WHO informal consultation on revision of guidelines on evaluation of similar biotherapeutic products, virtual meeting, 30 June - 2 July 2021.

The WHO informal consultation was held to promote the revision of WHO guidelines on evaluation of similar biotherapeutic products (SBPs) adopted by the Expert Committee on Biological Standardization (ECBS) in 2009. It was agreed in the past consultations that the evaluation principles in the guidelines are still valid, but a review was recommended to provide more clarity and case-by-case flexibility. The opportunity was therefore taken to review the experience and identify areas where the current guidance could be more permissive without compromising its basic principles, and where additional explanation could be provided regarding the possibility of reducing the amount of data needed for regulatory approval. The meeting participants applauded the leading role taken by the WHO in providing a much-needed streamlined approach for development and evaluation of SBPs which will provide efficient and cost-effective product development and increase patient access to treatments. It was recognized that the principles as currently described in the draft WHO guidelines are based on sound science and experience gained over the last fifteen years of biosimilar approvals. However, since these guidelines when finalised will constitute the global standard for biosimilar evaluation and assist national regulatory authorities in establishing revised guidance and regulatory practice in this complex area, it was felt that further revision and clarity on certain perspectives in specific areas was necessary to dispel uncertainties arising in the current revised version. This report describes the principles in the draft guidelines, including topics discussed and consensus reached.

Collaborative study for the establishment of Etanercept Biological Reference Preparation Batch 1.

Three preparations of the human tumour necrosis factor (TNF) receptor II Fc fusion protein (TNFR II-Fc) Etanercept were formulated and lyophilised at the National Institute for Biological Standards & Control (NIBSC) prior to evaluation in a collaborative study for their suitability to serve as a World Health Organization (WHO) International Standard (IS)/European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the potency assay of Etanercept. Seven laboratories tested the preparations using an in vitro cell-based bioassay (TNF-α neutralisation) prescribed by the Ph. Eur. monograph on Etanercept (2895). The results of this study indicated that the candidate preparation, coded 13/204, established as the first IS for Etanercept with an assigned potency for TNF neutralisation activity of 10 000 IU per ampoule was also suitable to serve as Ph. Eur. BRP batch 1. The results were compared to those obtained with different cell-based neutralisation assays that were used by further laboratories in the context of establishing the 1st WHO IS for Etanercept. Based on these analyses, preparation 13/204 was adopted by the Ph. Eur. Commission as Etanercept BRP batch 1 with an assigned potency of 10 000 IU per ampoule.

Collaborative study for the establishment of Infliximab Biological Reference Preparation Batch 1.

Two preparations of the chimeric anti-Tumour Necrosis Factor (TNF) monoclonal antibody Infliximab were formulated and lyophilised at the National Institute for Biological Standards & Control (NIBSC) prior to evaluation in a collaborative study for their suitability to serve as a World Health Organization (WHO) International Standard (IS)/European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the potency assay of Infliximab. Twenty-six laboratories tested the preparations using different in vitro cell-based bioassays (TNF-α neutralisation, antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and binding assays. Amongst them, 19 laboratories performed cell-based bioassays. The results of this study indicated that the candidate preparation coded 16/170 was suitable to serve as an International Standard for Infliximab based on the data obtained for biological activity. This candidate standard was established in 2017 as the first International Standard for Infliximab with an assigned potency for TNF neutralisation activity of 500 IU per ampoule. In the same study, the suitability of preparation 16/170 of Infliximab to serve as the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the Infliximab potency assay as described in the Ph. Eur. monograph on Infliximab concentrated solution (2928) was also evaluated. The corresponding analysis, based on the measurement of the inhibitory action of anti-human TNF (Infliximab) on the cytotoxic activity of TNF-alpha, was performed using data from a subset of 9 laboratories using the TNF-alpha-sensitive fibrosarcoma cell line WEHI-164. The results obtained were compared to those obtained from different cell-based neutralisation assays that were used by other laboratories in the context of establishing the 1st World Health Organization (WHO) International Standard (IS) for Infliximab. Based on the analyses, preparation 16/170 was adopted by the Ph. Eur. Commission in June 2018 as Infliximab BRP batch 1 with an assigned potency of 500 IU per ampoule.

Magnesium isotopes of the bulk solar wind from Genesis diamond-like carbon films.

