Risk factors for unilateral cranial cruciate ligament rupture diagnosis and for clinical management in dogs under primary veterinary care in the UK.

This study aimed to evaluate demographic risk factors associated with unilateral cranial cruciate ligament (CCL) rupture diagnosis and to explore demographic and clinical risk factors associated with management of unilateral CCL rupture in dogs under primary veterinary care in the UK. A retrospective cohort study design was used. Clinical records were automatically searched and manually verified for incident cases of unilateral CCL rupture during 2019 and additional clinical management information extracted. Multivariable logistic regression modelling was used to evaluate associations between risk factors and: (1) CCL rupture diagnosis; and (2) clinical management (surgical or non-surgical). The analysis included 1000 unilateral CCL rupture cases and a random selection of 500,000 non-cases. After accounting for confounding factors, dogs aged 6 to < 9 years, male neutered and female neutered dogs, insured dogs, and Rottweiler, Bichon Frise, and West Highland White terrier breeds, in particular, had increased odds of unilateral CCL rupture diagnosis. Insured dogs and dogs ≥ 20 kg had increased odds of surgical management, while dogs ≥ 9 years and dogs with one non-orthopaedic comorbidity at diagnosis with CCL rupture had reduced odds. These findings inform identification of at-risk dogs, with Rottweilers and Bichon Frise particularly predisposed. Additionally, they contribute to a greater understanding of the clinical rationales used in primary-care veterinary practices to decide between surgical or non-surgical management of unilateral CCL rupture.

Demography, disorders and mortality of pet hamsters under primary veterinary care in the United Kingdom in 2016.

BACKGROUND: Hamsters are popular pets worldwide but there is limited evidence on the overall health issues of pet hamsters. This study aimed to characterise the demography, disorder prevalence and mortality of pet hamsters in the United Kingdom. METHOD: The VetCompass study included anonymised clinical records of 16,605 hamsters. RESULTS: The most common hamster species were Syrian (golden) (Mesocricetus auratus) (n=12,197, 73.45%), Djungarian (winter white dwarf) (Phodopus sungorus) (2286, 13.77%) and Roborovski hamsters (Phodopus roborovskii) (1054, 6.35%). The most prevalent precise-level disorders recorded across all hamsters were a presentation categorised as 'wet tail' (n=293, 7.33%), disorder undiagnosed (292, 7.30%), bite injuries from other hamsters (235, 5.88%), overgrown nail(s) (165, 4.13%), overgrown incisor(s) (159, 3.98%) and traumatic injury (152, 3.80%). The most prevalent disorders groups across all species of hamster were traumatic injury (n=616, 15.41%), enteropathy (450, 11.26%), ophthalmological disorder (445, 11.13%), skin disorder (362, 9.05%) and mass (361, 9.03%). The median age at death across all hamsters was 1.75 years (interquartile range: 0.83 to 2.20, range: 0.01 to 3.65). The most common causes of death at a precise level were wet tail (7.88%, 95% confidence interval: 6.35 to 9.66), abdominal mass (6.40%, 95% confidence interval: 5.01 to 8.03), neoplasia (5.38%, 95% confidence interval: 4.11 to 6.90) and dyspnoea (3.99%, 95% confidence interval: 2.9 to 5.34). CONCLUSION: This study provides veterinary professionals, educators, welfare scientists and owners with an evidence base on pet hamster health. A greater understanding of the common disorders of pet hamsters can support veterinary professionals to communicate more effectively with owners on key issues and outcomes to expect from hamster ownership.

How much is that doodle in the window? Exploring motivations and behaviours of UK owners acquiring designer crossbreed dogs (2019-2020).

