissue.（4）The antitumor efficacy evaluation of ¹³¹I-hu4G4 in situ GBM model:¹³¹I-hu4G4 group significantly reduced tumor load and had obvious tumor inhibition effect on Day 6 after the first administration.（5）Morphological analysis,immunogenic death analysis and the changes of immune cells in the immune microenvironment of mouse brain glioma by flow cytometry and multiple immunofluorescences showed that the intracranial tumors in each group were intact,and no necrosis occurred.¹³¹I-hu4G4 group could significantly induce HMGB1 release and CRT expression in intracranial tumor cells of mice on the 6th day after the first administration.Moreover,flow cytometry and multiple immunofluorescence results showed that ¹³¹I-hu4G4 treatment not only significantly promoted the increase of CD8⁺T cell and CD4⁺T cell infiltration,but also promoted the polarization of M2 macrophages to M1 macrophages.Conclusions:1.Hu4G4,the self-developed humanized antibody targeting B7-H3,has good tumor targeting and specificity,and has low non-specific concentration in normal tissues,especially in the liver.It is suggested that the hu4G4 antibody may be used as a candidate antibody for the development of tumor radioimmunotherapy drugs.2.¹³¹I-hu4G4 showed excellent binding specificity,affinity and internalization for B7-H3.¹³¹I-hu4G4 can significantly inhibit the proliferation and migration of U87 cells,arrest the G2/M phase of the cell cycle,and promote cell apoptosis and cell immunogenic death.3.The antitumor effects on subcutaneous and in situ glioma models showed that ¹³¹I-hu4G4 showed a good therapeutic effect,good safety and no obvious toxicity.4.¹³¹I-hu4G4 can improve the immunogenicity of cells,reshape the microenvironment of tumor tissue,promote the polarization of M2 macrophages to M1 macrophages and the infiltration of CD8⁺T cells,and thus produce synergistic anti-tumor effects.