Brain pharmacokinetics of anti-transferrin receptor antibody affinity variants in rats determined using microdialysis.

Receptor-mediated transcytosis (RMT) is used to enhance the delivery of monoclonal antibodies (mAb) into the central nervous system (CNS). While the binding to endogenous receptors on the brain capillary endothelial cells (BCECs) may facilitate the uptake of mAbs in the brain, a strong affinity for the receptor may hinder the efficiency of transcytosis. To quantitatively investigate the effect of binding affinity on the pharmacokinetics (PK) of anti-transferrin receptor (TfR) mAbs in different regions of the rat brain, we conducted a microdialysis study to directly measure the concentration of free mAbs at different sites of interest. Our results confirmed that bivalent anti-TfR mAb with an optimal dissociation constant (KD) value (76 nM) among four affinity variants can have up to 10-fold higher transcytosed free mAb exposure in the brain interstitial fluid (bISF) compared to lower and higher affinity mAbs (5 and 174 nM). This bell-shaped relationship between KD values and the increased brain exposure of mAbs was also visible when using whole-brain PK data. However, we found that mAb concentrations in postvascular brain supernatant (obtained after capillary depletion) were almost always higher than the concentrations measured in bISF using microdialysis. We also observed that the increase in mAb area under the concentration curve in CSF compartments was less significant, which highlights the challenge in using CSF measurement as a surrogate for estimating the efficiency of RMT delivery. Our results also suggest that the determination of mAb concentrations in the brain using microdialysis may be necessary to accurately measure the PK of CNS-targeted antibodies at the site-of-actions in the brain.

Human Cerebrospinal Fluid Monoclonal LGI1 Autoantibodies Increase Neuronal Excitability.

OBJECTIVE: Leucine-rich glioma-inactivated 1 (LGI1) encephalitis is the second most common antibody-mediated encephalopathy, but insight into the intrathecal B-cell autoimmune response, including clonal relationships, isotype distribution, frequency, and pathogenic effects of single LGI1 antibodies, has remained limited. METHODS: We cloned, expressed, and tested antibodies from 90 antibody-secreting cells (ASCs) and B cells from the cerebrospinal fluid (CSF) of several patients with LGI1 encephalitis. RESULTS: Eighty-four percent of the ASCs and 21% of the memory B cells encoded LGI1-reactive antibodies, whereas reactivities to other brain epitopes were rare. All LGI1 antibodies were of IgG1, IgG2, or IgG4 isotype and had undergone affinity maturation. Seven of the overall 26 LGI1 antibodies efficiently blocked the interaction of LGI1 with its receptor ADAM22 in vitro, and their mean LGI1 signal on mouse brain sections was weak compared to the remaining, non-ADAM22-competing antibodies. Nevertheless, both types of LGI1 antibodies increased the intrinsic cellular excitability and glutamatergic synaptic transmission of hippocampal CA3 neurons in slice cultures. INTERPRETATION: Our data show that the patients' intrathecal B-cell autoimmune response is dominated by LGI1 antibodies and that LGI1 antibodies alone are sufficient to promote neuronal excitability, a basis of seizure generation. Fundamental differences in target specificity and antibody hypermutations compared to the CSF autoantibody repertoire in N-methyl-D-aspartate receptor encephalitis underline the clinical concept that autoimmune encephalitides are very distinct entities. Ann Neurol 2020;87:405-418.

A translational platform PBPK model for antibody disposition in the brain.

