A phase I trial of idiotypic vaccination with HMFG1 in ovarian cancer

Introduction: An extended phase I trial was conducted in a total of 26 patients with ovarian cancer. The objectives were to assess the safety and tolerability of idiotypic vaccination using the murine monoclonal antibody HMFG1 (anti-MUC1), and to develop robust assays to monitor humoral immune responses generated against either the antibody or MUC1. Material and methods: All patients had undergone standard debulking surgery (where appropriate) and at least one regimen of platinum-based chemotherapy. Eligibility criteria included: (a) residual disease at the end of chemotherapy, (b) relapsed disease, and (c) pathologically confirmed second complete remission following salvage chemotherapy. Patients received a priming dose of 25 mg of HMFG1 either intravenously or intraperitoneally, followed by up to six intradermal doses of HMFG1 in 10% Alhydrogel at intervals of 1 month. The three dose levels were 0.5 mg, 1 mg and 5 mg. We devised modifications of published protocols for the measurement of anti-idiotypic and anti-MUC1 antibody responses and also extended the use of the IAsys resonant mirror biosensor to measure the kinetics of the idiotypic network response in these patients. Results: There were no serious adverse events at any dose level. The trial confirmed that all doses could be administered safely with minimal toxicity. No clinical responses were seen in patients with evaluable disease. ELISA for anti-idiotypic antibodies (Ab2) showed significant levels in patients who completed the protocol. There were no significant differences in the levels of Ab2 generated by the different doses of antibody. These results were confirmed by biosensor assays for Ab2, which also showed affinity maturation of the Ab2 response as patients progressed through the vaccination protocol. Biosensor assays also demonstrated no difference in the affinity of Ab2 generated by different booster doses of HMFG1. ELISA for anti-MUC1 antibodies showed less consistent results, with very small but statistically significant rises in anti-MUC1 signals seen in 38% of patients who completed the vaccination regimen. Discussion: The clinical endpoints of safety and tolerability were met. The assays developed for this project have shown reproducibility and may provide surrogate endpoints to assess vaccination for future trials. The use of similar biosensors may be of particular relevance for monitoring of humoral immune responses in other vaccine trials. The low levels of anti-MUC1 antibodies generated may correspond with the lack of clinical efficacy in the few patients with evaluable disease.     

A defucosylated anti-PD-L1 monoclonal antibody 13-mG2a-f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. A strong correlation has been reported between PD-L1 expression in tumor cells and negative prognosis in cancer patients. Previously, we established an anti-PD-L1 monoclonal antibody (mAb), L₁Mab-13 (IgG₁, kappa), by immunizing mice with PD-L1-overexpressing CHO-K1 cells. L₁Mab-13 specifically reacts with endogenous PD-L1 in lung cancer cell lines in flow cytometry and Western blot applications, and stains a plasma membrane-like pattern in lung cancer tissues via immunohistochemical analysis. In this study, we investigated whether L₁Mab-13 reacts with oral cancer cell lines and exerts antitumor activities. Because L₁Mab-13 lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), we first converted the subclass of L₁Mab-13 from IgG₁ into IgG_2a (13-mG_2a), and further produced a defucosylated version (13-mG_2a-f) using FUT8-deficient ExpiCHO-S (BINDS-09) cells. Defucosylation of 13-mG_2a-f was confirmed using fucose-binding lectins, such as Aleuria aurantia and Pholiota squarrosa lectins. The dissociation constants (K_D) for 13-mG_2a-f in SAS and HSC-2 oral cancer cells were determined via flow cytometry to be 2.8 × 10^?9 M and 4.8 × 10^?9 M, respectively, indicating that 13-mG_2a-f possesses extremely high binding affinity. In vitro analysis demonstrated that 13-mG_2a-f showed moderate ADCC and CDC activities against SAS and HSC-2 oral cancer cells. In vivo analysis revealed that 13-mG_2a-f significantly reduced tumor development in SAS and HSC-2 xenografts in comparison to control mouse IgG, even after injection seven days post-tumor inoculation. Taken together, these data demonstrate that treatment with 13-mG_2a-f may represent a useful therapy for patients with PD-L1-expressing oral cancers.     

Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4

Programmed cell death-ligand 1 (PD-L1), which is a ligand of programmed cell death-1 (PD-1), is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1) expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb), L₁Mab-4 (IgG_2b, kappa), using cell-based immunization and screening (CBIS) method and investigated hPD-L1 expression in oral cancers. L₁Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4) in flow cytometry and stained oral cancers in a membrane-staining pattern. L₁Mab-4 stained 106/150 (70.7%) of oral squamous cell carcinomas, indicating the very high sensitivity of L₁Mab-4. These results indicate that L₁Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers.     