NASA's Genesis Mission returned solar wind (SW) to the Earth for analysis to derive the composition of the solar photosphere from solar material. SW analyses control the precision of the derived solar compositions, but their ultimate accuracy is limited by the theoretical or empirical models of fractionation due to SW formation. Mg isotopes are "ground truth" for these models since, except for CAIs, planetary materials have a uniform Mg isotopic composition (within ≤1‰) so any significant isotopic fractionation of SW Mg is primarily that of SW formation and subsequent acceleration through the corona. This study analyzed Mg isotopes in a bulk SW diamond-like carbon (DLC) film on silicon collector returned by the Genesis Mission. A novel data reduction technique was required to account for variable ion yield and instrumental mass fractionation (IMF) in the DLC. The resulting SW Mg fractionation relative to the DSM-3 laboratory standard was (-14.4‰, -30.2‰) ± (4.1‰, 5.5‰), where the uncertainty is 2Æ¡ SE of the data combined with a 2.5‰ (total) error in the IMF determination. Two of the SW fractionation models considered generally agreed with our data. Their possible ramifications are discussed for O isotopes based on the CAI nebular composition of McKeegan et al. (2011).

Herbal feed additives containing essential oil: 1. Impact on the nutritional worth of complete feed in vitro.

This study was taken up to assess the impact of supplementing herbal feed additives [HFAs; fruit of Myristica fragrans (Jayphall), seeds of Anethum sowa (Suva), fruit of Apium graveolens (Ajmo), fruit of Cuminum cyminum (Jeera), bark of Cinnamonum zeylanicum (Dalchini), or whole plant of Eclipta alba (Bhangro)] containing essential oils as active component on the nutrient utilization and methane production using wheat straw-based total mixed ration (TMR) as a substrate by in vitro gas production technique. The essential oil content was the highest (P < 0.01) in M. fragrans followed by E. alba and A. sowa. In addition to essential oils, these HFAs also contained saponins, tannins, and antioxidants. The HFAs were supplemented at 1-3% of substrate dry matter (DM). The data were analyzed by 6 × 4 factorial design. Irrespective of level of HFA, the net gas production (NGP) and metabolizable energy (ME) availability was the highest (P < 0.01) in TMR supplemented with C. zeylanicum comparable with E. alba, but higher than TMR supplemented with other HFAs. Supplementation of TMR with different HFAs did not affect the digestibility of neutral detergent fiber (NDF) and true organic matter (TOM) and partitioning factor (PF). The total volatile fatty acids (VFAs), acetate, propionate (P < 0.01), and butyrate (P < 0.05) production was the highest in TMR supplemented with A. sowa, and the lowest was observed in TMR supplemented with C. cyminum. The isobutyrate and valerate production was also the highest (P < 0.01) in diet supplemented with A. sowa, but isovalerate production was the highest (P < 0.01) in diet supplemented with C. zeylanicum. The A:P ratio was the best in TMR supplemented with A. sowa. The efficiency of rumen fermentation was the highest, and efficiency of conversion of hexose to methane was the lowest in diet supplemented with A. sowa as compared to all other supplements. The in vitro methane production expressed as either percent of NGP, ml/100 mg DM of substrate/24 h, or as ml/100 mg of digestible OM/24 h was the lowest in TMR supplemented with A. sowa. The ammonia nitrogen production from TMR supplemented with M. fragrans and A. sowa was comparable, but significantly (P < 0.01) lower than TMR supplemented with other HFAs. Irrespective of the nature of HFA, the NGP and ME availability were significantly (P < 0.01) higher in TMR supplemented with HFAs at all levels as compared to un-supplemented TMR. As compared to control, the digestibility of NDF and that of TOM was depressed slightly in all the HFA-supplemented TMRs. The supplementation of HFAs at 2% of substrate DM improved (P < 0.01) the production of total VFAs, acetate, and propionate, and that of isovalerate in comparison to the un-supplemented TMR. The acetate to propionate ratio increased (P < 0.01) with the increase in the level of supplementation of HFAs containing essential oils. The methane and ammonia productions were depressed significantly when TMR was supplemented at 2% level of HFAs as compared to control TMR. It was concluded that supplementation of TMR with A. sowa at 2% of substrate was fermented better as indicated by the production of total and individual VFA, methane, and ammonia as compared to TMR supplemented with other HFA or un-supplemented TMR.

Adenosine A1 receptor antagonist mitigates deleterious effects of sleep deprivation on adult neurogenesis and spatial reference memory in rats.

