Characterization of a novel T cell-engaging bispecific antibody for elimination of L1CAM-positive tumors.

Neural cell adhesion molecule L1 (L1CAM) is a cell-surface glycoprotein involved in cancer occurrence and migration. Up to today, L1CAM-targeted therapy appeared limited efficacy in clinical trials although quite a few attempts by monoclonal antibody (mAb) or chimeric antigen receptor T-cell therapy (CAR-T) have been reported. Therefore, the development of new effective therapies targeting L1CAM is highly desirable. It has been demonstrated that T cell-engaging bispecific antibody (TCE) plays an effective role in cancer immunotherapy by redirecting the cytotoxic activity of CD3+ T cells to tumor cells, resulting in tumor cell death. In this study, we designed and characterized a novel bispecific antibody (CE7-TCE) based on the IgG-(L)-ScFv format, which targets L1CAM and CD3 simultaneously. In vitro, CE7-TCE induced specific killing of L1CAM-positive tumor cells through T cells. In vivo, CE7-TCE inhibited tumor growth in human peripheral blood mononuclear cell/tumor cell co-grafting models. To overcome the adaptive immune resistance (AIR) that impairs the efficacy of TCEs, we conducted a combination therapy of CE7-TCE with Pembrolizumab (anti-PD1 mAb), which enhanced the anti-tumor activity of CE7-TCE. Our results confirmed the feasibility of using L1CAM as a TCE target for the treatment of solid tumors and revealed the therapeutic potential of CE7-TCE combined with immune checkpoint inhibitors.

Accumulation of PSA-NCAM marks nascent neurodegeneration in the dorsal hippocampus after neonatal hypoxic-ischemic brain injury in mice.

Neonatal hypoxia-ischemia (nHI) disrupts hippocampal GABAergic development leading to memory deficits in mice. Polysialic-acid neural-cell adhesion molecule (PSA-NCAM) developmentally declines to trigger GABAergic maturation. We hypothesized that nHI changes PSA-NCAM abundance and cellular distribution, impairing GABAergic development, and marking nascent neurodegeneration. Cell degeneration, atrophy, and PSA-NCAM immunoreactivity (IR) were measured in CA1 of nHI-injured C57BL6 mice related to: (i) cellular subtype markers; (ii) GAD65/67 and synatophysin (SYP), pre-synaptic markers; (iii) phospho-Ser396Tau, cytoskeletal marker; and (iv) GAP43, axonalregeneration marker. PSA-NCAM IR was minimal in CA1 of shams at P11. After nHI, PSA-NCAM IR was increased in injured pyramidal cells (PCs), minimal in parvalbumin (PV)+INs, and absent in glia. PSA-NCAM IR correlated with injury severity and became prominent in perikaryal cytoplasm at P18. GAD65/67 and SYP IRs only weakly related to PSA-NCAM after nHI. Injured phospho-Ser396Tau+ PCs and PV+INs variably co-expressed PSA-NCAM at P40. While PCs with cytoplasmic marginalized PSA-NCAM had increased perisomatic GAP43, those with perikaryal cytoplasmic PSA-NCAM had minimal GAP43. PSA-NCAM increased in serum of nHI-injured mice. Increased PSA-NCAM is likely a generic acute response to nHI brain injury. PSA-NCAM aberrant cellular localization may aggravate neuronal degeneration. The significance of PSA-NCAM as a biomarker of recovery from nHI and nascent neurodegeneration needs further study.

Facile Impedimetric Analysis of Neuronal Exosome Markers in Parkinson's Disease Diagnostics.

The egress of α-synuclein in neuronally derived exosomes predates the clinical presentation of Parkinson's disease (PD), offering a means of developing a predictive or prognostic test. Here, we report the reagentless impedimetric assay of two internal exosome markers (α-synuclein and syntenin-1) from neuronal exosomes. Exosomes were efficiently extracted from patient sera using anti-L1CAM conjugated zwitterionic polymer-modified magnetic beads prior to lysis and analyzed by electrochemical impedance spectroscopy. The quantification of α-synuclein level across 40 clinical samples resolved statistically significant differences between PD patients and healthy controls (HC).

CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing.