In this manuscript, we have presented the development of a novel platform physiologically-based pharmacokinetic (PBPK) model to characterize brain disposition of mAbs in the mouse, rat, monkey and human. The model accounts for known anatomy and physiology of the brain, including the presence of distinct blood-brain barrier and blood-cerebrospinal fluid (CSF) barrier. CSF and interstitial fluid turnover, and FcRn mediated transport of mAbs are accounted for. The model was first used to characterize published and in-house pharmacokinetic (PK) data on the disposition of mAbs in rat brain, including the data on PK of mAb in different regions of brain determined using microdialysis. Majority of model parameters were fixed based on literature reported values, and only 3 parameters were estimated using rat data. The rat PBPK model was translated to mouse, monkey, and human, simply by changing the values of physiological parameters corresponding to each species. The translated PBPK models were validated by a priori predicting brain PK of mAbs in all three species, and comparing predicted exposures with observed data. The platform PBPK model was able to a priori predict all the validation PK profiles reasonably well (within threefold), without estimating any parameters. As such, the platform PBPK model presented here provides an unprecedented quantitative tool for prediction of mAb PK at the site-of-action in the brain, and preclinical-to-clinical translation of mAbs being developed against central nervous system (CNS) disorders. The proposed model can be further expanded to account for target engagement, disease pathophysiology, and novel mechanisms, to support discovery and development of novel CNS targeting mAbs.

Targeted radioimmunotherapy for embryonal tumor with multilayered rosettes.

PURPOSE: We explored the use of intraventricular 131I-Omburtamab targeting B7-H3 in patients with ETMR. METHODS: Patients were enrolled in an IRB approved, phase 1, 3 + 3 dose escalation trial. Patients with CNS disease expressing the antibody target antigen B7-H3 were eligible. We report on a cohort of three patients with ETMR who were enrolled on the study. Three symptomatic children (ages 14 months, 3 and 3.5 years) had large parietal masses confirmed to be B7-H3-reactive ETMR. Patients received 2 mCi 131I-Omburtamab as a tracer followed by one or two therapeutic 131I-Omburtamab injections. Dosimetry was based on serial CSF, blood samplings and region of interest (ROI) on nuclear scans. Brain and spine MRIs and CSF cytology were done at baseline, 5 weeks after 131I-Omburtamab, and approximately every 3 months thereafter. Acute toxicities and survival were noted. RESULTS: Patients received surgery, focal radiation, and high dose chemotherapy. Patients 1 and 2 received 131I-Omburtamab (80 and 53 mCi, respectively). Patient 3 had a local recurrence prior to 131I-Omburtamab treated with surgery, external beam radiation, chemotherapy, then 131I-Omburtamab (36 mCi). 131I-Omburtamab was well-tolerated. Mean dose delivered by 131I-Omburtamab was 68.4 cGy/mCi to CSF and 1.95 cGy/mCi to blood. Mean ROI doses were 230.4 (ventricular) and 58.2 (spinal) cGy/mCi. Patients 1 and 2 remain in remission 6.8 years and 2.3 years after diagnosis, respectively; patient 3 died of progressive disease 7 months after therapy (2 years after diagnosis). CONCLUSIONS: 131I-Omburtamab appears safe with favorable dosimetry therapeutic index. When used as consolidation following surgery and chemoradiation therapy, 131I-Omburtamab may have therapeutic benefit for patients with ETMR.

Monoclonal antibody exposure in rat and cynomolgus monkey cerebrospinal fluid following systemic administration.

BACKGROUND: Many studies have focused on the challenges of small molecule uptake across the blood-brain barrier, whereas few in-depth studies have assessed the challenges with the uptake of antibodies into the central nervous system (CNS). In drug development, cerebrospinal fluid (CSF) sampling is routinely used as a surrogate for assessing CNS drug exposure and biomarker levels. In this report, we have studied the kinetic correlation between CSF and serum drug concentration-time profiles for five humanized monoclonal antibodies in rats and cynomolgus monkeys and analyzed factors that affect their CSF exposure. RESULTS: Upon intravenous (IV) bolus injection, antibodies entered the CNS slowly and reached maximum CSF concentration ( CSF T max ) in one to several days in both rats and monkeys. Antibody serum and CSF concentration-time curves converged until they became parallel after CSF T max was reached. Antibody half-lives in CSF ( CSF t ½ ) approximated their serum half-lives ( serum t ½ ). Although the intended targets of these antibodies were different, the steady-state CSF to serum concentration ratios were similar at 0.1-0.2% in both species. Independent of antibody target and serum concentration, CSF-to-serum concentration ratios for individual monkeys ranged by up to tenfold from 0.03 to 0.3%. CONCLUSION: Upon systemic administration, average antibodies CSF-to-serum concentration ratios in rats and monkeys were 0.1-0.2%. The CSF t ½ of the antibodies was largely determined by their long systemic t ½ ( systemic t ½ ).