BACKGROUND: Demand for intentional crosses of purebred dog breeds, often labelled 'designer crossbreeds' (e.g., Labrador Retriever X Poodle, the 'Labradoodle'), has recently increased in the UK. This study aimed to explore this phenomenon by comparing pre-purchase motivations, pre-purchase and purchase behaviours of UK owners of designer crossbred puppies purchased during 2019-2020 with those of owners of purebred puppies purchased during the same period. RESULTS: Data were collected in an online cross-sectional survey between November-December 2020. Responses from n = 6293 puppies (designer crossbred puppies: n = 1575; purebred puppies: n = 4718) were analysed. Perceived hypoallergenicity was cited as a motivator for breed/crossbreed choice by almost half of designer crossbreed owners (47.1%), six times more than purebred dog owners (7.86%; odds ratio [OR]: 9.12, 95% CI: 7.70-10.8). Designer crossbred puppies were more likely to have been acquired via a general selling website (e.g., Gumtree; 13.8%) compared to purebred puppies (7.67%; OR: 2.19, 95% CI: 1.77-2.71), or an animal-specific selling websites (e.g., Pets4Homes; 55.7%) compared to purebred puppies (37.4%; OR: 1.89, 95% CI: 1.65-2.17). Designer crossbreed owners were less likely to see their puppy in person prior to purchase than purebred owners (60.4% vs. 67.0%, respectively; OR: 0.74, 95% CI: 0.64-0.85), and at purchase, designer crossbred puppies were less likely to be seen with their mother (73.1% vs. 79.8%, respectively; OR: 0.82, 95% CI: 0.70-0.95), and littermates (67.7% vs. 78.1%, respectively; OR: 0.63, 95% CI: 0.55-0.73). Designer crossbreeds had a significantly higher purchase price, with 25.7% of designer crossbreed puppies costing £2000-£2999 compared to 15.1% of purebred puppies (X2 = 207.31, p <  0.001). CONCLUSIONS: The recent boom in designer crossbreeds in the UK has been fuelled by a desire for perceived hypoallergenic and generally healthy dogs that fit the lifestyles of households with children and limited experience with dogs. Some sought-after traits in designer crossbreeds are misconceptions that risk canine welfare, including relinquishment risk, if owner expectations are not met. Purchasing practices fuelling this boom support irresponsible breeding and selling practices, which combined with reduced pressure for health testing from buyers, may result in a higher disease burden and poorer future welfare for this growing designer dog population.

Epidemiology of periodontal disease in dogs in the UK primary-care veterinary setting.

OBJECTIVES: Periodontal disease is a frequent diagnosis of dogs and can have severe negative impacts on welfare. It was hypothesised that breeds with skull shapes that differ most in conformation from the moderate mesocephalic skull shape have higher odds of periodontal disease. MATERIALS AND METHODS: The cohort study included a random sample of dogs under primary veterinary care in 2016 from the VetCompass Programme database. Risk factor analysis used random effects multivariable logistic regression modelling. RESULTS: The study included a random sample of 22,333 dogs. The 1-year period prevalence for diagnosis with periodontal disease was 12.52% (95% CI: 12.09 to 12.97). Eighteen breeds showed increased odds compared with crossbred dogs. Breeds with the highest odds included Toy Poodle (odds ratio 3.97, 95% confidence intervals 2.21 to 7.13), King Charles Spaniel (odds ratio 2.63, 95% confidence interval 1.50 to 4.61), Greyhound (odds ratio 2.58, 95% confidence interval 1.75 to 3.80) and Cavalier King Charles Spaniel (odds ratio 2.39, 95% confidence interval 1.85 to 3.09). Four breeds showed reduced odds compared with crossbreds. Brachycephalic breeds had 1.25 times the odds (95% confidence interval 1.11 to 1.42) of periodontal disease compared with mesocephalic breeds. Spaniel types had 1.63 times the odds (95% confidence interval 1.42 to 1.87) compared with non-spaniel types. Increasing adult bodyweight was associated with progressively decreasing odds of periodontal disease. CLINICAL SIGNIFICANCE: The high prevalence identified in this study highlights periodontal disease as a priority welfare concern for predisposed breeds. Veterinarians can use this information to promote improved dental care in predisposed dogs, especially as these dogs age.

Frequency, breed predisposition and demographic risk factors for overweight status in dogs in the UK.

