Afuco™ Anti-Human TNF ADCC Recombinant Antibody (AVX-470), ADCC Enhanced (CAT#: AFC-601CL)

Anti-TNF ADCC Enhanced Antibody (AVX-470) is an ADCC enhanced antibody produced by our Afuco™ platform. AVX-470 is a novel polyclonal antibody specific for human TNF used for Inflammatory bowel disease (IBD). IBD is a chronic inflammatory disease of the gastrointestinal tract, which is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF).

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Neut

Figure 1 TNF-binding potency, TNF-binding affinity and TNF-neutralizing potency of AVX-470 and AVX-470m.

Figure 1 TNF-binding potency, TNF-binding affinity and TNF-neutralizing potency of AVX-470 and AVX-470m.

(A) TNF-binding potency of AVX-470 and control Ig measured in duplicate wells by ELISA on rhTNF-coated plates. Representative experiment. AVX-470 mean titer = 1.2 µg/mL in 8 experiments. (B) TNF-binding potency of AVX-470m and control Ig measured in duplicate wells by ELISA on rmTNF-coated plates. AVX-470m mean titer = 4.3 µg/mL in 3 experiments (C) TNF-binding affinity in a representative experiment of AVX-470A affinity-purified antibody and infliximab for human TNF analyzed in duplicate wells by competition ELISA. IC50 concentration is an estimate of affinity. AVX-470A mean IC50 = 260 pM from 2 experiments; infliximab mean IC50 = 280 pM from 2 experiments. (D) Affinity of AVX-470mA affinity-purified antibody and TN3 antibody for murine TNF in a representative experiment analyzed in duplicate wells by competition ELISA. AVX-470mA IC50 = 550 pM; TN3 IC50 = 26 pM. Means from 2 experiments. (E) TNF-neutralization potency of AVX-470 measured in an L929 cell-based assay. Representative experiment. AVX-470 mean IC50 = 22 µg/mL over 31 experiments (F) TNF-neutralization potency of AVX-470A and infliximab measured in an L929 cell-based assay. Representative of 2 separate experiments. AVX-470A mean IC50 = 50 ng/mL; infliximab mean IC50 = 56 ng/mL.

Bhol, K. C., Tracey, D. E., Lemos, B. R., Lyng, G. D., Erlich, E. C., Keane, D. M.,... & Fox, B. S. (2013). AVX-470: a novel oral anti-TNF antibody with therapeutic potential in inflammatory bowel disease. Inflammatory bowel diseases,19(11), 2273-2281.

Figure 2 Oral AVX-470m efficacy in murine IBD models as assessed by endoscopy.

Figure 2 Oral AVX-470m efficacy in murine IBD models as assessed by endoscopy.

(A) Representative endoscopic images in TNBS-colitis (Day 5). Preventative treatment with saline, AVX-470m (10 mg/day shown) or control Ig (3 mg/day). (B) Representative endoscopic images in DSS-colitis (Day 12). Preventative treatment with saline, AVX-470m (10 mg/day shown) or control Ig (3 mg/day). (C) Endoscopy scores in TNBS-colitis (Day 5). 8-12 mice per group. *, P <.05 compared with saline-treated group. (D) Endoscopy scores in DSS-colitis prevention (Day 12). 10-12 mice per group. *, P <.05 compared with control Ig group. (E) Endoscopy scores in established DSS-colitis study (Day 19). 12-15 mice per group. *, P <.05 compared with salinetreated group.

Bhol, K. C., Tracey, D. E., Lemos, B. R., Lyng, G. D., Erlich, E. C., Keane, D. M.,... & Fox, B. S. (2013). AVX-470: a novel oral anti-TNF antibody with therapeutic potential in inflammatory bowel disease. Inflammatory bowel diseases,19(11), 2273-2281.

Figure 3 Oral bovine Ig localizes in colon tissues, but with minimal systemic exposure, in mice with DSS-induced colitis.

Figure 3 Oral bovine Ig localizes in colon tissues, but with minimal systemic exposure, in mice with DSS-induced colitis.

(A) Representative sections of colon tissues showing localization of bovine Ig by IHC (brown staining). Colon sections were from mice with DSS-induced colitis on Day 19 treated with AVX-470m (10 mg/day) or control Ig (3 mg/day) or from normal mice without colitis (top right panel) treated for 28 days with AVX-470m (10 mg/day). Colon sections show mucosa and lamina propria regions except lower left panel shows muscularis mucosa (top) and submucosa (bottom) regions. (B) Pie chart of sera from oral bovine Ig-treated mice showing the percentage positive for bovine Ig by ELISA. Sera were obtained from individual mice on the day of sacrifice from TNBS and DSS colitis prevention studies and three established DSS-induced colitis studies with oral administration of AVX-470m or control Ig.

