A Human Anti-c-Met Fab Fragment Conjugated With Doxorubicin Inhibits Hepatocellular Carcinoma Growth In Vitro And In Vivo

Among primary liver cancers, hepatocellular carcinoma (HCC) represents themajor histological subtype, accounting for70%to85%of the total liver cancerburden worldwide. HCC is the fifth most common tumor worldwide, but due to itspoor prognosis, it ranks as the third most common cause of death from cancer. Theglobal incidence of HCC is greater than a million cases a year. Despite therapeuticadvances, the treatment to patients with HCC still falls short of affectivity orcredibility. In the last2decades, a new therapeutic modality directed against tumormolecular targets, defined as “targeted therapy,” has been developed rapidly.Targeted cancer therapy is promising to minimize the nonspecific toxicity and toimprove therapeutic efficiency compared to conventional chemotherapy.Antibody-targeted chemotherapy is designed for selective delivery of cytotoxic drugsto tumor cells by linking them to monoclonal antibodies, thereby enhancing antibodytherapeutic activity while minimizing the systemic effects of a cytotoxic drug. Severalmonoclonal antibodies (mAb) have established roles in cancer chemotherapy due totheir specificities for tumor-associated antigens and their manageable off-targettoxicities.Antibody drug conjugates (ADCs) are composed of three key elements that caninfluence the therapeutic index: antibody, linker, and drug. The choice of targetantigen (expression in cancer versus normal tissues) and the ability to internalize theantigen-antibody complex are crucial as they determine tumor cell–specific deliveryof cytotoxic drug. c-Met，the ligand of hepatocyte growth factor (HGF)，is involved inorgan regeneration, as demonstrated for liver and kidney, embryogenesis, hematopoiesis, muscle development, and in the regulation of migration and adhesionof normally activated B cells and monocytes. Furthermore, numerous studiesindicated the involvement of c-Met overexpression in malignant transformation andinvasiveness of malignant cells. c-Met was overexpression in hepatocellularcarcinoma tissues and cells and low or no expression in normal tissues. As a result, itcan be used as a target for antibody drug conjugate in HCC therapy.Doxorubicin (DOX) is an effective and widely used chemotherapeutic agent indifferent cancers. It may produce free radicals, leading to serious side effects,especially cardiomyopathy to congestive heart failure. Therefore, DOX is a preferredcandidate for drug targeting research to evaluate both effectiveness and toxicity.The aim of our study was preparing anti-c-Met Fab and conjugating it withdoxorubicin, bringing the drug target to the tumor tissue by virtue of the specificity ofthe antibody against c-Met on the surface of the cells. Consequently, the conjugatecan decrease the side effect of doxorubicin and increase the therapy effect of HCC.The research plan is as following:1. Constructing immunized human Fab antibody phage library2. Screening, expressing and purifying human anti-c-Met Fab antibody3. Conjugating doxorubicin with human anti-c-Met Fab antibody and identifying thecharacteristics of conjugation in vitro4. Observing the cytotoxicity of the conjugation on HCC cells in vitro5. Evaluating the anti-tumor effect of the conjugation on the mouse xenograft modelof HCC6. Observing the distribution and localization of the conjugation in the mousexenograft modelMethods1. An immunized phage-display Fab library was constructed from peripheral blood lymphocytes from40patients with HCC. The immunized antibody library was usedto select and produce antibodies that bound to c-Met protein of cell surface. Afterscreening, the clone with plasmid of pCOMB3X-MetFab showed highest aviditywith c-Met protein was sequenced and chosen for the next experiment.2. The plasmid pCOMB3X-MetFab was transformed into competent E.coli Top10F’,and the positive insert was identified by PCR amplification of the Fab fragmentfrom bacterial colony. After optimizing the parameters of both expression andpurification, large-scale anti-c-Met Fab was produced by the Protein L affinitychromatography.3. The MetFab and DOX were conjugated via chemical synthesis and confirmed byHPLC. The binding efficacy of the conjugate (MetFab-DOX) was assessed invarious cell lines that are either