Sleep deprivation (SD) upsurges intracellular levels of adenosine, impairs adult neuronal cell proliferation (NCP) and cognition while caffeine, a non-selective adenosine A1 receptor (A1R) antagonist improves cognition and adult NCP during SD. We examined the selective antagonistic effects of adenosine A1R using 8-cyclopentyl-1,3-dimethylxanthine (8-CPT) on impairment of spatial reference memory and adult NCP during 48h SD. Adult male Sprague Dawley rats were sleep deprived for 48h, using an automatic cage vibrating stimulus based on animal activity. Spatial reference memory was tested as a measure of cognitive performance employing Morris Water Maze. Rats were given 8-CPT dissolved in 50% dimethyl sulfoxide (DMSO), twice daily (10mg/kg, i.p.) along with 5-bromo-2-deoxyuridine (BrdU) (50mg/kg/day, i.p.). The rats treated with 8-CPT showed significantly short mean latency and path-length to reach the platform compared to the SD rats. Consistent with these findings, 8-CPT-treated group was found to have significantly increased the number of BrdU, Ki-67 and doublecortin (DCX) positive cells. However, no significant difference was seen in NeuN expression in the Dentate Gyrus (DG). Brain-derived neurotropic factor (BDNF) expression in the DG and CA1 region was observed to decrease significantly after SD and be rescued by 8-CPT treatment. Furthermore, latency to reach platform showed a negative correlation with number of BrdU, DCX type-1 cells and BDNF expression in DG. Thus, it may be concluded that treatment with 8-CPT, an adenosine A1R antagonist during SD mitigates SD induced decline in spatial reference memory and adult NCP possibly via up regulation of BDNF levels in DG and CA1 regions.

A genetic screen for increasing metabolic flux in the isoprenoid pathway of Saccharomyces cerevisiae: Isolation of SPT15 mutants using the screen.

A genetic screen to identify mutants that can increase flux in the isoprenoid pathway of yeast has been lacking. We describe a carotenoid-based visual screen built with the core carotenogenic enzymes from the red yeast Rhodosporidium toruloides. Enzymes from this yeast displayed the required, higher capacity in the carotenoid pathway. The development also included the identification of the metabolic bottlenecks, primarily phytoene dehydrogenase, that was subjected to a directed evolution strategy to yield more active mutants. To further limit phytoene pools, a less efficient version of GGPP synthase was employed. The screen was validated with a known flux increasing gene, tHMG1. New mutants in the TATA binding protein SPT15 were isolated using this screen that increased the yield of carotenoids, and an alternate isoprenoid, α-Farnesene confirming increase in overall flux. The findings indicate the presence of previously unknown links to the isoprenoid pathway that can be uncovered using this screen.

Caffeine and modafinil given during 48 h sleep deprivation modulate object recognition memory and synaptic proteins in the hippocampus of the rat.

We aimed to evaluate the effect of caffeine/modafinil on sleep deprivation (SD) induced alterations in recognition memory and synaptic proteins. The data revealed a beneficial effect of caffeine/modafinil against deficit in the familiar object retrieval performance and object exploration ratio after 48 h SD. Caffeine treatment prevented the SD induced down-regulation of synaptophysin and synapsin I proteins with no change in PSD-95 protein in hippocampus. However, modafinil administration improved the down-regulation of synaptophysin, synapsin I and PSD-95 proteins in hippocampus. Hence, caffeine/modafinil can serve as counter measures in amelioration of SD induced consequences at behavioural and protein levels.

Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T-cells.

BACKGROUND AND PURPOSE: In 2006, a life-threatening 'cytokine storm', not predicted by pre-clinical safety testing, rapidly occurred in all six healthy volunteers during the phase I clinical trial of the CD28 superagonist monoclonal antibody (mAb) TGN1412. To date, no unequivocal explanation for the failure of TGN1412 to stimulate profound cytokine release in vitro or in vivo in species used for pre-clinical safety testing has been established. Here, we have identified a species difference almost certainly responsible for this disparate immunopharmacology. EXPERIMENTAL APPROACH: Polychromatic flow cytometry and intracellular cytokine staining were employed to dissect the in vitro immunopharmacology of TGN1412 and other therapeutic mAbs at the cellular level to identify differences between humans and species used for pre-clinical safety testing. KEY RESULTS: In vitro IL-2 and IFN-γ release from CD4+ effector memory T-cells were key indicators of a TGN1412-type response. This mechanism of cytokine release differed from that of other therapeutic mAbs, which can cause adverse reactions, because these other mAbs stimulate cytokine release primarily from natural killer cells. In contrast to humans, CD28 is not expressed on the CD4+ effector memory T-cells of all species used for pre-clinical safety testing, so cannot be stimulated by TGN1412. CONCLUSIONS AND IMPLICATIONS: It is likely that activation of CD4+ effector memory T-cells by TGN1412 was responsible for the cytokine storm. Lack of CD28 expression on the CD4+ effector memory T-cells of species used for pre-clinical safety testing of TGN1412 offers an explanation for the failure to predict a 'cytokine storm' in humans.