Widespread brain distribution and activity following i.c.v. infusion of anti-ß-secretase (BACE1) in nonhuman primates.

BACKGROUND AND PURPOSE: The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood-brain barrier. I.c.v. delivery is a promising route to the brain; however, it is unclear how efficiently antibodies delivered i.c.v. penetrate the cerebrospinal spinal fluid (CSF)-brain barrier and distribute throughout the brain parenchyma. EXPERIMENTAL APPROACH: We evaluated the pharmacokinetics and pharmacodynamics of an inhibitory monoclonal antibody against ß-secretase 1 (anti-BACE1) following continuous infusion into the left lateral ventricle of healthy adult cynomolgus monkeys. KEY RESULTS: Animals infused with anti-BACE1 i.c.v. showed a robust and sustained reduction (~70%) of CSF amyloid-ß (Aß) peptides. Antibody distribution was near uniform across the brain parenchyma, ranging from 20 to 40 nM, resulting in a ~50% reduction of Aß in the cortical parenchyma. In contrast, animals administered anti-BACE1 i.v. showed no significant change in CSF or cortical Aß levels and had a low (~0.6 nM) antibody concentration in the brain. CONCLUSION AND IMPLICATIONS: I.c.v. administration of anti-BACE1 resulted in enhanced BACE1 target engagement and inhibition, with a corresponding dramatic reduction in CNS Aß concentrations, due to enhanced brain exposure to antibody.

Monoclonal antibody with conformational specificity for a toxic conformer of amyloid ß42 and its application toward the Alzheimer's disease diagnosis.

Amyloid ß-protein (Aß42) oligomerization is an early event in Alzheimer's disease (AD). Current diagnostic methods using sequence-specific antibodies against less toxic fibrillar and monomeric Aß42 run the risk of overdiagnosis. Hence, conformation-specific antibodies against neurotoxic Aß42 oligomers have garnered much attention for developing more accurate diagnostics. Antibody 24B3, highly specific for the toxic Aß42 conformer that has a turn at Glu22 and Asp23, recognizes a putative Aß42 dimer, which forms stable and neurotoxic oligomers more potently than the monomer. 24B3 significantly rescues Aß42-induced neurotoxicity, whereas sequence-specific antibodies such as 4G8 and 82E1, which recognizes the N-terminus, do not. The ratio of toxic to total Aß42 in the cerebrospinal fluid of AD patients is significantly higher than in control subjects as measured by sandwich ELISA using antibodies 24B3 and 82E1. Thus, 24B3 may be useful for AD diagnosis and therapy.

Preclinical Pharmacokinetics Evaluation of Anti-heparin-binding EGF-like Growth Factor (HB-EGF) Monoclonal Antibody Using Cynomolgus Monkeys via (89)Zr-immuno-PET Study and the Determination of Drug Concentrations in Serum and Cerebrospinal Fluid.

PURPOSE: Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KHK2866 is a humanized anti-HB-EGF monoclonal antibody IgG that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. The phase I study of KHK2866 was discontinued because of neuropsychiatric toxicity. In this study, the pharmacokinetics of KHK2866 was evaluated by (89)Zr-immuno-PET study and the determination of drug concentrations in serum and cerebrospinal fluid using cynomolgus monkeys was performed in order to predict neurotoxicity in a reverse-translational manner. METHODS: KHK2866 was radiolabeled with (89)Zr for preclinical evaluations in normal cynomolgus monkeys and its distribution was analyzed. Furthermore, as a separate study, KHK2866 concentrations in serum and cerebrospinal fluid were determined after administration of a single dose. RESULTS: PET studies with monkeys revealed (89)Zr-KHK2866 accumulation in the liver, spleen and joints of multiple parts, but not in brain. In addition, the pharmacokinetic analyses in serum and CSF demonstrated a low penetration of KHK2866 into the brain. CONCLUSIONS: These studies indicate the difficulty of prediction for neuropsychiatric toxicity of monoclonal antibodies in human by means of pharmacokinetic evaluations using cynomolgus monkeys.

