Rats were anaesthetised around 9 a.m. in a plastic box with isoflurane (Forene, Abott, Wiesbaden, Germany) at a rising concentration up to 4% using an evaporator (Forane Vapor 19.3, Dräger AG, Lübeck, Germany). Animals were weighed, individually marked at the tail and the neck region was shaved and disinfected with 70% ethanol. Then, using a syringe with a 27-gauge needle, either fremanezumab or isotype control antibody (30 mg/kg) was subcutaneously injected, evenly distributed 2 cm left and right from the midline and 5 cm caudal of the occiput. As active anti-CGRP antibody, fremanezumab (Teva Pharmaceuticals, Redwood City, CA, USA) diluted in saline (10 mg/mL) was used, alternatively a human IgG2 antibody (Teva Pharmaceuticals) targeting keyhole limpet hemocyanin (KLH) as the isotype control antibody, to demonstrate that effects seen only in the fremanezumab group are specifically due to targeting CGRP, was used. The examiners were blinded to the identity of the antibodies. Animals were placed back in their cage, where they recovered from anaesthesia usually within 2–3 min. During the following days, they were inspected every day to register any unusual behaviour. Body weight of the animals was measured 1, 3 or 10 days after control antibody or fremanezumab administration.
Fremanezumab
Manufactured by Teva Pharmaceuticals
Sourced in United States
Fremanezumab is a monoclonal antibody developed by Teva Pharmaceuticals. It is designed to target the calcitonin gene-related peptide (CGRP), which is involved in the pathophysiology of migraine. The core function of Fremanezumab is to neutralize the activity of CGRP, thereby reducing the frequency and severity of migraine attacks.
Automatically generated - may contain errors
Lab products found in correlation
Isotype control antibody, by Teva Pharmaceuticals (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Calcitonin, by Bachem (1 mentions)
Lamp1, by Abcam (1 mentions)
αcgrp, by Bachem (1 mentions)
Goat anti human 594, by Thermo Fisher Scientific (1 mentions)
Amylin, by Bachem (1 mentions)
6 protocols using fremanezumab
1
Subcutaneous Anti-CGRP Antibody Administration in Rats
2
Receptor Binding and Trafficking Assay
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Human αCGRP, amylin, adrenomedullin, intermedin and calcitonin were
purchased from Bachem. Erenumab, a CGRP receptor monoclonal antibody
(Amgen; lot 1093104), fremanezumab, a CGRP ligand monoclonal antibody
(Teva Pharmaceuticals; lot E15204A001) and isotype control IgG2
antibody (prepared in-house) were used. For flow cytometry, the
following antibodies were used: Anti-human IgG Fc APC (Biolegend),
anti-human IgG Fc BV421 Biolegend), anti-HA.11 PE (Biolegend), Human
c-Myc Alexa Fluor 647-conjugated Antibody (R&D systems) and
anti-myc-FITC (Sigma). For imaging experiments, the following
antibodies were used: Early endosomal marker (early endosomal antigen
1 (EEA1), Abcam), lysosomal marker (lysosomal-associated membrane
protein 1 (LAMP1), Abcam), late endosome marker (Ras-related protein
Rab11, Cell Signaling), goat anti-human 594 and goat anti-rabbit Alexa
Fluor 647 (Invitrogen). The small molecule CGRP receptor antagonist
telcagepant (MedChemExpress) was used.
purchased from Bachem. Erenumab, a CGRP receptor monoclonal antibody
(Amgen; lot 1093104), fremanezumab, a CGRP ligand monoclonal antibody
(Teva Pharmaceuticals; lot E15204A001) and isotype control IgG2
antibody (prepared in-house) were used. For flow cytometry, the
following antibodies were used: Anti-human IgG Fc APC (Biolegend),
anti-human IgG Fc BV421 Biolegend), anti-HA.11 PE (Biolegend), Human
c-Myc Alexa Fluor 647-conjugated Antibody (R&D systems) and
anti-myc-FITC (Sigma). For imaging experiments, the following
antibodies were used: Early endosomal marker (early endosomal antigen
1 (EEA1), Abcam), lysosomal marker (lysosomal-associated membrane
protein 1 (LAMP1), Abcam), late endosome marker (Ras-related protein
Rab11, Cell Signaling), goat anti-human 594 and goat anti-rabbit Alexa
Fluor 647 (Invitrogen). The small molecule CGRP receptor antagonist
telcagepant (MedChemExpress) was used.