238U/235U variations in meteorites: extant 247Cm and implications for Pb-Pb dating.

The 238U/235U isotope ratio has long been considered invariant in meteoritic materials (equal to 137.88). This assumption is a cornerstone of the high-precision lead-lead dates that define the absolute age of the solar system. Calcium-aluminum-rich inclusions (CAIs) of the Allende meteorite display variable 238U/235U ratios, ranging between 137.409 +/- 0.039 and 137.885 +/- 0.009. This range implies substantial uncertainties in the ages that were previously determined by lead-lead dating of CAIs, which may be overestimated by several million years. The correlation of uranium isotope ratios with proxies for curium/uranium (that is, thorium/uranium and neodymium/uranium) provides strong evidence that the observed variations of 238U/235U in CAIs were produced by the decay of extant curium-247 to uranium-235 in the early solar system, with an initial 247Cm/235U ratio of approximately 1.1 x 10(-4) to 2.4 x 10(-4).

Measurement of neutralising antibodies to type I interferons by gene expression assays specific for type 1 interferon-inducible 6-16 mRNA.

Human type I interferon products have been approved for the treatment of several diseases, though neutralising antibodies against them may develop and reduce therapeutic efficacy. Traditionally, potencies of human interferons (IFNs) and of neutralising antibodies (NAbs) against them are quantified by antiviral assays. These are being increasingly replaced by less cumbersome and faster bioassay methods. Since IFNs exert their biological effects by binding to receptors on target cells and stimulating the expression of IFN-inducible genes, measurement of transcribed mRNAs can form the basis of functional bioassays. In this study we have used two approaches, quantitative reverse transcription-polymerase chain reaction (qPCR) and branched DNA (bDNA), to develop efficient, sensitive and robust non-viral assays to quantify type I IFNs per se and NAbs in sera from patients treated with either IFNbeta or IFNalpha2a. We show the rapid (4h) induction of the type I IFN-inducible 6-16 mRNA in A549 lung carcinoma cells is sensitively and reproducibly concentration-dependent for both IFNbeta and IFNalpha2a stimulation, is quantifiable by either approach, and is readily adaptable for the detection and measurement of NAbs against type I IFNs. Quantitative neutralisation of IFN-stimulated 6-16 mRNA expression was achieved in both assays when sera from patients receiving IFNbeta or IFNalpha2a therapy known to contain NAbs against these IFNs were tested. Their rapid and potentially automatable performance strongly suggests these assays could be used in a clinical setting to monitor the development of neutralising antibodies in patients receiving IFN therapy.

Detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals.

Assessment of the unwanted immunogenicity of therapeutic biologicals in recipients is an important consideration in the evaluation of these medicinal products. Proper planning of immunogenicity studies with appropriately devised strategies is critical if valid and meaningful conclusions concerning the unwanted immunogenicity are to be derived. An essential requisite for such studies is the need for conducting carefully selected and validated procedures. Several techniques are available for detection, characterization and measurement of antibodies elicited in an immune response. These include various formats of immunoassays, surface plasmon resonance and biological assays. None of these assays alone can provide sufficient information on the characteristics of the induced antibodies. A combination of methods is therefore usually necessary for a detailed understanding of the quantity and type(s) of antibodies generated against a therapeutic product. This manuscript considers the benefits and limitations of the various techniques available for antibody detection and outlines a brief strategy for the assessment of unwanted immunogenicity of therapeutic products.

Immune responses in advanced colorectal cancer following repeated intradermal vaccination with the anti-CEA murine monoclonal antibody, PR1A3: results of a phase I study.