OBJECTIVES: To evaluate the prevalence and risk factors for overweight status in dogs under primary veterinary care in the UK. MATERIALS AND METHODS: A retrospective study design was used to estimate the 1-year (2016) period prevalence of overweight status. The clinical records were randomly ordered and manually validated for dogs with overweight status during 2016. Univariable and multivariable logistic regression modelling were used to evaluate associations between risk factors (breed, brachycephalic status, adult bodyweight, bodyweight relative to breed-sex mean, age, sex-neuter and insurance) and overweight status. RESULTS: There were 1580 of 22,333 dogs identified as overweight during 2016. The estimated 1-year period prevalence for overweight status recorded in dogs under veterinary care was 7.1% (95% confidence interval 6.7-7.4). After accounting for confounding factors, eight breeds showed increased odds of overweight status compared with crossbred dogs. The breeds with the highest odds included the Pug (OR 3.12, 95% confidence interval 2.31 to 4.20), Beagle (OR 2.67, 1.75 to 4.08), Golden Retriever (OR 2.58, 1.79 to 3.74) and English Springer Spaniel (OR 1.98, 1.31 to 2.98). Being neutered, middle-aged and insured were additionally associated with overweight status. CLINICAL SIGNIFICANCE: Targeted overweight prevention strategies should be prioritised for predisposed breeds, such as Pugs and Beagles. The findings additionally raise questions about further preventative efforts following neutering. The prevalence estimate suggests veterinary professionals are underreporting overweight status and therefore could be missing key welfare opportunities.

Unravelling the health status of brachycephalic dogs in the UK using multivariable analysis.

Brachycephalic dog breeds are regularly asserted as being less healthy than non-brachycephalic breeds. Using primary-care veterinary clinical data, this study aimed to identify predispositions and protections in brachycephalic dogs and explore differing inferences between univariable and multivariable results. All disorders during 2016 were extracted from a random sample of 22,333 dogs within the VetCompass Programme from a sampling frame of 955,554 dogs under UK veterinary care in 2016. Univariable and multivariable binary logistic regression modelling explored brachycephaly as a risk factor for each of a series of common disorders. Brachycephalic dogs were younger, lighter and less likely to be neutered than mesocephalic, dolichocephalic and crossbred dogs. Brachycephalic differed to non-brachycephalic types in their odds for 10/30 (33.33%) common disorders. Of these, brachycephalic types were predisposed for eight disorders and were protected for two disorders. Univariable and multivariable analyses generated differing inference for 11/30 (30.67%) disorders. This study provides strong evidence that brachycephalic breeds are generally less healthy than their non-brachycephalic counterparts. Results from studies that report only univariable methods should be treated with extreme caution due to potential confounding effects that have not been accounted for during univariable study design or analysis.

The use of hydrolysed diets for vomiting and/or diarrhoea in cats in primary veterinary practice.

OBJECTIVE: To describe responses of cats prescribed a hydrolysed diet with or without concurrent medication for chronic vomiting and/or diarrhoea of undetermined aetiology. MATERIALS AND METHODS: Anonymised records of 512,213 cats under UK veterinary care in 2016 from the VetCompass database were searched using relevant terms for hydrolysed diets. The records of 5000 (90%) of 5569 cats with evidence of receiving a hydrolysed diet were randomly reviewed for gastrointestinal indication, prior and concurrent medication and response after hydrolysed dietary intervention. A poor response was defined as evidence of receiving antibiotic or glucocorticoid treatment for vomiting/diarrhoea at visits after the onset of the diet or death from gastrointestinal signs for at least 6 months follow-up. RESULTS: Of 977 cats prescribed a hydrolysed diet for chronic vomiting/diarrhoea, 697 (71%) were first prescribed the diet without concurrent antibiotics or glucocorticoids while 280 (29%) first received the diet with these medications. Thirty-four per cent of cats in the former group and 61% in the latter had a poor response. Cats older than 6 years and cats prescribed antibiotic and/or glucocorticoid for vomiting/diarrhoea before and concurrently with the diet had higher odds of poor response. CLINICAL SIGNIFICANCE: Although variations in our observations may reflect severity of signs or prescribing habits of primary-care veterinary surgeons, our study suggests there is merit in trialling a hydrolysed diet first as a sole therapy in cats with chronic vomiting/diarrhoea when diagnostic investigations do not reveal a cause, before resorting to antibiotic and/or glucocorticoid therapy for cases that respond poorly.

Associations between neutering and early-onset urinary incontinence in UK bitches under primary veterinary care.