BACKGROUND: Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. METHODS: CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. RESULTS: All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. CONCLUSION: Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

Development of a Theranostic Convergence Bioradiopharmaceutical for Immuno-PET Based Radioimmunotherapy of L1CAM in Cholangiocarcinoma Model.

PURPOSE: Cholangiocarcinoma is a malignancy of bile duct with a poor prognosis. Conventional chemotherapy and radiotherapy are generally ineffective, and surgical resection is the only curative treatment for cholangiocarcinoma. L1-cell adhesion molecule (L1CAM) has been known as a novel prognostic marker and therapeutic target for cholangiocarcinoma. This study aimed to evaluate the feasibility of immuno-PET imaging-based radioimmunotherapy using radiolabeled anti-L1CAM antibody in cholangiocarcinoma xenograft model. EXPERIMENTAL DESIGN: We prepared a theranostic convergence bioradiopharmaceutical using chimeric anti-L1CAM antibody (cA10-A3) conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator and labeled with 64Cu or 177Lu and evaluated the immuno-PET or SPECT/CT imaging and biodistribution with 64Cu-/177Lu-cA10-A3 in various cholangiocarcinoma xenograft models. Therapeutic efficacy and response monitoring were performed by 177Lu-cA10-A3 and 18F-FDG-PET, respectively, and immunohistochemistry was done by TUNEL and Ki-67. RESULTS: Radiolabeled cA10-A3 antibodies specifically recognized L1CAM in vitro, clearly visualized cholangiocarcinoma tumors in immuno-PET and SPECT/CT imaging, and differentiated the L1CAM expression level in cholangiocarcinoma xenograft models. 177Lu-cA10-A3 (12.95 MBq/100 µg) showed statistically significant reduction in tumor volumes (P < 0.05) and decreased glucose metabolism (P < 0.01). IHC analysis revealed 177Lu-cA10-A3 treatment increased TUNEL-positive and decreased Ki-67-positive cells, compared with saline, cA10-A3, or 177Lu-isotype. CONCLUSIONS: Anti-L1CAM immuno-PET imaging using 64Cu-cA10-A3 could be translated into the clinic for characterizing the pharmacokinetics and selecting appropriate patients for radioimmunotherapy. Radioimmunotherapy using 177Lu-cA10-A3 may provide survival benefit in L1CAM-expressing cholangiocarcinoma tumor. Theranostic convergence bioradiopharmaceutical strategy would be applied as imaging biomarker-based personalized medicine in L1CAM-expressing patients with cholangiocarcinoma.

Extracellular vesicles reveal abnormalities in neuronal iron metabolism in restless legs syndrome.

STUDY OBJECTIVES: Determine abnormalities in levels of iron-management proteins in neuronal origin-enriched extracellular vesicles (nEVs) in restless legs syndrome (RLS). METHODS: We used immunoprecipitation for neuronal marker L1CAM to isolate nEVs from the serum of 20 participants with RLS from a study including magnetic resonance imaging (MRI) determinations of iron deposition in the substantia nigra and hematologic parameters and 28 age- and sex-matched Controls. RESULTS: RLS compared with Control participants showed higher levels of nEV total ferritin but similar levels of transferrin receptor and ferroportin. Western blot analysis showed that heavy- but not light-chain ferritin was increased in nEVs of RLS compared with Control participants. In RLS but not Control participants, nEV total ferritin was positively correlated with systemic iron parameters; the two groups also differed in the relation of nEV total ferritin to MRI measures of iron deposition in substantia nigra. CONCLUSIONS: Given the neuronal origin and diversity of EV cargo, nEVs provide an important platform for exploring the underlying pathophysiology and possible biomarkers of RLS.

L1CAM Immunopositivity in Anaplastic Supratentorial Ependymomas: Correlation With Clinical and Histological Parameters.