BACKGROUND AND AIMS: The aim was to determine the toxicity, clinical and immune responses to the murine monoclonal anti-carcinoembryonic antigen (CEA) antibody, PR1A3, in patients with advanced colorectal cancer. MATERIALS AND METHODS: Fifteen patients with advanced colorectal cancer received either 0.5-, 1.0- or 5.0-mg doses of PR1A3 mixed with 10% w/v Alum adjuvant (Superfos Biosector, Denmark) intradermally at 4-week intervals for 3 months. Patient serum was assessed for anti-idiotypic (Ab2), anti-anti-idiotypic (Ab3) and human anti-mouse antibody (HAMA) reactivity. Peripheral blood mononuclear cell (PBMC) proliferation with phytohaemagglutinin (PHA), CEA and PR1A3, stimulated IL-2, IL-4 and IFN-gamma levels and PR1A3-stimulated IL-2 receptor expression during immunotherapy were determined. Comparisons were made with 16 age-matched controls without malignant disease. RESULTS: Hyperimmune sera from 12 of the 15 patients showed Ab2 reactivity with no detectable Ab3 responses. Strong HAMA reactivity was recorded in 7 of the 15 cases with no adverse clinical effect. Delayed-type hypersensitivity (DTH) responses developed in 12 of the 15 patients. Pre-treatment PBMC proliferation with PHA was subnormal in each patient compared with controls, becoming normal (or supranormal) in all patients during immunisation (P<0.001). PBMC proliferation with CEA and PR1A3 increased during immunotherapy (P<0.001) along with stimulated production of IL-2, IFN-gamma and IL-2 receptor expression. Progressive disease was observed in 14 of the 15 patients with minimal toxicity. CONCLUSION: PR1A3 generated limited idiotypic responses but robust DTH reactivity in most patients. In vitro PBMC proliferation with mitogens and recall antigens is greatly increased during the course of immunisation, with a shift in stimulated cytokine profile.

In vitro function of platelet concentrates prepared after filtration of whole blood or buffy coat pools.

BACKGROUND/METHOD: Data on the quality of platelet concentrates (PC) produced by the buffy coat method and stored beyond 5 days in plasma are limited. We therefore evaluated the quality of PCs prepared by leucocyte depletion of whole blood (Terumo WBSP, n = 10) or a buffy coat pool (Pall Autostop, n = 10), and stored for 7 days in plasma by assessing platelet parameters and markers of platelet activation. RESULTS: In both types of PC, levels of glucose decreased during storage but were not totally depleted (> 11 mM on day 7). In contrast, lactate levels increased on storage and was consistently < 20 mM throughout, with pH maintained at > 6.8 in all units. Hypotonic shock response scores were > 47% in all units at day 7. On day 1, markers of platelet activation were significantly higher in WBSP PC, but by day 7 were similar for percentage CD63+ and CD62P + (40%) with levels of platelet microparticles and annexin V binding two-fold higher in WBSP. The expression of CD61 did not alter during storage and the percentage of platelets expressing CD42b was > 88% in all units on day 7. RANTES (Regulated on activation, normal, T-cell expressed and secreted) and TGFbeta released from platelets by day 7 was < 800 ng/ml and 90 ng/ml, respectively. C3a(desarg) increased throughout storage in both types of PC, but without a commensurate increase in the terminal complex SC5b-9 or activation of factor XII. CONCLUSION: Our data indicates that the in vitro characteristics of PCs prepared using these methods is maintained over storage for 7 days in plasma and is not associated with significant deterioration of platelet function. ONE SENTENCE SUMMARY: In vitro function of platelet concentrates prepared by either filtration of whole blood, or pooled buffy coats.

Kinetics of development and characteristics of antibodies induced in cancer patients against yeast expressed rDNA derived granulocyte macrophage colony stimulating factor (GM-CSF).

We have determined the presence and kinetics of granulocyte macrophage colony stimulating factor (GM-CSF) antibodies induced after repeated administration of a yeast expressed GM-CSF product in prostate cancer patients with minimal recurrent disease using a panel of assays for detection and characterization of antibodies. Results showed that all 15 prostate cancer patients treated with GM-CSF developed GM-CSF reactive antibodies during the course of therapy. Most patients (87%) developed GM-CSF reactive antibodies within 3 months while in other patients (13%), these antibodies were induced after additional cycles of GM-CSF treatment. For most patients, the timing of occurrence of these antibodies was the same regardless of whether the ELISA or surface plasmon resonance (SPR) assays were used for detection. However, in two patients, the recognition of GM-CSF reactive antibodies by SPR assays preceded their detection by ELISA. A significant number of patients (n=9, 60%) developed GM-CSF antibodies which neutralized the biological activity of GM-CSF in vitro in a cell-line based bioassay. These antibodies also recognized GM-CSF protein from different expression systems including the non-glycosylated protein from E. coli indicating that the antibody response is directed towards the amino acid backbone of the protein. A significant effect of GM-CSF antibodies on PSA modulation was not observed in this small cohort of patients despite an alteration in PSA levels in some treated patients. The study design used here did not allow conclusions regarding the relationship between neutralizing antibodies and the PSA levels which were used as a marker for clinical outcome. Implementation of a clinical strategy which permits monitoring for antibody development and for levels of a relevant pre-determined clinical marker at appropriate time-points is necessary for assessing the impact of antibody development on the therapeutic efficacy of the protein.

