saporin

170 entries

Effective antitumor therapy based on a novel antibody-drug conjugate targeting the Tn carbohydrate antigen.

Sedlik C, Heitzmann A, Viel S, Ait Sarkouh R, Batisse C, Schmidt F, De La Rochere P, Amzallag N, Osinaga E, Oppezzo P, Pritsch O, Sastre-Garau X, Hubert P, Amigorena S, Piaggio E (2016) Effective antitumor therapy based on a novel antibody-drug conjugate targeting the Tn carbohydrate antigen. Oncoimmunology 5:e1171434. doi: 10.1080/2162402X.2016.1171434

Summary: Scientists wanted to study the potential of Chi-Tn, a monoclonal antibody against a glycol-peptidic tumor-associated antigen, as an anticancer antibody-drug conjugate. They demonstrated that Chi-Tn specifically targeted tumor cells in vivo, using flow cytometry and deconvolution microscopy to show that Chi-Tn is rapidly internalized. Chi-Tn-SAP (ATS Custom Services) effectively killed Tn-positive cells, but had no effect on Tn-negative cells. Saporin (Cat. #PR-01) was used as control. The cytotoxicity of the Chi-Tn-SAP correlated with the level of tumoral Tn expression.

Related Products: Saporin (Cat. #PR-01), Custom Conjugates

Saporin Safety

Over the years, ATS has frequently been asked about Saporin’s safety for use in the lab as well as when used clinically. Residual awareness of alternate Ribosome-Inactivating Proteins (RIPs) and ‘toxins’ such as Ricin have caused some researchers new to the use of RIPs to question the belief that Saporin is safe. Unlike Type 2 RIPs (such as Ricin), Type I RIPs, like Saporin have no binding chain and consequently no means of entering the physiological space necessary for the protein to act as a toxin. The following is a review of safety in handling and potential toxicity within the human body for systemic events not related to normal research applications of Saporin conjugates, including Substance P-Saporin (SP-SAP), which is a therapeutic under development for the treatment of chronic pain.

The acute LD50 for saporin in mice (25 g) is 6.8 mg/kg;[1] that would translate in humans (75 kg) to 510 mg! A concentration of about 100 nM is the threshold to see even a vague hint of saporin toxicity. In human blood, that would correspond to 24 mg injected systemically into a person. The fermentation process to produce recombinant saporin has a titer of 2 mg/L meaning that the production broth itself contains no more than 67 nM concentration of saporin. Furthermore, the final protein concentrations from production batches of recombinant Saporin used in our drug are 4 mg/ml, meaning 6 mL of final material would need to accidentally end up in a human before the ‘hint of toxicity’ threshold would potentially be met.

The toxicology studies of SP-SAP contained within ATS’s IND prior to the current human Phase I clinical trial evaluated effects related to the intended method of administration, intrathecal local injection. SP-SAP is not expected to ever be a self-administered therapy, so the effects of gross off-target events, such as accidental auto-injection, swallowing, spillage, or immersion were not considered.

The table below[2] highlights antibody-saporin conjugates approved by the FDA for Phase I/II clinical trials in humans. The therapeutics listed below were administered intravenously and imply what the FDA accepted as non-toxic levels of saporin-based conjugates in these studies.

Looking more closely at the study by French et al.,[3] several milligrams of antibody conjugate were repeatedly injected into human patients under a FDA regulated clinical trial and peak serum levels tested, demonstrating rapid clearing of saporin from the system.

As a company that specializes in Saporin, our two-plus decades of experience working with the protein in research, preclinical, and clinical environments has taught us that with minimal standard laboratory precautions users are not at any real risk of toxic effects. Even our CSO, after 30+ years of working with Saporin exhibits undetectable levels of Saporin antibodies in his blood!

References:

  1. Thorpe PE et al. An immunotoxin composed of monoclonal anti-thy 1.1 antibody and a ribosome-inactivating protein from Saponaria officinalis: potent antitumor effects in vitro and in vivo. J Natl Cancer Inst 75:151-159, 1985.
  2. Polito L et al. Immunotoxins and other conjugates containing saporin-s6 for cancer therapy. Toxins (Basel) 3(6):697-720, 2011.
  3. French RR et al. Response of B-cell lymphoma to a combination of bispecific antibodies and saporin. Leuk Res 20(7):607-17, 1996.

Functional characterization of a mouse model for central post-stroke pain.