Supratentorial ependymomas (ST EPNs) are molecularly characterized, of which the RELA fusion positive tumors are the most common and aggressive subgroup. Moreover, histologically, anaplastic ST EPN (ST-AE) often mimic other central nervous system primary high-grade tumors resulting in a diagnostic dilemma. We aimed to study a cohort of ST-AE; evaluate the expression of two RELA fusion-associated markers-L1CAM and p65 (NF-κB); and correlate their expression with clinical and histological parameters. Cases of ST-AE diagnosed in our department from January 2011 to June 2016 (n = 72) were reviewed. A battery of immunohistochemical markers was employed. A total of 65 confirmed ST-AE were included in the study. Age ranged from 9 months to 60 years. There was a slight predominance in the pediatric population (57%). Male-to-female ratio was 1:1.16. Histomorphological features were varied and mimicked other high-grade tumors in several cases. L1CAM immunopositive tumors constituted 26% of cases and were predominantly seen in young children, in the frontoparietal location, and exhibited clear cell morphology with calcification. A consistent pattern of L1CAM immunopositivity was noted in paired primary and recurrent tumor samples. Our study portrays the varied clinical and histomorphological spectrum of ST-AE. The study emphasizes the association of L1CAM immunopositivity with a wide spectrum of histological parameters, literature on which is scant till date. Since ST EPN-RELA are tumors with aggressive behavior, such a correlation would be clinically relevant, particularly when there is limited access to molecular testing.

L1CAM induces perineural invasion of pancreas cancer cells by upregulation of metalloproteinase expression.

Pancreas cancer cells have a tendency to invade along nerves. Such cancerous nerve invasion (CNI) is associated with poor outcome; however, the exact mechanism that drives cancer cells to disseminate along nerves is unknown. Immunohistochemical analysis of human pancreatic ductal adenocarcinoma (PDAC) specimens showed overexpression of the L1 cell adhesion molecule (L1CAM) in cancer cells and in adjacent Schwann cells (SC) in invaded nerves. By modeling the neural microenvironment, we found that L1CAM secreted from SCs acts as a strong chemoattractant to cancer cells, through activation of MAP kinase signaling. L1CAM also upregulated expression of metalloproteinase-2 (MMP-2) and MMP-9 by PDAC cells, through STAT3 activation. Using a transgenic Pdx-1-Cre/KrasG12D /p53R172H (KPC) mouse model, we show that treatment with anti-L1CAM Ab significantly reduces CNI in vivo. We provide evidence of a paracrine response between SCs and cancer cells in the neural niche, which promotes cancer invasion via L1CAM secretion.

Disseminated tumor cells are not associated with established risk factors, L1CAM immunoreactivity and outcome in endometrial carcinoma.

PURPOSE: The presence of disseminated tumor cells (DTC) in the bone marrow of endometrial carcinoma patients has been demonstrated previously. In contrast to breast cancer, no prognostic significance or association with clinicopathological features was revealed for endometrial carcinoma so far. The aim of this study was to investigate DTC in a large patient cohort with in-depth pathology review data available and to study DTC occurrence in the context of L1CAM and long-term disease specific follow-up. METHODS: Patients treated for endometrial carcinoma at the Tuebingen University Women's hospital between 2003 and 2013 were identified. Cases with previous expert central pathology review including L1CAM immunohistochemistry and bone marrow aspirates available were selected. The presence of DTC and L1CAM expression was studied immunohistochemically. RESULTS: In 395 cases with a confirmed diagnosis of endometrial carcinoma, bone marrow aspirates were available. DTC were detected in 17.2%. The presence of DTC was independent from tumor histology, grade, lymphovascular space involvement (LVSI), FIGO stage, myoinvasion, L1CAM immunoreactivity, and nodal metastasis. DTC occurred less frequently in cases with a microcystic elongated and fragmented (MELF) pattern of invasion (2.2 vs. 21.8%, p = 0.0003). Disease progression was distributed equally among patients with and without DTC present. CONCLUSIONS: We were able to confirm previous findings of DTC presence in a large well-characterized cohort of endometrial carcinoma patients. DTC are detectable in almost one-fifth of endometrial carcinoma and occur less frequently with a MELF pattern of invasion. Further studies investigating the role of DTC in endometrial carcinoma are warranted.

Treatment with a recombinant human IgM that recognizes PSA-NCAM preserves brain pathology in MOG-induced experimental autoimmune encephalomyelitis.