Application of VP1 protein to develop monoclonal antibody against foot-and-mouth disease virus Asia1 type

In order to develop an anti-FMDV Asia1 type monoclonal antibody (mAb), BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5E1 and 5E2, were then further optimized. The result indicated that prepared anti-FMDV Asia1 mAbs had no cross-reactivity with Swine vesicular disease (SVD) and FMDV O, A and C type antigen. Their titers in abdomen liquor were 1:5×10⁶, 1:2×10⁶ and 1:5×10⁶, respectively. 1B8 was found to be of IgG₁ subtype, 5E1 and 5E2 belonged to IgG_2b subtype. In this study, the prepared mAbs are specific for detecting FMDV type Asia1, and is potentially useful for pen-side diagnosis. Foudation items: The National high Technology Research and Development Program of China (No.2006AA10A204); The National science & Technology Pillar Program (No. 2006BAD06A17)     

Reactivity of an anti-(human gastric carcinoma) monoclonal antibody with core-related peptides of gastrointestinal mucin

Summary A murine anti-(human gastric carcinoma) monoclonal antibody, GL-013 (IgG1), which reacts with a high-molecular-mass glycoprotein from colorectal tumour tissue [Yang and Price (1989) Anticancer Res 9: 1707], was examined for reactivity against a panel of purified and partially purified antigens associated with tumours of the gastrointestinal tract. These included carcinoembryonic antigen (CEA), normal cross-reacting antigen, Y-hapten glycoproteins, and perchloric acid extracts and glycolipid preparations from colorectal tumours. While the GL-013 antibody failed to bind to these antigens, it was found to react strongly with synthetic peptides with sequences based upon that reported for the protein core of a human gastrointestinal mucin [Barnd et al. (1989) Proc Natl Acad Sci USA 86: 7159; Gum et al. (1989) J Biol Chem 264: 6480]. In control tests, a series of other anti-(colorectal tumour) antibodies (IgG1 and IgG3), with broad reactivity towards gastrointestinal carcinomas, as well as an anti-CEA antibody, (IgG1) failed to react with the synthetic peptides. It is concluded that the anti-(gastric carcinoma) monoclonal antibody GL-013 binds to a threonine-rich peptide epitope expressed within the protein core of gastrointestinal mucins. Present address: Cancer Research Institute, China Medical University, Shenyang, Liaoning, People's Republic of China     

Characterization and application of a murine monoclonal antibody that reacts specifically with the serogroup D1Salmonella

A murine hybridoma cell line that produces monoclonal antibody (mAb) against the serogroup D1 Salmonella lipopolysaccharide (LPS) antigen was established. The trisaccharide tyvelose alpha 1----3 mannose alpha 1----4 rhamnose was shown to be involved in the reactive epitope of the mAb since this mAb reacted strongly with strains of serogroup D1 Salmonella but not with Salmonella strains from the O serogroups of A, B, and D2, and sodium meta-periodate was found to destroy the reactivity of the serogroup D1 O-antigen with the mAb. As such this mAb was found to be a useful serotyping reagent for the identification of serogroup D1 Salmonella, and for the differentiation of strains of serogroups D1 and D2 Salmonella which have identical flagellar H antigens.     

Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody

Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb specifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody recognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis demonstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses.     

Construction and characterization of a high-affinity humanized SM5-1 monoclonal antibody

SM5-1 is a mouse monoclonal antibody which has a high specificity for melanoma, hepatocellular carcinoma, and breast cancer, making it a promising candidate for cancer targeting therapy. We have therefore attempted to construct a humanized antibody of SM5-1 to minimize its immunogenicity for potential clinical use. Using a molecular model of SM5-1 built by computer-assisted homology modeling, framework region (FR) residues of potential importance to the antigen binding were identified. Then, a humanized version of SM5-1 was generated by transferring these mouse key FR residues onto a human framework that was selected based on homology to the mouse framework, together with the mouse complementarity-determining region (CDR) residues. This humanized antibody retained only six murine residues outside of the CDRs but was shown to possess affinity and specificity comparable to that of the parental antibody, suggesting that it might have the potential to be developed for future clinical use.     