OBJECTIVES: To investigate association between neutering and early-onset urinary incontinence in bitches under primary veterinary care in the UK. MATERIALS AND METHODS: A retrospective cohort study of bitches within VetCompass born between January 1, 2010 and December 31, 2012 that were followed until March 31, 2018. The clinical records were automatically searched and manually validated for incontinence cases. Incidence risk and rate over the study period were calculated. Cox regression modelling separately evaluated the hazard of urinary incontinence and association with neutering: (1) from the date of birth for all bitches, both neutered and entire; and, (2) from the date of neutering for the neutered subset. Other variables considered included breed, bodyweight and veterinary practice group. RESULTS: Overall, 492 bitches were identified with early-onset urinary incontinence from a total of 72,971 included in the study period. Incidence risk was 0.68% (95% confidence intervals 0.62 to 0.74), while incidence rate increased with age. After accounting for confounding factors, increased hazard of early-onset urinary incontinence was identified in: (1) neutered bitches, with the effect increasing with age; and, (2) bitches neutered before 6 months, within the first 2 years following neutering. In both models, increased hazard was additionally associated with increasing bodyweight and breed. CLINICAL SIGNIFICANCE: Neutering itself and early-age neutering (<6 months) are major risk factors for early-onset urinary incontinence. These results should be taken into account in making evidence-based recommendations on neutering and its timing.

Spaying and urinary incontinence in bitches under UK primary veterinary care: a case-control study.

OBJECTIVES: To evaluate associations between spaying and urinary incontinence in bitches under primary veterinary care in the UK. MATERIALS AND METHODS: A case-control study was nested within the study population of 333,910 bitches, which included all bitches within the VetCompass database with an electronic patient record in 2016 or in both 2015 and 2017. The electronic records were searched automatically for urinary incontinence cases, which were manually reviewed for inclusion. All non-cases were included as controls. Additional demographic and clinical information was extracted on cases and controls. RESULTS: The study included 427 incident cases and 1708 controls that were presented between November 1, 2014 and October 31, 2017. Prior spaying was associated with increased odds of urinary incontinence (odds ratio: 3.01; 95% CIs: 2.23 to 4.05). Increased odds of urinary incontinence were additionally associated with increasing age and increasing bodyweight. Age at spay was not associated with urinary incontinence. CLINICAL SIGNIFICANCE: The findings support spaying as a major risk factor associated with urinary incontinence, but age at spay appears to be of less clinical importance. These results will help assist clinicians in making evidence-based recommendations on spaying while taking other considerations for urinary incontinence into account.

Phase I trial results of iodine-131-labeled antitenascin monoclonal antibody 81C6 treatment of patients with newly diagnosed malignant gliomas.

PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine-131 ((131)I)-labeled 81C6 antitenascin monoclonal antibody (mAb) administered clinically into surgically created resection cavities (SCRCs) in malignant glioma patients and to identify any objective responses with this treatment. PATIENTS AND METHODS: In this phase I trial, newly diagnosed patients with malignant gliomas with no prior external-beam therapy or chemotherapy were treated with a single injection of (131)I-labeled 81C6 through a Rickham reservoir into the resection cavity. The initial dose was 20 mCi and escalation was in 20-mCi increments. Patients were observed for toxicity and response until death or for a minimum of 1 year after treatment. RESULTS: We treated 42 patients with (131)I-labeled 81C6 mAb in administered doses up to 180 mCi. Dose-limiting toxicity was observed at doses greater than 120 mCi and consisted of delayed neurotoxicity. None of the patients developed major hematologic toxicity. Median survival for patients with glioblastoma multiforme and for all patients was 69 and 79 weeks, respectively. CONCLUSION: The MTD for administration of (131)I-labeled 81C6 into the SCRC of newly diagnosed patients with no prior radiation therapy or chemotherapy was 120 mCi. Dose-limiting toxicity was delayed neurologic toxicity. We are encouraged by the survival and toxicity and by the low 2.5% prevalence of debulking surgery for symptomatic radiation necrosis.

Dosimetry and dose-response relationships in newly diagnosed patients with malignant gliomas treated with iodine-131-labeled anti-tenascin monoclonal antibody 81C6 therapy.