Primary rhabdomyosarcoma of the larynx.

Sarcomas constitute fewer than 1% of the head and neck cancers. They represent less than 1% of laryngeal cancers. Primary rhabdomyosarcoma of the larynx is an extremely rare malignancy. The available literature on this medical oddity is in the form of isolated case reports only. The purpose of this article is to add another case of primary rhabdomyosarcoma of a rare site, the larynx, of which only 36 cases have so far been reported in the world literature. The present patient, an eighteen-year-old boy is only the third case being reported from India among all reported cases of rhabdomyosarcoma of the larynx in the world literature.

Strategies for detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals.

An important aspect of evaluating the safety of therapeutic biologicals is the assessment of the unwanted immunogenicity of such biologicals in recipients. Properly planned immunogenicity studies with appropriately devised strategies are critical if valid conclusions concerning the unwanted immunogenicity are to be derived. Such studies need to be conducted using carefully selected and validated procedures. Several techniques are available for detection and measurement of immunogenicity including immunoassays, radioimmunoprecipitation assays (RIPAs), surface plasmon resonance (SPR) and bioassays. A combination of methods for characterization of the induced antibodies is usually necessary for a detailed understanding of the type(s) of antibodies generated against a therapeutic product. This review considers the benefits and limitations of the various techniques available for antibody detection and outlines a strategy for the assessment of unwanted immunogenicity of therapeutic products.

Immunogenicity of GM-CSF products in cancer patients following immunostimulatory therapy with GM-CSF.

There is a high risk of developing neutralising and non-neutralising antibodies when GM-CSF is used as an immunomodulatory agent in non-immunocompromised patients. The presence of neutralising antibodies may seriously hamper the clinical response of the patients. This must be taken into account when designing protocols if the biological activity of the exogenously administered GM-CSF is not to be impaired and the endogenous production of GM-CSF is not to be inactivated. Assessment of production of neutralising antibodies during cytokine therapy is important for predicting the clinical response to progressive therapy. Use of validated assays is imperative for evaluation of antibodies generated following therapy with a particular protein.

Anti-cytokine autoantibodies in autoimmunity: preponderance of neutralizing autoantibodies against interferon-alpha, interferon-omega and interleukin-12 in patients with thymoma and/or myasthenia gravis.

We have screened for spontaneous anticytokine autoantibodies in patients with infections, neoplasms and autoimmune diseases, because of their increasingly reported co-occurrence. We tested for both binding and neutralizing autoantibodies to a range of human cytokines, including interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-alpha2 (IFN-alpha2), IFN-omega, IFN-beta, IFN-gamma, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF), in plasmas or sera. With two notable exceptions described below, we found only occasional, mostly low-titre, non-neutralizing antibodies, mainly to GM-CSF; also to IL-10 in pemphigoid. Strikingly, however, high-titre, mainly IgG, autoantibodies to IFN-alpha2, IFN-omega and IL-12 were common at diagnosis in patients with late-onset myasthenia gravis (LOMG+), thymoma (T) but no MG (TMG-) and especially with both thymoma and MG together (TMG+). The antibodies recognized other closely related type I IFN-alpha subtypes, but rarely the distantly related type I IFN-beta, and never (detectably) the unrelated type II IFN-gamma. Antibodies to IL-12 showed a similar distribution to those against IFN-alpha2, although prevalences were slightly lower; correlations between individual titres against each were so modest that they appear to be entirely different specificities. Neither showed any obvious correlations with clinical parameters including thymoma histology and HLA type, but they did increase sharply if the tumours recurred. These antibodies neutralized their respective cytokine in bioassays in vitro; although they persisted for years severe infections were surprisingly uncommon, despite the immunosuppressive therapy also used in most cases. These findings must hold valuable clues to autoimmunizing mechanisms in paraneoplastic autoimmunity.