Gritsch S, Bali K, Kuner R, Vardeh D (2016) Functional characterization of a mouse model for central post-stroke pain. Mol Pain 12:1744806916629049. doi: 10.1177/1744806916629049

Summary: While clinical evidence has pointed toward central pain pathway dysfunction in central post-stroke pain (CPSP), the underlying mechanisms have not been defined. In this work the authors created a mouse model of CPSP through lesions of the thalamic ventral posterolateral nucleus. In order to examine the role of neurokinin-1 receptor-expressing (NK1R) neurons in lamina I/III of the spinal cord in the development and maintenance of CPSP the authors administered 1 μmol intrathecal injections of SSP-SAP (Cat. #IT-11). Saporin (Cat. #PR-01) was used as a control. While the NK1R+ neurons in the spinal cord were not involved in establishing CPSP, the data indicate that sensory changes in the mice are comparable to those observed in human patients with CPSP.

Related Products: SSP-SAP (Cat. #IT-11), Saporin (Cat. #PR-01)

Roles of isolectin B4-binding afferents in colorectal mechanical nociception.

La J, Feng B, Kaji K, Schwartz E, Gebhart G (2016) Roles of isolectin B4-binding afferents in colorectal mechanical nociception. Pain 157:348-354. doi: 10.1097/j.pain.0000000000000380

Summary: Primary afferent neurons are often classified as peptidergic or non-peptidergic. One characteristic of the non-peptidergic neurons is that they bind isolectin-B4. In the spinal cord these neurons terminate mainly in inner lamina II. Non-peptidergic neurons in the spinal cord have been found to be involved in various aspects of pain response. In this work the authors examined the role of non-peptidergic neurons in the viscerosensory system. Rats received 1.5 μg of intrathecal recombinant IB4-SAP (Cat. #IT-10) between the L5 and L6 vertebrae. Saporin (Cat. #PR-01) was used as a control. While IHC demonstrated that a majority of viscerosensory L6 colon DRG neurons are IB4+, they do not play a significant role in colorectal mechano-nociception.

Related Products: IB4-SAP (Cat. #IT-10), Saporin (Cat. #PR-01)

Safety of the Toxin

Q: Our QA group wants to know about the safety of the toxin in your conjugates. What precautions should we take in handling saporin products?

A: Saporin is a Type 1 ribosome-inactivating protein (RIP), due to its N-glycosidase activity, from the seeds of Saponaria officinalis. It was first described by Fiorenzo Stirpe and his colleagues in 1983 in an article that illustrated the unusual stability of the protein.[1] Among the RIPs are some of the most toxic molecules known, including ricin and abrin (the latter is the poison preferred by the characters in the movie The Blue Lagoon). These toxins contain a second protein strand that inserts the RIP into a cell, making it able to enzymatically inactivate the ribosomes, shutting down protein synthesis and resulting in cell death and eventually causing death of the victim.

Saporin does not possess a cell-binding chain[2] and has no method of internalization without a targeting agent to escort it into a cell. It is this fact that also adds to the safety of its use in the lab. Autoclaving or exposure to 0.2 M NaOH is sufficient to decontaminate material that has been in contact with Saporin and its conjugates. The LD50 for Saporin in mice is 4-8 mg/kg.[3] With an average person, let’s say 75 kg, that would be more than what you might have in your freezer, let alone be able to inject in yourself. Targeted Saporin, if targeted to a human epitope, should be handled more carefully, but due to logistics, it’s difficult to imagine an effect.  Hundreds of articles in the scientific literature (search “Saporin” in Pub Med) have demonstrated tremendous specificity in targeting neuronal cells with many different Saporin conjugates and by many different scientists.

References

  1. Stirpe F et al. Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort) of Agrostemma githago L. (corn cockle) and of Asparagus officinalis (asparagus) and from the latex of Hura crepitans L. (sandbox tree). Biochem J 216:617-625, 1983.
  2. Barthelemy I et al. The expression of saporin, a ribosome-inactivating protein from the plant Saponaria officinalis, in Escherichia coli. J Biol Chem 268(9):6541-6548, 1993.
  3. Stirpe F et al. Hepatotoxicity of immunotoxins made with saporin, a ribosome-inactivating protein from Saponaria officinalis. Virchows Arch B Cell Pathol Incl Mol Pathol 53(5):259-271, 1987.

Alterations in the rostral ventromedial medulla after the selective ablation of μ-opioid receptor expressing neurons.