positive or negative for c-Met by FACS, ELISA andimmunofluorescence observation. The cellular distribution of free DOX andMetFab-DOX was observed by fluorescence microscopy.4. HCC cells with c-Met-positive expression and NIH3T3cells with c-Met-negativeexpression were treated with doxorubicin and MetFab-DOX. Cytotoxicity ofMetFab-DOX was analyzed by the MTT method in vitro and compared with that ofdoxorubicin.5. The mouse xenograft model of HCC was constructed and treated with doxorubicinand MetFab-DOX. The anti-tumor effect in vivo was evaluated and the side effectof each group was observed.6. The localization of conjugation was confirmed by immunofluorescence staining offrozen tumor tissue section from the mouse xenograft model of HCC in vitro.Furthermore, the localization of conjugation was confirmed by optical tumorimaging in vivo with cy5.5labeled MetFab. The tissue distribution of conjugationwas confirmed in a nude mouse xenograft model of HCC by spectrofluorometermehtod. The drug concentrations of doxorubicin in organs at different times werecompared between doxorubicin treated and MetFab-DOX treated mice. Results1. The immunized antibody library, with a diversity of2.0×109, was constructed andused to select and produce anti-Met Fab fragment. One clone, with plasmid ofpCOMB3X-MetFab showed highest avidity with c-Met protein was sequenced andchosen for the next experiment. The optimal Fab antibody production was carriedout by using the SB culture medium adding2%glucose before induction,25℃induction temperature, and1mmol/L IPTG. The expected protein was purified fromthe sonication supernatant with high purity by Protein L affinity chromatographywith adding350mmol/L NaCl in binding buffer and150mmol/L NaCl in elutionbuffer. The final yield was5mg per liter liquid of bacteria.2. The MetFab and DOX were conjugated via chemical synthesis and confirmed byHPLC. The drug release patterns of the conjugate were analyzed under pH7.2andpH4.0, respectively. The conjugation was stable at pH7.2, and the accumulationrelease rate was12.1%after96h and16.9%after240h. While the conjugation canrelease doxorubicin quickly at pH4.0, and the accumulation release rate was81.3%after96h.3. FACS result also demonstrated that the MetFab-DOX could specifically recognizeand bind c-Met protein in live cells with various c-Met expression levels, butcouldn’t bind NIH3T3cells with c-Met negative expression. Immunofluorescencemicroscopy observation and ELISA analysis showed that the affinity ofMetFab-DOX to the c-Met expression HCC cells was smaller compared withMetFab. The entry and partition routes of MetFab-DOX were distinct from that offree DOX. The MetFab-DOX conjugate was clearly distributed in the membrane,cytoplasm and perinuclear zone after incubation, while free doxorubicin quicklylocated into the nulei.4. MetFab-DOX only specifically bound to HCC cell lines expressing c-Met and hadcytotoxic effect on these cells, but not to the NIH3T3cells that does not expressc-Met to a substantial level. 5. Experiments in vivo confirmed that MetFab-DOX exerted anti-tumor effectsimilarly as free DOX. The tumor inhibition rate was65.4%in MetFab-DOXgroup, but significantly reduced the side effects of free DOX(P<0.05), such as theweight loss, the pathological changes in heart, lung and kidney.6. Frozen tumor tissue sectioning showed that MetFab-DOX could bind the tumortissue after24h injection into mice bearing HCC xenograft. Optical tumor imagingin vivo demonstrated that MetFab could target the tumor tissue. After48h ofinjection, the fluorescent signal of MetFab-Cy5.5was still strong in the tumor site.Doxorubicin concentrations detected by spectrofluorometer in the tumor tissue andplasma were higher in mice given MetFab-DOX than in those given free DOX,while the condition was verse in the heart and kidney.ConclusionAs a conclusion, a human anti-Met Fab was screened and conjugated withdoxorubicin successfully. The MetFab-DOX had cytotoxity effect on HCC cells withc-Met expression in vitro. Furthermore, MetFab-DOX effectivly targeted HCC cellswith c-Met expression in mice bearing HCC xenograft, had similar anti-tumor effectas doxorubicin in vivo, changed the distribution of doxorubicin in vivo and reducedthe side effects of doxorubicin effectively. MetFab-DOX may have therapeuticpotential for targeted treatent of HCC.