Management and outcome of CSF-JC virus PCR-negative PML in a natalizumab-treated patient with MS.

OBJECTIVE: To describe the diagnosis and management of a 49-year-old woman with multiple sclerosis (MS) developing a progressive hemiparesis and expanding MRI lesion suspicious of progressive multifocal leukoencephalopathy (PML) 19 months after starting natalizumab. RESULTS: Polyomavirus JC (JCV)-specific qPCR in CSF was repeatedly negative, but JCV-specific antibodies indicated intrathecal production. Brain biopsy tissue taken 17 weeks after natalizumab discontinuation and plasmapheresis was positive for JCV DNA with characteristic rearrangements of the noncoding control region, but histology and immunohistochemistry were not informative except for pathologic features compatible with immune reconstitution inflammatory syndrome. A total of 22 months later, the clinical status had returned close to baseline level paralleled by marked improvement of neuroradiologic abnormalities. CONCLUSIONS: This case illustrates diagnostic challenges in the context of incomplete suppression of immune surveillance and the potential of recovery of PML associated with efficient immune function restitution.

Two-compartment model of radioimmunotherapy delivered through cerebrospinal fluid.

PURPOSE: Radioimmunotherapy (RIT) using (131)I-3F8 injected into cerebrospinal fluid (CSF) was a safe modality for the treatment of leptomeningeal metastases (JCO, 25:5465, 2007). A single-compartment pharmacokinetic model described previously (JNM 50:1324, 2009) showed good fitting to the CSF radioactivity data obtained from patients. We now describe a two-compartment model to account for the ventricular reservoir of (131)I-3F8 and to identify limiting factors that may impact therapeutic ratio. METHODS: Each parameter was examined for its effects on (1) the area under the radioactivity concentration curve of the bound antibody (AUC[C(IAR)]), (2) that of the unbound antibody AUC[C(IA)], and (3) their therapeutic ratio (AUC[C(IAR)]/AUC[C(IA)]). RESULTS: Data fitting showed that CSF kBq/ml data fitted well using the two-compartment model (R = 0.95 ± 0.03). Correlations were substantially better when compared to the one-compartment model (R = 0.92 ± 0.11 versus 0.77 ± 0.21, p = 0.005). In addition, we made the following new predictions: (1) Increasing immunoreactivity of (131)I-3F8 from 10% to 90% increased both (AUC[C(IAR)]) and therapeutic ratio ([AUC[C(IAR)]/AUC[C(IA)]] by 7.4 fold, (2) When extrapolated to the clinical setting, the model predicted that if (131)I-3F8 could be split into 4 doses of 1.4 mg each and given at ≥24 hours apart, an antibody affinity of K(D) of 4 × 10(-9) at 50% immunoreactivity were adequate in order to deliver ≥100 Gy to tumor cells while keeping normal CSF exposure to <10 Gy. CONCLUSIONS: This model predicted that immunoreactivity, affinity and optimal scheduling of antibody injections were crucial in improving therapeutic index.

A 4-week intrathecal toxicity and pharmacokinetic study with trastuzumab in cynomolgus monkeys.

Trastuzumab is indicated for the treatment of patients with breast cancer overexpressing human epidermal growth factor 2 (HER2). Women with HER2-positive tumors have an increased risk of brain metastases. The blood-brain barrier and blood-cerebrospinal fluid (CSF) barrier may prevent trastuzumab from reaching appropriate concentrations in the brain and CSF following standard intravenous administration. To evaluate the potential of effects on the central nervous system, a 4-week toxicology study with weekly intrathecal administration of trastuzumab was performed in cynomolgus monkeys at doses of 0, 3, or 15 mg. No trastuzumab-related effects on body weight, clinical signs, neurological function, clinical pathology, or anatomic pathology were noted. The applied doses and CSF concentrations achieved in the current study exceeded those reported in patients after intrathecal administration. The results support future studies for further evaluation of intrathecal application of trastuzumab in patients with brain metastases in HER2-positive breast cancer.

Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid.

OBJECTIVE: Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic. METHODS: We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy- and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections. RESULTS: Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immunoreactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells. INTERPRETATION: The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.

Rituximab levels in cerebrospinal fluid of patients with neurological autoimmune disorders.

BACKGROUND: Rituximab, a monoclonal antibody against the B-cell-specific surface protein CD20, is being evaluated for treatment of multiple sclerosis and neuromyelitis optica. Both diseases are restricted to the brain and cerebrospinal fluid (CSF). Whereas the ability of rituximab to deplete B cells in peripheral blood and tissue is well known, little information is available about the ability of rituximab to penetrate the barriers separating brain and CSF from the serum compartment. OBJECTIVE: To measure rituximab levels in serum and CSF of rituximab-treated patients and correlate them with CSF and response markers. METHODS: Fourteen paired serum/CSF samples of patients with autoimmune nervous system disorder were analyzed for up to 43 weeks after rituximab application. RESULTS: Rituximab remains detectable within the CSF after i.v. application for up to 24 weeks. Furthermore, levels of rituximab in CSF correlate significantly with the integrity of the blood CSF barrier.

Effect of infliximab in progressive neuro-Behçet's syndrome.

Recent studies have shown the beneficial effect of infliximab in ocular manifestation of Behçet's disease. The current studies examined the efficacy of infliximab in progressive neuro-Behçet's syndrome (NB) refractory to methotrexate (MTX). Five male patients with progressive NB with sustained elevation of cerebrospinal fluid (CSF) IL-6 (over 20 pg/ml) despite administration of MTX and steroid, were given intravenous infusion of 5 mg/kg infliximab at weeks 0, 2, 6, and 14 with MTX (10-17.5 mg/week) and prednisolone (<10 mg/day) at the same doses. The clinical responses were judged by neuropsychiatric findings, revised Wechsler adult intelligence scale (WAIS-R), and brain magnetic resonance imaging (MRI) scans at 24 weeks. In all the 5 patients, CSF IL-6 were markedly decreased by 1/2-1/37 on the next day of the first infusion and remained below 20 pg/ml before the last infusion at 14 weeks, whereas CSF TNF-alpha were not significantly changed at any time point. At 24 weeks from the initial infusion, none of the 5 patients showed exacerbation (3 patients significantly improved). Nor did the atrophy in midbrain, pons and medulla on brain MRI scans show significant progression. These results suggest that infliximab might have a beneficial effect in the treatment of progressive NB by reducing CSF IL-6 levels but not TNF-alpha. Since infliximab has been shown to have cytotoxic effects on monocytes/macrophages, the rapid fall of CSF IL-6 after the infusion suggest that infliximab might directly act on such inflammatory cells producing IL-6.

Defining limits of treatment with humanized neutralizing monoclonal antibody for West Nile virus neurological infection in a hamster model.

A potent anti-West Nile virus (anti-WNV)-neutralizing humanized monoclonal antibody, hE16, was previously shown to improve the survival of WNV-infected hamsters when it was administered intraperitoneally (i.p.), even after the virus had infected neurons in the brain. In this study, we evaluated the therapeutic limit of hE16 for the treatment of WNV infection in hamsters by comparing single-dose peripheral (i.p.) therapy with direct administration into the pons through a convection-enhanced delivery (CED) system. At day 5 after infection, treatments with hE16 by the peripheral and the CED routes were equally effective at reducing morbidity and mortality. In contrast, at day 6 only the treatment by the CED route protected the hamsters from lethal infection. These experiments suggest that hE16 can directly control WNV infection in the central nervous system. In support of this, hE16 administered i.p. was detected in a time-dependent manner in the serum, cerebrospinal fluid (CSF), cerebral cortex, brain stem, and spinal cord in CSF. A linear relationship between the hE16 dose and the concentration in serum was observed, and maximal therapeutic activity occurred at doses of 0.32 mg/kg of body weight or higher, which produced serum hE16 concentrations of 1.3 microg/ml or higher. Overall, these data suggest that in hamsters hE16 can ameliorate neurological disease after significant viral replication has occurred, although there is a time window that limits therapeutic efficacy.