Harasawa I, Johansen J, Fields H, Porreca F, Meng I (2016) Alterations in the rostral ventromedial medulla after the selective ablation of μ-opioid receptor expressing neurons. Pain 157:166-173. doi: 10.1097/j.pain.0000000000000344

Summary: The rostral ventromedial medulla (RVM) has both excitatory and inhibitory control over nociceptive neurons in the medullary dorsal horn and spinal cord. Previous work has demonstrated that elimination of mu-opioid receptor-expressing neurons in the RVM reduces stress and injury-induced behavioral hypersensitivity, but the effect of losing these cells on the descending inhibitory system has not been examined. The authors administered 1.2 pmol of Dermorphin-SAP (Cat. #IT-12) to each side of the RVM of rats. Saporin (Cat. #PR-01) was used as a control. Characterization of RVM neurons in lesioned animals showed a reduction in on- and off-cells, but no change in the number of neutral cells. These data indicate that mu-opioid receptor-expressing cells in the RVM are not needed for analgesia produced by activation of RVM neurons.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Saporin (Cat. #PR-01)

Cognitive and motor deficits in a rodent model of Parkinson’s disease displaying concurrent dopamine and acetylcholine loss

Ostock CY, Conti MM, Larose T, Meadows S, Bishop C (2015) Cognitive and motor deficits in a rodent model of Parkinson’s disease displaying concurrent dopamine and acetylcholine loss. Neuroscience 2015 Abstracts 676.26/D33. Society for Neuroscience, Chicago IL.

Summary: Dopamine (DA) loss in Parkinson’s disease (PD) is frequently accompanied by degeneration of acetylcholine neurons within the basal forebrain (BF) and the pedunculopontine nucleus (PPN). Recently, Ach neurons in these nuclei have been implicated in both the motor and non-motor symptoms of PD. However, few rodent models of PD actually account for Ach loss in both the BF and PPN. Here, we evaluated the effects of concurrent BF and PPN Ach loss alone and in combination with striatal DA loss on motor and cognitive performance in a rat model of PD. Sprague-Dawley rats (N = 44) received bilateral: striatal 6-OHDA lesions to deplete DA (DA-lesioned; n = 14), BF (192 IgG-Saporin) and PPN (anti-ChAT Saporin) saporin lesions to deplete Ach (Ach-lesioned; n = 10), combined 6-OHDA + saporin lesions (dual-lesioned; n = 6) , or sham lesions (n = 14). Following recovery from surgery, rats underwent a battery of motor and cognitive behavioral tests. Results indicated that Ach-lesioned and dual-lesioned rats displayed spatial memory deficits on the Morris Water Maze and Spontaneous Alternation tests. DA and Ach lesions alone impaired stepping for the forepaw adjusting steps and vibrissae-elicited paw placement tests and this deficit was exacerbated in dual-lesioned rats. However, only rats with Ach or dual lesions showed motor deficits on the rotarod tests. Collectively, these findings demonstrate that Ach loss may exacerbate cognitive and motor symptoms in PD and highlight the importance of including Ach loss in preclinical models of PD.

Related Products: 192-IgG-SAP (Cat. #IT-01), Anti-ChAT-SAP (Cat. #IT-42), Saporin (Cat. #PR-01)

Immunolesions of melanopsin receptive neurons attenuates the hormonal reproductive axis in the adult but has no effect on growth in immature Peking ducks

Alenciks E, Frazier K, Porter A, Fraley G (2015) Immunolesions of melanopsin receptive neurons attenuates the hormonal reproductive axis in the adult but has no effect on growth in immature Peking ducks. Neuroscience 2015 Abstracts 613.05/R20. Society for Neuroscience, Chicago IL.