Increased tumor cell reactivity and complement-dependent cytotoxicity with mixtures of monoclonal antibodies against different gangliosides

Melanomas and other cancers of neuroectodermal origin express multiple cell-surface gangliosides in patterns that vary significantly even within the same tumor type. Monoclonal antibodies (mAb) against four of these gangliosides (GM2, GD2, 9-O-acetyl-GD3 and GD3) were tested alone and in combination on 14 tumor cell lines (7 melanomas, 3 neuroblastomas, 3 sarcomas and 1 astrocytoma) using flow cytometry and complement-dependent cytotoxicity (CDC) assays. Increased tumor cell recognition and CDC resulting from the combination of three or four mAb were found in 14/14 tested cell lines, and this was most striking when each mAb was used at suboptimal concentration. At these concentrations, the average mean fluorescence intensity of the 14 cell lines with individual mAb was between 3.0 and 6.8 and increased to 10.8 and 18.8 with the three- and four-mAb mixtures. The average percentage CDC-specific release with individual mAb was 2.0%–8.3%, and 12.3% and 16.6% with the three- and four-mAb combinations. The number of cell lines showing significant mean fluorescence intensity and CDC increased from 2–8/14 with single mAb to 13–14/14 with the mixtures of three or four mAb. Our experimental results support the rationale for active immunization with a polyvalent ganglioside vaccine or passive therapy with a combination of mAb to different gangliosides in patients with tumors of neuroectodermal origin. In addition, our studies have demonstrated that 9-O-acetyl-GD3 is a surprisingly effective target for immune attack, although it is a minor constituent of these cells.     

A monoclonal antibody reacting with the cell envelope of spirochaetes isolated from cases of intestinal spirochaetosis in pigs and humans

Abstract A monoclonal antibody (mAb) designed BJL/AC1 was prepared against the cell envelope of an intestinal spirochaete (strain 3295) that was isolated from a pig with intestinal spirochaetosis. The mAb reacted with a band of approximately 29 kDa in cell envelope preparations from 13 porcine and 11 human spirochaetes isolated from cases of intestinal spirochaetosis, but did not react with preparations made from a range of other intestinal spirochaetes. Immunogold labelling demonstrated that the reactive epitope was located on the cell envelope of the strains causing intestinal spirochaetosis. The mAb was used in an indirect immunofluorescence test to detect spirochaetes in the faeces of pigs with experimentally induced intestinal spirochaetosis. The mAb should prove to be a useful reagent for detection and identification of spirochaetes that are specifically associated with intestinal spirochaetosis.     

Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus

The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (K_d, ∼10⁻¹¹ M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R² = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R² = 0.834). These results suggest that the BV–KinExA method described here provides a suitably accurate K_d evaluation of mAbs against proteins on the cell surface.     

A new monoclonal antibody, 4-1a,that binds to the amino terminus of human lipoprotein lipase

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5–25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL–GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.     

Non-uniform staining of myofibril a bands by a monoclonal antibody to skeletal muscle myosin S1 heavy chain

A monoclonal antibody specific for the S1 fragment of skeletal muscle myosin has been identified. The antibody does not inhibit actin-activated Mg2+-ATPase or K+-EDTA-activated ATPase of myosin, indicating that it is not related to the portion of the S1 which carries the ATPase activity. In the absence of relaxing medium, antibody binding to the myosin filament is restricted to narrow regions on each side of the bare zone region of the filament, and to a narrow region at the tapered ends of the filament. This restricted antibody binding is not altered by the attachment of the myosin cross-bridges to the actin filaments. In the presence of relaxing medium, antibody binding occurs along the entire length of the cross-bridge-bearing region of the filament. The restricted binding to only small regions of the filament in the absence of relaxing medium suggests that the molecular packing of the myosin in different portions of the filament may be different, resulting in differences in the availability of the antigenic site on the S1 for antibody binding. The change in availability of the antigenic sites along the filament in the presence of relaxing medium may reflect a perturbation in the molecular packing of the filament, or a conformational change resulting from the binding of MgATP, both of which could affect the availability of the antigenic sites on the S1 for antibody.     