3
Subcutaneous Administration of Anti-CGRP mAb
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Administration of mAbs was performed as previously reported [27 (link),28 (link),29 (link)]. In short, either 30 mg/kg of the anti-CGRP mAb fremanezumab, or the isotype control mAb (both Teva Pharmaceuticals, Redwood City, CA, USA) diluted in saline (10 mg/mL), was subcutaneously injected into the neck of the animals under short isoflurane anesthesia. fremanezumab binds to an epitope that is identical in human α-CGRP, β-CGRP, and rat β-CGRP. There is one similar amino acid change within the epitope for rat α-CGRP, which has a minor effect on overall binding affinity. The operator was blinded to the identity of the antibodies. The animals were individually marked, placed back in their cages, and inspected daily regarding their health state and behavior until they were used for further procedures.
4
Subcutaneous Injection of Anti-CGRP Antibody in Rats
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The animals were divided into two groups, one group that received isotype control antibody and the other anti-CGRP antibody, fremanezumab (Teva Pharmaceuticals, Redwood City, CA, USA). The rats were transferred from their housing cage in a plastic box and anesthetized around 9 a.m. with an increasing concentration up to 4% isoflurane applied by an evaporator (Forane Vapor 19.3, Dräger AG, Lübeck, Germany). The animals were weighed, individually marked at the tail, and shaved in the neck region. After disinfecting the skin with 70% ethanol, 30 mg/kg anti-CGRP antibody or the same dose of isotype control antibody diluted in saline (10 mg/mL) was subcutaneously injected using a syringe with a 27G needle into the shaved area 2 cm left and right from the midline and 5 cm caudal of the occiput. Examiners performing the injections were blinded to the treatment. The animals were placed back into the housing cage where they recovered from anesthesia within 2–3 min. The animals were inspected two times every following day.
5
Subcutaneous CGRP Antibody Administration in Rats
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The rats were anaesthetized around 9 a.m. in a plastic box with a concentration of isoflurane increasing up to 4% (Forene, Abott, Wiesbaden, Germany), applied with an evaporator (Forane Vapor 19.3, Dräger AG, Lübeck, Germany). The animals were weighed, and the neck region was shaved and disinfected with 70% ethanol. Then, 30 mg/kg anti-CGRP antibody fremanezumab or isotype control antibody (Teva Pharmaceuticals, Redwood City, CA, USA) diluted in saline (10 mg/mL) was subcutaneously injected in an even distribution 2 cm left and right from the midline and 5 cm from the caudal of the occiput, using a syringe with a 27-gauge needle. A human IgG2 antibody-targeting keyhole limpet hemocyanin (KLH) was used as the isotype control antibody to assess the specific effect of the targeting CGRP. The examiners were blinded as to the identity of the antibodies. The rats were marked at their tail for identification and placed back in their cage, where they recovered from the anaesthesia usually within 2–3 min. The animals were inspected two times on every following day with regard to any unusual behaviour.
6
Preparation and Characterization of Anti-CGRP F(ab')2
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Preparation of F(ab 0 ) 2 Fragment Antibody F(ab 0 ) 2 fragment antibody was used as a tool to investigate the selective targeting of peripheral CGRP without the possible immunomodulatory effects of the fragment crystallizable (Fc) region of the full-length antibody in the tested animal models. Briefly, fremanezumab (fully humanized anti-CGRP monoclonal antibody; Teva Pharmaceuticals, Redwood City, CA) and isotype control antibody (Teva Pharmaceuticals) were digested with pepsin, and affinity chromatography purification of F(ab 0 ) 2 using Kappa-Select columns was performed. Purity and integrity of the F(ab 0 ) 2 were analyzed by SDS-PAGE under reducing and nonreducing conditions. Similar binding affinity of anti-CGRP F(ab 0 ) 2 to CGRP compared with full-length anti-CGRP was confirmed by surface plasmon resonance.
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