Ratio of trastuzumab levels in serum and cerebrospinal fluid is altered in HER2-positive breast cancer patients with brain metastases and impairment of blood-brain barrier.

Patients receiving trastuzumab for HER2-overexpressing metastatic breast cancer seem to suffer from an increased risk of brain metastases, even in cases with responsive disease. To evaluate whether trastuzumab is able to penetrate the blood-brain barrier, we measured trastuzumab levels in the serum and in cerebrospinal fluid of metastatic breast cancer patients with brain metastases receiving trastuzumab for HER2-overexpressing metastatic breast cancer. In a pilot study, metastatic breast cancer patients with brain metastases and HER2-overexpressing tumors (HercepTest; Dako, Copenhagen, Denmark) were included. At different time points, trastuzumab levels in the serum and cerebrospinal fluid were measured using a newly developed immunoenzymatic test for trastuzumab. Six out of eight patients were evaluable for determination of trastuzumab level in the serum and cerebrospinal fluid. Before radiotherapy, median trastuzumab level in the serum was 52 054 ng/ml compared with 124 ng/ml in cerebrospinal fluid (ratio 420 : 1). After completion of radiotherapy, median trastuzumab level was 20 185 ng/ml in the serum and 226 ng/ml in cerebrospinal fluid, respectively (ratio 76 : 1). With concomitant meningeal carcinomatosis, trastuzumab level in the serum after radiotherapy was 17 431 and 356 ng/ml in cerebrospinal fluid (ratio 49 : 1). For the first time, we present clinical evidence that trastuzumab levels in cerebrospinal fluid are increased under conditions of an impaired blood-brain barrier such as meningeal carcinomatosis or radiotherapy. This evidence supports the concept of continuing trastuzumab therapy in patients with brain metastases treated by radiotherapy. Monitoring of trastuzumab levels in the serum and cerebrospinal fluid may enable individualized therapy strategies in metastatic breast cancer patients with brain metastases, and lead to a better understanding of trastuzumab pharmacokinetics in the cerebrospinal fluid and serum.

Continuous delivery of a monoclonal antibody against Reissner's fiber into CSF reveals CSF-soluble material immunorelated to the subcommissural organ in early chick embryos.

The subcommissural organ (SCO) is an ependymal differentiation located in the dorsal midline of the caudal diencephalon under the posterior commissure. SCO cells synthesize and release glycoproteins into the cerebrospinal fluid (CSF) forming a threadlike structure known as Reissner's fiber (RF), which runs caudally along the ventricular cavities and the central canal of the spinal cord. Numerous monoclonal antibodies have been raised against bovine RF and the secretory material of the SCO. For this study, we selected the 4F7 monoclonal antibody based on its cross-reactivity with chick embryo SCO glycoproteins in vivo. E4 chick embryos were injected with 4F7 hybridoma cells or with the purified monoclonal antibody into the ventricular cavity of the optic tectum. The hybridoma cells survived, synthesized and released antibody into the CSF for at least 13 days after the injection. E5 embryos injected with 4F7 antibody displayed precipitates in the CSF comprising both the monoclonal antibody and anti-RF-positive material. Such aggregates were never observed in control embryos injected with other monoclonal antibodies used as controls. Western blot analysis of CSF from E4-E6 embryos revealed several immunoreactive bands to anti-RF (AFRU) antibody. We also found AFRU-positive material bound to the apical surface of the choroid plexus primordia in E5 embryos. These and other ultrastructural evidence suggest the existence of soluble SCO-related molecules in the CSF of early chick embryos.