A single peripheral dose of CNS-binding IgMs promote remyelination and preserve axons in a number of animal models of neurologic disease. A myelin-binding recombinant human IgM (rHIgM22) is presently in a safety trial in MS patients following an acute MS exacerbation. rHIgM22 (directed against oligodendrocytes) or rHIgM12 (directed against neurons) were administered to mice with MOG-induced experimental autoimmune encephalomyelitis (EAE) with study endpoints: clinical deficits and brain and spinal cord pathology. IgMs were administered at a therapeutic dose of 100 µ g intra peritoneal at the time of immunization (day -1, 0, +$1), disease onset (15 days) or peak of the disease (28 days). Disease course was not worsened by either human IgM regardless of the time of treatment. Of note, the human IgM that recognizes a carbohydrate epitope on gangliosides and NCAM, rHIgM12, reduced brain pathology when given at time of immunization or at onset of disease, but did not reduce clinical deficits or spinal cord disease burden. Hence, treatment with rHIgM12 resulted in marked reduction in meningeal inflammation. Data consistent with the hypothesis that in the EAE model this molecule has an immune-modulatory effect. Treatment with an anti-CD4 blocking IgG prevented both clinical course and CNS pathology. This pre-clinical study further supports the safety of therapeutic CNS-binding human IgMs in the presence of autoimmunity and clearly differentiates them from IgGs directed against MOG or aquaporin-4 that worsen neurologic disease.

Circulating levels of autoantibodies against L1-cell adhesion molecule as a potential diagnostic biomarker in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common malignant disease worldwide, especially in China. We aimed to determine the level of autoantibodies against L1CAM in patients with ESCC. METHODS: Levels of circulating autoantibodies against L1CAM antigens were determined by an enzyme-linked immunosorbent assay in cohort 1 (191 patients with ESCC and 94 normal controls) and validated in cohort 2 (47 patients with ESCC and 47 normal controls). Receiver-operating characteristics were employed to calculate diagnostic accuracy. Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. RESULTS: In cohorts 1 and 2, levels of autoantibodies against L1CAM were all significantly higher in sera of patients with ESCC compared to normal controls (P < 0.05). Detection of autoantibodies against L1CAM provided a sensitivity of 26.2%, a specificity of 90.4%, and an area under the curve (AUC) of 0.603 (95% CI 0.535-0.672) in diagnosing ESCC in cohort 1, and a sensitivity of 27.7%, a specificity of 91.5%, and an AUC of 0.628 (95% CI 0.516-0.741). Similar results were observed in the diagnosis of early stage ESCC (25.2% sensitivity, 90.4% specificity, and an AUC of 0.611 (95% CI 0.533-0.689) in cohort 1, and 33.3% sensitivity, 91.5% specificity, and an AUC of 0.636 (95% CI 0.439-0.832) in cohort 2). Moreover, positive rates of autoantibodies against L1CAM had no statistical correlation with clinical outcome of ESCC (P > 0.05). CONCLUSIONS: Our results suggest that circulating autoantibodies against L1CAM is a potential biomarker for the early detection of ESCC.

Preclinical Assessment of CD171-Directed CAR T-cell Adoptive Therapy for Childhood Neuroblastoma: CE7 Epitope Target Safety and Product Manufacturing Feasibility.

PURPOSE: The identification and vetting of cell surface tumor-restricted epitopes for chimeric antigen receptor (CAR)-redirected T-cell immunotherapy is the subject of intensive investigation. We have focused on CD171 (L1-CAM), an abundant cell surface molecule on neuroblastomas and, specifically, on the glycosylation-dependent tumor-specific epitope recognized by the CE7 monoclonal antibody. EXPERIMENTAL DESIGN: CD171 expression was assessed by IHC using CE7 mAb in tumor microarrays of primary, metastatic, and recurrent neuroblastoma, as well as human and rhesus macaque tissue arrays. The safety of targeting the CE7 epitope of CD171 with CE7-CAR T cells was evaluated in a preclinical rhesus macaque trial on the basis of CD171 homology and CE7 cross reactivity. The feasibility of generating bioactive CAR T cells from heavily pretreated pediatric patients with recurrent/refractory disease was assessed. RESULTS: CD171 is uniformly and abundantly expressed by neuroblastoma tumor specimens obtained at diagnoses and relapse independent of patient clinical risk group. CD171 expression in normal tissues is similar in humans and rhesus macaques. Infusion of up to 1 × 108/kg CE7-CAR+ CTLs in rhesus macaques revealed no signs of specific on-target off-tumor toxicity. Manufacturing of lentivirally transduced CD4+ and CD8+ CE7-CAR T-cell products under GMP was successful in 4 out of 5 consecutively enrolled neuroblastoma patients in a phase I study. All four CE7-CAR T-cell products demonstrated in vitro and in vivo antitumor activity. CONCLUSIONS: Our preclinical assessment of the CE7 epitope on CD171 supports its utility and safety as a CAR T-cell target for neuroblastoma immunotherapy. Clin Cancer Res; 23(2); 466-77. ©2016 AACR.