Establishment of an immunoscreening system using recombinant VP1 protein for the isolation of a monoclonal antibody that blocks JC virus infection

Polyomavirus JC (JCV) infection causes the fatal human demyelinating disease, progressive multifocal leukoencephalopathy. Although the initial interaction of JCV with host cells occurs through direct binding of the major viral capsid protein (VP1) with cell-surface molecules possessing sialic acid, these molecules have not yet been identified. In order to isolate monoclonal antibodies which inhibit attachment of JCV, we established an immunoscreening system using virus-like particles consisting of the VP1. Using this system, among monoclonal antibodies against the cell membrane fraction from JCV-permissive human neuroblastoma IMR-32 cells, we isolated a monoclonal antibody designated as 24D2 that specifically inhibited attachment and infection of JCV to IMR-32 cells. The antibody 24D2 recognized a single molecule of around 60 kDa in molecular weight in the IMR-32 membrane fraction. Immunohistochemical staining with 24D2 demonstrated immunoreactivity in the cell membrane of JCV-permissive cell lines and glial cells of the human brain. These results suggested that the molecule recognized by 24D2 plays a role in JCV infection, and that it might participate as a receptor or a co-receptor in JCV attachment and entry into the cells.     

Treatment of human lung carcinoma xenografts with a combination of 131I-labelled monoclonal antibody Po66 and doxorubicin

 Po66, a mouse monoclonal antibody, is directed against an intracytoplasmic antigen present in human lung squamous cell carcinoma cells. In previous work it was found that the co-administration of ¹²⁵I-radiolabelled Po66 and doxorubicin strongly enhanced the uptake of radioactivity by the tumour. The present-work was designed to evaluate, in a tumour-bearing mouse model of lung carcinoma, the ability of ¹³¹I-labelled Po66 to retard tumour growth when injected alone, or in combination with doxorubicin (8 mg kg ^– 1 at 1-week intervals). A single dose of 550 μCi ¹³¹I-Po66 alone had no effect on tumour growth, whereas three fractionated doses of 250 μCi ¹³¹I-Po66 decreased it over two doubling times from 14.5±1.5 days for untreated control mice to 24.8±2.7 days. Mice treated with doxorubicin alone had a double tumour doubling time of 22.6±4.9 days, compared to 35.2±2.9 days (1.55-fold increase) in mice treated with doxorubicin and a single dose of 550 μ Ci ¹³¹I-Po66. Doxorubicin combined with three fractionated doses of 250 μCi ¹³¹I-Po66 provoked a twofold decrease in tumour growth compared to mice treated with doxorubicin alone. The administration of fractionated doses of ¹³¹I-Po66 simultaneously with doxorubicin resulted in a highly delayed mortality, which was not observed when ¹³¹I-Po66 was administered after doxorubicin. Thus, in a non-small-cell lung tumour model, a ¹³¹I-radiolabelled monoclonal antibody, directed against an intracellular antigen, significantly potentiated the effect of chemotherapy. Such a therapeutic approach could be used as an adjuvant therapy and improve the effect of chemotherapy on distant small metastases. Received: 20 June 1996 / Accepted: 3 October 1996     

Protruding disordered loop of gC1qR is specifically exposed and related to antiapoptotic property in germ cell lineage

We established a monoclonal antibody (MAb), 5G9, with the use of a fixed seminoma tissue from an archival paraffin-embedded specimen, as an immunogen. Without antigen retrieval, positive 5G9-immunohistochemical staining was confined mostly to primordial germ cells, spermatogonia and various germ cell tumors. 5G9 recognized a mitochondrial 32-kD protein with an isoelectric point of pH 4.2, identified as a multifunctional ubiquitous protein, receptor for globular head of C1q (gC1qR), whose epitope was mapped in a disordered loop connecting the β3 and the β4 strands. Reflecting the ubiquitous distribution of gC1qR, with antigen retrieval, 5G9 was found reactive to a wide range of normal and tumor tissues. Since several co-precipitated and phosphorylated bands were observed in various human cell lines but not in germ cell tumor cell lines by in vitro phosphorylation assay, we speculate that the epitope of gC1qR is specifically unmasked in the germ cell lineage. By reducing gC1qR by siRNA, a significant increase was observed in the number of apoptotic cells in ITO-II and TCam-2 cell lines, but to a lesser extent in the Colo201 colon cancer cell line, showing an antiapoptotic property of gC1qR in the germ cells. Since protein–protein interaction is partially preserved by fixation, archival paraffin-embedded specimens can be a valuable source of immunogens for generating monoclonal antibodies (MAbs) that recognize tissue-specific protein conformation.     