Bhol, K. C., Tracey, D. E., Lemos, B. R., Lyng, G. D., Erlich, E. C., Keane, D. M.,... & Fox, B. S. (2013). AVX-470: a novel oral anti-TNF antibody with therapeutic potential in inflammatory bowel disease. Inflammatory bowel diseases,19(11), 2273-2281.

Inhib

Figure 4 Oral AVX-470m inhibits colon inflammation and inflammatory marker expression in established DSS-induced colitis.

Figure 4 Oral AVX-470m inhibits colon inflammation and inflammatory marker expression in established DSS-induced colitis.

(A) H&E stained colon sections were scored for the presence of inflammatory aggregates and the degree of diffuse inflammation on a scale of 0-4; magnification 400×. (B) Representative IHC staining of Day 19 colon sections for the macrophage marker CD68. Shown are representative sections from normal mice (No DSS), or mice treated with saline, AVX-470m or control Ig. (C) IHC scores for the saline and AVX-470m (10 mg/day) treatment groups on a scale of 0-4 for a panel of inflammation markers: MPO (myeloperoxidase, a neutrophil granulocyte marker); CD3, a pan T-cell marker; and cytokines TNF, IFNγ, IL-1β, IL-6 and IL-12p40 (which detects IL-12 and IL-23). (D) Representative IHC staining for TNF in Day 19 colon sections. Shown are representative sections from normal mice (No DSS), or mice treated with saline, AVX-470m or control Ig. (E) Relative mRNA expression levels of a panel of inflammation markers as measured by qRT-PCR for the saline and AVX-470m (10 mg/day) treatment groups. *, P <.05 for treatment groups compared with the saline-treated group. Results are expressed as mean ± SEM.

Bhol, K. C., Tracey, D. E., Lemos, B. R., Lyng, G. D., Erlich, E. C., Keane, D. M.,... & Fox, B. S. (2013). AVX-470: a novel oral anti-TNF antibody with therapeutic potential in inflammatory bowel disease. Inflammatory bowel diseases,19(11), 2273-2281.

FuncS

Figure 5 Measurement of bovine Ig in stool homogenates after 4 weeks of treatment with AVX-470 or placebo.

Figure 5 Measurement of bovine Ig in stool homogenates after 4 weeks of treatment with AVX-470 or placebo.

(A) Dose-dependent increase in the percentage of patients with detectable levels of bovine Ig in stool at Week 4. Bovine Ig in the placebo group was presumably from dietary sources. Patient numbers varied between the groups: placebo, n = 8; AVX-470 0.2g/day, n = 8; 1.6g/day, n = 11; 3.5g/day, n = 6.

Harris, M. S., Hartman, D., Lemos, B. R., Erlich, E. C., Spence, S., Kennedy, S.,... & Fox, B. S. (2016). AVX-470, an orally delivered anti-tumour necrosis factor antibody for treatment of active ulcerative colitis: results of a first-in-human trial. Journal of Crohn's and Colitis, 10(6), 631-640.

Inhib

Figure 6 Serum human anti-bovine Ig antibody (HABA) levels. Mean percentage inhibition of optical density over time for AVX-470 groups and pooled placebo.

Figure 6 Serum human anti-bovine Ig antibody (HABA) levels. Mean percentage inhibition of optical density over time for AVX-470 groups and pooled placebo.

Weeks 1-4 represent the treatment period; Week 5 corresponds to the follow-up visit 1 week post-cessation of treatment (A) pooled placebo; (B) AVX-470 0.2g/day; (C) AVX-470 1.6g/day; (D) AVX-470 3.5g/day. Each line represents an individual patient. Week 0 = baseline samples collected prior to the first dose of AVX-470. Screening cut point = 0.162 OD (dotted line) based on 55 normal serum samples.

Harris, M. S., Hartman, D., Lemos, B. R., Erlich, E. C., Spence, S., Kennedy, S.,... & Fox, B. S. (2016). AVX-470, an orally delivered anti-tumour necrosis factor antibody for treatment of active ulcerative colitis: results of a first-in-human trial. Journal of Crohn's and Colitis, 10(6), 631-640.