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 (KD) 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.

Elevated sL1-CAM levels in BALF and serum of IPF patients.

BACKGROUND AND OBJECTIVE: IPF is a form of interstitial pneumonia of unknown origin that has a poor prognosis for which current treatments are limited. Recent studies have shown that EMT plays a role in IPF and tumour metastasis. L1-CAM has also been linked to EMT during tumour development and tumour metastasis. Our aim was to determine prospectively the level of L1-CAM in IPF patients. METHODS: Forty consecutive Chinese patients (with IPF, 16; LC, 12; and CC, 12), but no apparent lung or other organ's diseases were enrolled. Soluble L1-CAM (sL1-CAM), TGF-ß1, PDGF, γ-INF levels in BALF and serum sL1-CAM were measured using ELISA. RESULTS: BALF sL1-CAM levels of IPF, LC and CC patients were 10.87 ± 0.88 ng/mL, 6.34 ± 0.67 ng/mL and 5.43 ± 0.65 ng/mL, respectively. BALF sL1-CAM concentration of IPF patients was significantly higher than that in LC and in CC patients. Besides, serum sL1-CAM levels in patients with IPF, LC and CC were 9.60 ± 1.41 ng/mL, 9.82 ± 0.72 ng/mL and 5.41 ± 1.07 ng/mL, respectively. The serum sL1-CAM levels in patients with IPF and LC were significantly higher than those in patients with CC (P < 0.001, respectively). CONCLUSIONS: The concentrations of sL1-CAM both in BALF and in serum of patients with IPF are markedly increased compared with controls. This indicates that L1-CAM might be involved in the pathogenesis of IPF as well as that of LC.

Single chain fragment variable antibodies developed by using as target the 3rd fibronectin type III homologous repeat fragment of human neural cell adhesion molecule L1 promote cell migration and neuritogenesis.

L1CAM plays important roles during ontogeny, including promotion of neuronal cell migration and neuritogenesis, and stimulation of axonal outgrowth, fasciculation and myelination. These functions are at least partially exerted through a 16-mer amino acid sequence in the third fibronectin type III-like repeat of L1, which associates with several interaction partners, including integrins, other adhesion molecules and growth factor receptors. Here, using the Tomlinson I library for phage display, we obtained two single-chain variable fragment antibodies (scFvs) against this peptide sequence of human L1, hereafter called H3 peptide. Both scFvs recognize the H3 peptide and the extracellular domain of L1, as tested by enzyme-linked immunosorbent assay (ELISA), Western blot analysis and immunofluorescence staining of L1 expresssing cells. Furthermore, both scFvs reduce U-87 MG cell adhesion to fibronectin, while stimulating cell migration. Application of scFvs to human neuroblastoma SK-N-SH cells promote process outgrowth. Similar to triggering of endogenous L1 functions at the cell surface, both scFvs activate the signal transducers Erk and Src in these cells. Our results indicate that scFvs against a functionally pivotal domain in L1 trigger its regeneration-beneficial functions in vitro, encouraging thoughts on therapy of neurodegenerative diseases in the hope to ameliorate human nervous system diseases.

Antibody therapy to human L1CAM in a transgenic mouse model blocks local tumor growth but induces EMT.