PURPOSE: The objective of this study was to perform the dosimetry and evaluate the dose-response relationships in newly diagnosed patients with malignant brain tumors treated by direct injections of (131)I-labeled 81C6 monoclonal antibody (MAb) into surgically created resection cavities (SCRCs). METHODS AND MATERIALS: Absorbed doses to the 2-cm-thick shell as measured from the margins of the resection cavity interface were estimated for 42 patients with primary brain tumors. MR images were used to assess the enhanced-rim volume as a function of time after radiolabeled MAb therapy. Biopsy samples were obtained from 15 patients and 1 autopsy. RESULTS: The average absorbed dose [range] to the 2-cm shell region was 32 [3-59] Gy. For the endpoint of minimal time to MR contrast enhancement, the optimal absorbed dose and initial dose-rate were 43 +/- 16 Gy and 0. 41 +/- 0.10 Gy/h, respectively. There was a correlation between the absorbed dose and dose rate to the shell region and biopsy outcome (tumor recurrence, radionecrosis, and tumor recurrence and/or radionecrosis). In this Phase I study, the maximum tolerated dose (MTD) was 120 mCi. At this MTD, the estimated average absorbed dose and initial dose rate to the 2-cm shell were 41 [9-89] Gy and 0.51 [0.24-1.13] Gy/h, respectively. These values are in agreement with the optimal values based on the time to MR lesion rim enhancement. CONCLUSIONS: The average absorbed dose to the 2-cm shell region varied considerably and mainly depended on cavity volume. In future clinical trials, the administered activity of (131)I-labeled 81C6 MAb may be adjusted based on cavity volume in order to deliver the optimal absorbed dose of 43 Gy rather than giving a fixed administered activity.

125I-labeled anti-epidermal growth factor receptor-vIII single-chain Fv exhibits specific and high-level targeting of glioma xenografts.

A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice. The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors. The scFv molecule, designed as V(H)-(Gly4-Ser)3-V(L), was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer. Size-exclusion chromatography of purified scFv demonstrated a protein monomer of Mr 26,000. Labeling was performed using N-succinimidyl 5-[125I]iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5-2.0 mCi/mg, with yields of 35-50% and 45-70%, respectively. The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65-80% when SIPC was used and 50-55% when Iodogen was used. The affinity (K(A)) of MRI(scFv) for EGFRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M(-1) by BIAcore analysis, and it was 1.0 x 10(8) +/- 0.1 x 10(8) M(-1) and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70%. The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.deltaEGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [125I]SIPC-MR1(scFv) and [131I]SIPC-anti-Tac(scFv) as a control. When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration. Specific: control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.

Iodine-131-labeled antitenascin monoclonal antibody 81C6 treatment of patients with recurrent malignant gliomas: phase I trial results.

PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine 131 (131I)-labeled 81C6 monoclonal antibody (mAb) in brain tumor patients with surgically created resection cavities (SCRCs) and to identify any objective responses to this treatment. METHODS: In this phase I trial, eligible patients were treated with a single injection of 131I-labeled 81C6. Cohorts of three to six patients were treated with escalating dosages of 131I (starting dose of 20 mCi with a 20-mCi escalation in subsequent cohorts) administered through an Ommaya reservoir in the SCRC. Patients were followed up for toxicity and response until death or for a minimum of 1 year after treatment. The SCRC patients, who were previously irradiated, were followed up without additional treatment unless progressive disease was identified. RESULTS: We administered 36 treatments of 131I doses up to 120 mCi to 34 previously irradiated patients with recurrent or metastatic brain tumors. Dose-limiting toxicity was reached at 120 mCi and was limited to neurologic or hematologic toxicity. None of the patients treated with less than 120 mCi developed significant neurologic toxicity; one patient developed major hematologic toxicity (MHT). The estimated median survival for patients with glioblastoma multiforme (GBM) and for all patients was 56 and 60 weeks, respectively. CONCLUSION: The MTD for administration of 131I-labeled 81C6 into the SCRCs of previously irradiated patients with recurrent primary or metastatic brain tumors was 100 mCi. The dose-limiting toxicity was neurologic toxicity. We are encouraged by the minimal toxicity and survival in this phase I trial. Radiolabeled mAbs may improve the current therapy for brain tumor patients.

In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison with its murine parent.