Characterization of a monoclonal antibody reactive with a glycolipid antigen expressed by tumorigenic and certain immortalized,non-tumorigenic rat esophageal epithelial cell lines

A monoclonal antibody (mAb 5G) was produced against a tumorigenic rat esophageal epithelial cell line, designated B2T. Using an enzyme-linked immunosorbent assay, immunofluorescence assay (IFA), thin-layer chromatography (TLC) and immunoperoxidase staining, it was found that mAb 5G reacted specifically with a glycolipid antigen expressed by three tumorigenic rat esophageal epithelial cell lines, and two out of the three nontumorigenic, immortalized rat esophageal epithelial cell lines tested; but did not react with primary cultures of normal rat esophageal epithelial cells or fibroblasts. mAb 5G did not bind to rat respiratory tract carcinoma cell lines, to immortalized rat tracheal epithelial cell lines, or to primary cultures of normal rat tracheal epithelial cells. In addition, mAb 5G did not react with any of the human or mouse cell lines tested. In IFA experiments, mAb 5G stained imprints prepared from in vivo propagated B2T tumor tissues, but did not react with normal rat esophageal, tracheal, lung, liver, and kidney tissues. The antigen was identified by TLC as a neutral glycolipid, consisting of two bands, withR _F = 0.45 and 0.41, which migrated in proximity to the ceramide trihexoside standard on TLC plates. Densitometric scanning of the antigen bands indicated that the tumorigenic rat esophageal cell lines possessed 50%–90% more mAb-5G-reactive antigen than the nontumorigenic esophageal cell lines. The results show that mAb 5G reacts specifically with a glycolipid antigen expressed by tumorigenic and certain non-tumorigenic, immortalized rat esophageal epithelial cell lines that might be at the late stages of transformation and early malignancy.     

Phase I trial of chimeric (human-mouse) monoclonal antibody L6 in patients with non-small-cell lung,colon, and breast cancer

We report a single institution phase I trial of chimeric (mouse-human) monoclonal antibody (chL6) directed against a tumor-associated cell surface antigen expressed in non-small cell lung, colon, and breast cancer. The results of the study were contrasted with a previous trial of murine L6. ChL6 was administered intravenously to 18 patients with advanced cancer as a single, 4–16 infusion in doses ranging from 350 mg/m² to 700 mg/m². One patient received four weekly doses of 350 mg/m². Patients were followed for side effects, localization of antibody to tumor cells, pharmacokinetics and the development of antibodies against chL6. Side effects associated with treatment were chills, fever, and nausea, which lasted 24–48 hours. Platelet count and absolute leukocyte count fell immediately after treatment, but returned to pretreatment levels by day 7. Localization of chL6 to tumor cells in vivo was seen at 350 mg/m² and saturation at 700 mg/m² and 350 mg/m² per week×4. The pharmacokinetics of this antibody appeared similar to its murine analogue. Human antibodies against chL6 were detected in only 4 of 18 patients. These antibodies were directed against murine variable regent and their titers were lower than those occurring in most patients who received murine L6 in an earlier trial. No tumor reductions were seen. Chimeric L6 appears to be a suitable antibody for delivering anti-tumor agents because of its low immunogenicity and favorable in vivo tumor binding characteristics.     

Generation,characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (K_D) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.     

运用氨基酸分析法和DOE法优化抗PD-1单克隆抗体生产用培养基

本研究采用氨基酸分析法结合DOE设计法优化并获得高表达抗PD-1单克隆抗体生产用基础和补料培养基。通过对市售多种基础和补料培养基进行筛选,获得细胞生长状况较优的基础培养基和抗体表达较高的补料培养基,利用氨基酸分析法检测较优基础培养基和补料培养基中氨基酸消耗情况,确定影响细胞生长和抗体表达的关键氨基酸种类,利用DOE分析软件设计分别在较优基础和补料培养基中添加不同浓度的氨基酸种类及浓度,根据细胞生长及抗体表达,优化得到抗PD-1单克隆抗体的基础和补料培养基组合。最终优化后基础培养基配方为:Hycell CHO培养基中添加1.04 mmol/L L-天冬酰胺和0.76 mmol/L L-谷氨酰胺。最终优化后补料培养基配方为:OPM CHOCD Feed1补料培养基中添加38.7 mmol/L L-组氨酸,75.0 mmol/L L-酪氨酸,64.0 mmol/L L-丝氨酸,49.2 mmol/L L-谷氨酰胺和18.7 mmol/L L-半胱氨酸。经过3 L反应器培养验证,优化后的培养基比未优化时,最大活细胞密度(PVCD)提高了62.7%,抗PD-1单克隆抗体表达量提高了71.5%,且活性无明显差异。