Summary: Several light sensitive receptors have been described in the avian brain that are thought to regulate the reproductive axis independently from the eyes and pineal gland. Recently, our lab has described the presence of 3 photoneuroendocrine systems in the Pekin duck: rhodopsin, opsin 5, & melanopsin. We set out to test the hypothesis that melanopsin receptive neurons are necessary to maintain seasonal reproductive status along with growth and development in the Pekin drake. To accomplish these goals we first investigated 50-week-old Pekin drakes that were housed in the aviary at Hope College under long day length (18 hrs lights on) conditions in floor pens. To specifically lesion melanopsin-receptive neurons, 3 μl of an anti-melanopsin-saporin conjugate (MSAP, 100 ng/ul) was injected into the lateral ventricle (n = 10). Control drakes were injected with 3 ul of equimolar unconjugated anti-melanopsin and saporin (SAP, n = 10). The drakes were returned to the aviary after complete recovery. Reproductive behaviors were analyzed weekly in a test pen with adult hens. After 4 weeks, birds were euthanized and body weights were measured, and brains, pituitaries, and testes collected and stored for analyses. To test melanopsin’s effect on immature ducks the same surgery was performed on a group of 10 day old ducks (n= 10). Ducks were weighed weekly starting at 3 days of age. After a final weight was obtained at 50 days of age, ducks were euthanized and a blood sample was collected and sent out for an avian panel. Mature MSAP-treated drakes had significantly (p< 0.001) reduced relative teste weights compared to SAP controls. qRT-PCR analyses (n= 3 per treatment) of anterior pituitary showed a significant reduction (p< 0.001) in both LH-beta and FSH mRNA’s. Immunoctyochemical analyses (n= 3 per treatment) showed a significant reduction in melanopsin and GnRH-immunoreactivities. Immature drake BW did not differ significantly between MSAP and SAP animals at any of the measured days. The data appeared to drift toward significance near the end of the sampling period (p = 0.297). Blood panel results revealed no significant differences between MSAP and SAP animals in any CBC component. Serum glutamic-oxaloacetic transaminase (SGOT) (p= 0.022) and creatine phosphokinase (CPK) values were significantly elevated (p = 0.006) in MSAP animals compared to controls. Although melanopsin neurons in the PMM appear to have an important role in adult drakes, their importance in the growth of immature ducks is still unclear. However, these data underscore the importance of the photoneuroendocrine system in maintaining the reproductive axis along with growth and development in seasonally breeding birds.

Related Products: Melanopsin-SAP (Cat. #IT-44), Saporin (Cat. #PR-01)

Lack of effects on growth and body weight gain after elimination of the leptin receptor from the brain of immature Pekin drakes

Porter LM, Alenciks E, Frazier K, Porter A, Fraley GS (2015) Lack of effects on growth and body weight gain after elimination of the leptin receptor from the brain of immature Pekin drakes. Neuroscience 2015 Abstracts 613.04/R19. Society for Neuroscience, Chicago IL.

Summary: The presence of the hormone leptin (LEP) is a controversial topic in the field of avian physiology. While LEP is well understood in mammals, the hormone has not been definitively verified in avian species. Although the hormone remains elusive, the leptin receptor (LEPR) has been identified and sequenced in multiple avian species. Its role, however, remains unclear. To attempt to deduce the role of the leptin system in birds, we focused on altering the leptin receptor expression in the brain of immature Pekin ducks. We hypothesized that eliminating the LEPR-expressing neurons of the hypothalamus would elicit an increase in body weight, as is the case for mammals. To test this hypothesis, we injected stereotaxically 3 ul of a solution containing a monoclonal antibody (anti-LEPR) conjugated to saporin (LSAP, 100 ng/ul) was injected into the lateral ventricle of 10 day old Pekin ducks (LSAP, N = 10). Control group animals (SAP) were injected with unconjugated antibody and saporin at equimolar concentrations to the LSAP. Ducks were weighed weekly starting at 3 days of age. After a final weight was obtained at 50 days of age, ducks were euthanized and a blood sample was collected and sent out for an avian panel to assay serum glucose and free fatty acids. We found that the elimination of LEPR had no significant effect on the body weights of the ducks (p>0.05). In addition, The CBC panel did not reveal any significant differences in the overall health of the ducks in each treatment group. Our data indicates LEPR may not play a significant role in the regulation of body weight or growth in juvenile ducks.

Related Products: Custom Conjugates, Saporin (Cat. #PR-01)

Retrograde Transport

Q: I’m trying to find out if enough Anti-DBH-SAP will be retrogradely transported and taken up by non targeted sympathetic neurons by bulk fluid-phase endocytosis. Does saporin become degraded after it kills the neuron or does it enter the extracellular matrix?

A: It is very unlikely that a targeted toxin such as Anti-DBH-SAP is freed from the targeted neuron in a meaningful condition. There has never been a reported identification of a targeted toxin, functionally or not, after it has eliminated its targeted neuron. Current evidence indicates that effective suicide transport agents undergo endocytosis at nerve terminals followed by retrograde axonal transport of the endocytic vesicles containing the toxin. Experiments using vincristine have shown that the retrograde axonal transport of suicide transport toxins utilizes the fast transport system (microtubules). However, it is not known what determines whether or not a specific toxin-ligand undergoes axonal transport after internalization.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

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