The mutant version of the epidermal growth factor receptor EGFRvIII has been found on gliomas and other tumors, but not on normal tissues. Radioiodinated murine (mu) L8A4 monoclonal antibody (MAb) specifically targets EGFRvIII xenografts in vivo when labeled using N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC). A chimeric (ch) MAb consisting of the variable region of muL8A4 and the constant domains of human IgG2 has been developed that has an affinity and radioiodinated immunoreactive fraction comparable to muL8A4. In vitro, both MAbs were internalized and processed by EGFRvIII expressing cell lines (U87MG delta EGFR or NR6M) at similar rates (maximum intracellular retention, 35-40%). In paired-label tissue distribution studies in athymic mice bearing U87MG delta EGFR tumor xenografts, the ch:mu L8A4 uptake ratio in normal tissues rose to greater than 2:1, whereas in tumor, the ratio remained 1:1 throughout the experiment. These results indicate that chL8A4 exhibits similar binding and internalization properties as its murine parent, but suggest different intracellular processing and/or deposition of catabolites in normal tissues for chL8A4.

L-amino acid oxidase (LOX) modulation of melphalan activity against intracranial glioma.

These studies evaluated the efficacy of sequential pretreatment with L-amino acid oxidase (LOX) and LOX antiserum in the modulation of melphalan activity against intracranial glioma in athymic nude mice. LOX produced statistically significant (P < 0.01) depletion of the large neutral amino acids isoleucine, leucine, methionine, phenylalanine, tyrosine, and valine in murine plasma at doses of 100 and 200 micrograms administered intravenously. Polyclonal anti-LOX antibody was successfully produced in mice, rabbits, and goats subsequent to immunization with LOX. Staphylococcal protein A-purified rabbit anti-LOX serum inhibited approximately 50% of LOX activity in vitro relative to control samples. This antiserum was used in vivo to inactivate LOX after it had depleted the large neutral amino acids, thereby preventing LOX-mediated catabolism of melphalan. Inoculation of three mice with rabbit anti-LOX serum after the treatment with LOX (100 micrograms) reduced LOX activity by 100%, 89%, and 100% at 6 h compared with reductions of 80%, 59%, and 52% over the same period in animals receiving LOX alone. In three separate studies using groups of eight to ten mice bearing intracranial human glioma xenografts, pretreatment with LOX followed by anti-LOX serum increased the antitumor activity of melphalan as compared with treatments with melphalan plus LOX, melphalan plus anti-LOX serum, or melphalan alone.

Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display.

EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 x 10(6) members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7-10 ng/ml (110-160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37 degrees C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

Intrathecal 131I-labeled antitenascin monoclonal antibody 81C6 treatment of patients with leptomeningeal neoplasms or primary brain tumor resection cavities with subarachnoid communication: phase I trial results.

We aimed to determine the maximum tolerated dose (MTD) of 131I-labeled 81C6 in patients with leptomeningeal neoplasms or brain tumor resection cavities with subarachnoid communication and to identify any objective responses. 81C6 is a murine IgG monoclonal antibody that reacts with tenascin in gliomas/carcinomas but does not react with normal adult brain. 131I-labeled 81C6 delivers intrathecal (IT) radiation to these neoplasms. This study was a Phase I trial in which patients were treated with a single IT dose of 131I-labeled 81C6. Cohorts of three to six patients were treated with escalating doses of 131I (starting dose, 40 mCi; 20 mCi escalations) on 10 mg 81C6. MTD is defined as the highest dose resulting in serious toxicity in no more than two of six patients. Serious toxicity is defined as grade III/IV nonhematological toxicity or major hematological toxicity. We treated 31 patients (8 pediatric and 23 adult). Eighteen had glioblastoma multiforme. Patients were treated with 131I doses from 40 to 100 mCi. Hematological toxicity was dose limiting and correlated with the administered 131I dose. No grade III/IV nonhematological toxicities were encountered. A partial response occurred in 1 patient and disease stabilization occurred in 13 (42%) of 31 patients. Twelve patients are alive (median follow-up, > 320 days); five are progression free >409 days median posttreatment. The MTD of a single IT administration of 131I-labeled 81C6 in adults is 80 mCi 131I-labeled 81C6. The MTD in pediatric patients was not reached at 131I doses up to 40 mCi normalized for body surface area.

Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas.

