Biodistribution And Radioimmunoimage Of Iodine-125-Labeled Anti-human Mutant CD40 Monoclonal Antibody 5H6 In Nude Mice Bearing An Ovary Tumor

In 1978,it was first reported by Goldenberg that RII(radioimmunoimaging)of tumor was done successfully with antibody of CEA(carcinoembryonic antigen).In 1980,Van Nagel firstly used polyclone antibody of CEA to detect ovarian cancer by RII.From then on,RII technique was introduced into clinic and broke a new way of radio diagnosis of ovarian cancer.The prognosis of ovarian cancer was very poor.Case fatality(around 30%) was the first among gynecological malignant tumors.But early diagnosis of ovarian cancer was very difficult.The reason for this dilemma was versatile:ovary was deep in pelvic cavity and could not be seen directly,the early ovarian cancer patients usually were absent of symptoms.RII has provided efficient tools for early diagnosis of ovarian cancer.CD40, a member of tumor necrosis factor receptor(TNFR)super family,is a 48KD type I transmembrane glycoprotein.Although three anti-CD40 monoclonal antibodies have entered preclinical trials,further studies are needed to warrant their safety due to wide expression of CD40 in normal tissues.CD40 mutant(CD40mu),a C-to-A transition (H78Q),is discovered in some cell lines and freshly isolated tumor cells.This amino acid located in the 2nd cysteine rich extramembrane region of CD40,a region very important to ligate with CD40L(CD154).Transfected results confirmed this mutant has changed partial epitope of CD40.However,the expression pattern of CD40mu and its biological function remains obscure.5H6 is a new antibody against CD40mu produced by our institute,and its epitope is quite different from antibody against normal human CD40.It can recognize CD40mu expressed by kinds of tumor cell lines(HO8910,K562)and fresh tumor tissues, can not recognize CD40 expressed by vascular endothelial cell line(Eahy 926)and normal tissues.This specificity has its potential application value. 1.Preparation of iodine-125 labeled anti-human CD40mu monoclonal antibody 5H6 and its binding properties in ovary cell line HO8910 in vitroThe mAb 5H6 was labeled with Na ¹²⁵I using chloramine-T method.The labeling efficiency and radiochemical purity of ¹²⁵I-5H6 were measured by paper chromatography. The stability of ¹²⁵I-5H6 in vitro was monitored by trichloroacetic acid(TCA)precipitation. The dissociation constant(K_d)of ¹²⁵I-5H6 from HO8910 cells and the numbers of maximum binding sites(B_max)were obtained by Scatchard analysis.Immunoreactivity of ¹²⁵I-5H6 to HO8910 cells was determined by "Lindmo" assay and its improved method. Internalization and retention of ¹²⁵I-5H6 by HO8910 cells were studied by cell binding experiments.The labeling efficiency of ¹²⁵I-5H6 was(85.4±5.2)%and its radiochemical purity was(99.2+0.5)%.Radiochemical purity of ¹²⁵I-5H6 was(95.3±0.8)%after 24 hours in 37℃plasma.Immunoreactivity of ¹²⁵I-5H6 was(38.6±5.4)%.At 4℃,K_d of ¹²⁵I-5H6 to HO8910 cells was 0.711±0.06 nmol/L and B_maxwas(2.17±0.08)×10⁵ sites/cell.The high affinity of ¹²⁵I-5H6 with HO8910 cells would benefit further animal study in vivo.2.Biodistribution and radioimmunoimage of ¹²⁵I-SH6 in nude mice bearing an ovary tumorAnimal models were established using 4-6 weeks female BALB/c nude mice.In order to block thyroid,1%KI 200μL/d was intragastric administration three days before the test till the end.30～45μCi/200μL ¹²⁵I-5H6 was intravenous injection.At 1,3,6,24,48,96h tissues of blood,heart,lung,liver,spleen,kidney,pancreas,stomach, intestine,muscle,bone,skin,brain,thyroid and tumor were weighed and detected its radioactivity count.%ID/g(%injected dose/gram)were calculated,and analysis of distribution was done.300～400μCi/200μL ¹²⁵I-5H6 was intravenous injection and radioimmunoimages was obtained by SPECT.T/NT(tumor/none tumor)was analyzed by ROI(region of interest)technique.Radioisotope uptake peaked at 48 hours for tumors, %ID/g was(16.29±1.17).¹²⁵I-5H6 can target the tumor with high specificity in vivo.Via planar gamma camera imaging,¹²⁵I-5H6 could provide good radioimmunoimages.In conclusion,the mAb 5H6 was labeled with Na ¹²⁵I using chloramine-T method. ¹²⁵I-5H6 possessed good labeling efficiency and perfect radiochemical purity.¹²⁵I-5H6 had suitable stability in vitro and its immunoreactivity was not low.The affinity of ¹²⁵I-5H6 with HO8910 cells was high in vitro and ¹²⁵I-5H6 could target the tumor with high specificity in vivo.Via planar gamma camera imaging,¹²⁵I-5H6 could provide good radioimmunoimages.Our study had laid basic experimental foundation for radioimmage and target therapy using different radio isotope iodine(¹²³I,¹²⁵I,¹³¹I)labeled mAb 5H6. ¹²⁵I-labeled mAb 5H6 was promising and could provide a new antibody-based agent for radioimmunodetection and radioimmunotherapy of patients bearing HO8910 tumors.