L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers, confers bad prognosis and augments cell motility, invasion and metastasis. Results from xenograft mouse models suggested that L1CAM antibodies might be promising tools for cancer therapy. Here, we generated human L1CAM-transgenic mice to study therapeutic efficacy and putative side effects in a model system. We established three transgenic lines (M2, M3 and F4) expressing the human L1CAM transgene in brain, kidney and colon with decreasing intensity (M2, M3 > F4). The expression pattern was similar to that of L1CAM in humans. No interference of the transgene with the expression of endogenous L1CAM was observed. Immunohistochemical analysis revealed correct expression of the transgene in mouse cortex and collective duct of the kidney. Injection of (125)I-labeled L1CAM antibodies resulted in specific enrichment in the kidney but not in the brain. The injection of the therapeutic anti-human L1CAM mAb L1-9.3/2a into transgenic mice even at high doses did not cause behavioral changes or other side effects. Similar results were obtained using a mouse specific L1CAM mAb in normal mice. Tumor therapy experiments were performed using syngeneic mouse tumor cells (RET melanoma and Panc02 pancreatic adenocarcinoma) transduced with human L1CAM. MAb L1-9.3/2a efficiently and specifically attenuated local tumor growth in both model systems without apparent side effects. The therapeutic effect was dependent on immune effector mechanisms. Analysis of Panc02-huL1CAM tumors after therapy showed elevated levels of EGF and evidence of immune-induced epithelial-mesenchymal transition. The results suggest that our transgenic mice are valuable tools to study L1CAM-based antibody therapy.

Function-triggering antibodies to the adhesion molecule L1 enhance recovery after injury of the adult mouse femoral nerve.

L1 is among the few adhesion molecules that favors repair after trauma in the adult central nervous system of vertebrates by promoting neuritogenesis and neuronal survival, among other beneficial features. In the peripheral nervous system, L1 is up-regulated in Schwann cells and regrowing axons after nerve damage, but the functional consequences of this expression remain unclear. Our previous study of L1-deficient mice in a femoral nerve injury model showed an unexpected improved functional recovery, attenuated motoneuronal cell death, and enhanced Schwann cell proliferation, being attributed to the persistent synthesis of neurotrophic factors. On the other hand, transgenic mice over-expressing L1 in neurons led to improved remyelination, but not improved functional recovery. The present study was undertaken to investigate whether the monoclonal L1 antibody 557 that triggers beneficial L1 functions in vitro would trigger these also in femoral nerve repair. We analyzed femoral nerve regeneration in C57BL/6J mice that received this antibody in a hydrogel filled conduit connecting the cut and sutured nerve before its bifurcation, leading to short-term release of antibody by diffusion. Video-based quantitative analysis of motor functions showed improved recovery when compared to mice treated with conduits containing PBS in the hydrogel scaffold, as a vehicle control. This improved recovery was associated with attenuated motoneuron loss, remyelination and improved precision of preferential motor reinnervation. We suggest that function-triggering L1 antibodies applied to the lesion site at the time of injury over a limited time period will not only be beneficial in peripheral, but also central nervous system regeneration.

Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161 compared to lutetium-177 in an ovarian cancer model.

PURPOSE: The L1 cell adhesion molecule (L1CAM) is considered a valuable target for therapeutic intervention in different types of cancer. Recent studies have shown that anti-L1CAM radioimmunotherapy (RIT) with (67)Cu- and (177)Lu-labelled internalising monoclonal antibody (mAb) chCE7 was effective in the treatment of human ovarian cancer xenografts. In this study, we directly compared the therapeutic efficacy of anti-L1CAM RIT against human ovarian cancer under equitoxic conditions with the radiolanthanide (177)Lu and the potential alternative (161)Tb in an ovarian cancer therapy model. METHODS: Tb was produced by neutron bombardment of enriched (160)Gd targets. (161)Tb and (177)Lu were used for radiolabelling of DOTA-conjugated antibodies. The in vivo behaviour of the radioimmunoconjugates (RICs) was assessed in IGROV1 tumour-bearing nude mice using biodistribution experiments and SPECT/CT imaging. After ascertaining the maximal tolerated doses (MTD) the therapeutic impact of 50 % MTD of (177)Lu- and (161)Tb-DOTA-chCE7 was evaluated in groups of ten mice by monitoring the tumour size of subcutaneous IGROV1 tumours. RESULTS: The average number of DOTA ligands per antibody was 2.5 and maximum specific activities of 600 MBq/mg were achieved under identical radiolabelling conditions. RICs were stable in human plasma for at least 48 h. (177)Lu- and (161)Tb-DOTA-chCE7 showed high tumour uptake (37.8-39.0 %IA/g, 144 h p.i.) with low levels in off-target organs. SPECT/CT images confirmed the biodistribution data. (161)Tb-labelled chCE7 revealed a higher radiotoxicity in nude mice (MTD: 10 MBq) than the (177)Lu-labelled counterpart (MTD: 12 MBq). In a comparative therapy study with equitoxic doses, tumour growth inhibition was better by 82.6 % for the (161)Tb-DOTA-chCE7 than the (177)Lu-DOTA-chCE7 RIT. CONCLUSIONS: Our study is the first to show that anti-L1CAM (161)Tb RIT is more effective compared to (177)Lu RIT in ovarian cancer xenografts. These results suggest that (161)Tb is a promising candidate for future clinical applications in combination with internalising antibodies.