Despite molecular biological advances in understanding human cancers, translation into therapy has been less forthcoming; targeting neoplastic cells still requires that tumor-specific markers, preferably those on the cell surface, be identified. The epidermal growth factor receptor (EGFR) exists in a deletion-mutant form, EGFRvIII, which has been identified by genetic and immunological means in a subset of gliomas and non-small cell lung carcinomas. Specific polyvalent antisera to the extracellular portion of the variant were readily induced, but immunization using a synthetic linear peptide representing the unique EGFRvIII primary sequence has been unsuccessful in mice or macaques. We report here five specific monoclonal antibodies (mAbs) developed through long-term immunization protocols using the EGFRvIII-specific synthetic peptide and the intact variant in different formats that maintained secondary and tertiary conformation. These mAbs identify the EGFRvIII on the cell surface with relatively high affinity (KA range, 0.13 to 2.5 x 10(9) M-1) by live cell Scatchard analysis. These mAbs are specific for EGFRvIII as determined by RIA, ELISA, Western blot, analytical flow cytometry, autophosphorylation, and immunohistochemistry. Isolating specific mAbs enabled us to analyze normal and neoplastic human tissue and establish that EGFRvIII is truly tumor specific for subsets of breast carcinomas and for previously reported non-small cell lung carcinomas and gliomas. Also, this receptor is not expressed by any normal human tissues thus far examined, including elements of the peripheral, central nervous, and lymphoid systems. With mAbs, we identified a higher incidence of EGFRvIII positivity in gliomas than previously described and identified an EGFRvIII-positive subset of breast tumors; also, we observed that the EGFRvIII epitope is not expressed in normal tissues, and we demonstrated the localizing and therapeutic potential of the mAbs for tumors expressing this epitope. Our observations strongly warrant development of this mAb-antigen system as therapy for breast, lung, and central nervous system tumors.

Phase I studies of treatment of malignant gliomas and neoplastic meningitis with 131I-radiolabeled monoclonal antibodies anti-tenascin 81C6 and anti-chondroitin proteoglycan sulfate Me1-14 F (ab')2--a preliminary report.

The advent of monoclonal antibody (MAb) technology has made Ehrlich's postulate of the 'magic bullet' an attainable goal. Although specific localization of polyvalent antibodies to human gliomas was demonstrated in the 1960s, the lack of specific, high affinity antibody populations and of defined target antigens of sufficient density precluded therapeutic applications. Not until the identification of operationally specific tumor-associated antigens (present in tumor tissue but not normal central nervous system tissue); production of homogeneous, high affinity MAbs to such antigens; and the use of compartmental administration (intrathecal or intracystic), has the promise of passive immunotherapy of primary and metastatic central nervous system neoplasms been recognized. We report here preliminary data from Phase I studies of the compartmental administration of the anti-tenascin MAb 81C6 and F(ab2)2 fragments of MAb Me1-14, which recognizes the proteoglycan chondroitin sulfate-associated protein of gliomas and melanomas, to patients with primary central nervous system tumors or tumors metastatic to the central nervous system. Phase I dose escalation studies of intracystically administered 131I-labeled anti-tenascin MAb 81C6 to either spontaneous cysts of recurrent gliomas or surgically created cystic resection cavities have resulted in striking responses. Of five patients with recurrent cystic gliomas treated, four had partial responses, clinically or radiographically. Similarly, in patients with surgically created resection cavities, a partial response at the treatment site and extended stable disease status has been obtained following intracystic administration of 131I-labeled 81C6. No evidence of hematologic or neurologic toxicity has been observed in either patient population, with the exception of transient exacerbation of a pre-existing seizure disorder in a single patient. Dosimetry calculations indicated high intracystic retention for four to six weeks with little or no systemic dissemination; estimated total doses intracystically ranged from 12,700-70,290 rad. Intrathecal administration of labeled MAbs to patients with neoplastic meningitis is more difficult to assess in terms of clinical responsiveness. Of patients so treated with either 131I-labeled 81C6 or 131I-labeled Me1-14 (F(ab)2, cerebrospinal fluid and radiographic responses have been achieved, and survival prolongation through maintenance of stable disease has been observed in several cases. Initial results from pHase I dose escalation trials are encouraging in terms of the proportion of cases of disease stabilization and partial and complete responses obtained. Importantly, neurotoxicity has been virtually nonexistent, and hematologic toxicity rare and rapidly responsive to treatment.(ABSTRACT TRUNCATED AT 400 WORDS)