L1CAM promotes enrichment of immunosuppressive T cells in human pancreatic cancer correlating with malignant progression.

Regulatory T cell (T-reg) enrichment in the tumor microenvironment is regarded as an important mechanism of tumor immune escape. Hence, the presence of T-regs in highly malignant pancreatic ductal adenocarcinoma (PDAC) is correlated with short survival. Likewise, the adhesion molecule L1CAM is upregulated during PDAC progression in the pancreatic ductal epithelium also being associated with poor prognosis. To investigate whether L1CAM contributes to enrichment of T-regs in PDAC, human CD4(+)CD25(+)CD127(-)CD49d(-) T-regs and CD4(+)CD25(-) T-effector cells (T-effs) were isolated by magnetic bead separation from blood of healthy donors. Their phenotype and functional behavior were analyzed in dependence on human premalignant (H6c7) or malignant (Panc1) pancreatic ductal epithelial cells, either exhibiting or lacking L1CAM expression. T cells derived from blood and tumors of PDAC patients were analyzed by flow cytometry and findings were correlated with clinical parameters. Predominantly T-regs but not T-effs showed an increased migration on L1CAM expressing H6c7 and Panc1 cells. Whereas proliferation of T-regs did not change in the presence of L1CAM, T-effs proliferated less, exhibited a decreased CD25 expression and an increased expression of CD69. Moreover, these T-effs exhibited a regulatory phenotype as they inhibited proliferation of autologous T cells. Accordingly, CD4(+)CD25(-)CD69(+) T cells were highly abundant in PDAC tissues compared to blood being associated with nodal invasion and higher grading in PDAC patients. Overall, these data point to an important role of L1CAM in the enrichment of immunosuppressive T cells in particular of a CD4(+)CD25(-)CD69(+)-phenotype in PDAC providing a novel mechanism of tumor immune escape which contributes to tumor progression.

[Comparison of site-mutated and error-prone PCR methods for constructing the secondary phage antibody libraries].

OBJECTIVE: To compare site-mutated PCR and error-prone PCR methods in constructing the secondary phage antibody libraries derived from a primary extracellular domain of cell adhesion molecule L1 (L1-ecd) binding single-chain variable fragment (scFv) antibody. METHODS: Secondary mutant phage libraries were established by transfecting the mutated phage at the DNA level to E.coli TG1 with designed site-mutated PCR or error-prone PCR primers. Using the selected phagemid as the template, the mutated plasmid was amplified by PCR and then constructed with restriction enzyme cutting and ligation. Phage-based ELISA was used to calculate the ratios of the positive monoclones from the two libraries and the results were statistically compared using the Pearson x(2); method. RESULTS: The size of the two libraries were 1.4×10(6); pfu/mL (site-mutated library) and 2.5×10(6); pfu/mL (error-prone library), respectively. The ratios of positive clones were 32.5% and 35.5%, respectively. The P value was 0.67, showing no significant difference. CONCLUSION: These two methods can be widely used to obtain antibodies with a high affinity on the basis of the existing phage antibody.