Figure 7 Changes in serum CRP and IL-6 were also most pronounced in the 3.5g/day dose group

Figure 7 Changes in serum CRP and IL-6 were also most pronounced in the 3.5g/day dose group

(A) Changes in CRP levels for each patient at Week 4 vs baseline (mean ± SE). Log-transformed changes for each treatment group were compared by 2-sided, 2-sample t -tests († p = 0.052, 3.5g/day vs placebo). (B) Raw change (mean ± SE) in IL-6 levels compared by ANCOVA as a function of treatment adjusting for baseline for all dose groups (p = 0.069) and AVX-470 3.5g/day vs placebo (†† p = 0.015).

Harris, M. S., Hartman, D., Lemos, B. R., Erlich, E. C., Spence, S., Kennedy, S.,... & Fox, B. S. (2016). AVX-470, an orally delivered anti-tumour necrosis factor antibody for treatment of active ulcerative colitis: results of a first-in-human trial. Journal of Crohn's and Colitis, 10(6), 631-640.


Specifications

  • Host Species
  • Cow
  • Derivation
  • Cow
  • Type
  • ADCC enhanced antibody
  • Species Reactivity
  • Human
  • Applications
  • Neut, FuncS
  • Related Disease
  • Ulcerative Colitis

Product Property

  • Purity
  • >95% as determined by analysis by SDS-PAGE
  • Storage
  • ≤1 year at -20°C.

Applications

  • Application Notes
  • AVX-470 and AVX-470m TNF-Binding and Neutralization Activities
    The ability of AVX-470 and AVX-470m to bind to TNF was measured in a direct enzyme-linked immunosorbent assay (ELISA). Microtiter plates were coated with human or mouse TNF at 1 µg/mL and binding was detected using horseradish peroxidase-conjugated sheep anti-bovine antibody using standard techniques. Titers, expressed as µg/mL, were defined as the concentration of test article resulting in an absorbance value of 0.2. The ability of antibody to neutralize TNF was determined using the murine fibroblast cell line L929. These cells are killed by TNF, especially in the presence of actinomycin D, and a neutralizing anti-TNF antibody will prevent this cytotoxicity15. IC50 values were calculated as the concentration of AVX-470/AVX-470m resulting in 50% inhibition of TNF-mediated killing of L929 cells. Affinity of AVX-470A and AVX-470mA for TNF by Competition ELISA Serial dilutions of human or mouse TNF were incubated with defined levels of anti-TNF antibodies or control bovine Ig. The levels of TNF-specific antibodies used in these assays were previously determined to be in the linear portions of titration curves in direct ELISAs: AVX-470A (35 ng/mL), AVX-470mA (50 ng/mL), infliximab (2 ng/mL) and TN3 (TN3-19.12, 10 ng/mL). The antibody/TNF dilution mixtures were then added to the TNF-coated plates which were incubated, washed, incubated with horseradish peroxidase-labeled anti-bovine, human or hamster IgG secondary antibodies, washed and read on a plate reader. Data were analyzed using Gen5 Data Analysis Software and affinities were estimated as dissociation constants (KD) calculated from the amount of soluble TNF required to inhibit 50% of the binding of the antibodies to plate-bound TNF. Mouse models of colitis Animal experiments were conducted in the AAALAC-approved facility of Biomodels, LLC with ethical review approval from Biomodels' Institutional Animal Care and Use Committee. Colitis was induced in male C57BL/6 mice by exposure to 3% DSS in drinking water from Day 0 to Day 512 or by intrarectal administration of 4 mg TNBS in 50% ethanol on Day 0. Treatments with vehicle (saline), AVX-470m (1-10 mg/day), control Ig (3 or 10 mg/day), or prednisolone (3 mg/kg/day) were administered in volumes of 0.2 mL/mouse twice a day by oral gavage. Treatments with etanercept (10 mg/kg) were administered by intraperitoneal injection every other day. In prevention studies, treatment began one day before the induction of colitis and continued through day 3 for TNBS colitis or through day 12 for DSS colitis. In treatment studies, treatment began on day 6 and continued through day 19. Colitis severity was assessed using video endoscopy (Karl Storz Endoskope, Tuttlingen, Germany) a method that provides a robust clinical readout of disease severity14,16. Images were scored by a blinded observer: 0 = normal, 1 = loss of vascularity, 2 = loss of vascularity and friability, 3 = friability and mucosal erosions, and 4 = ulcerations and bleeding.

Target

  • Alternative Names
  • TNF; DIF; TNFA; TNFSF2; TNF-alpha; tumor necrosis factor

Related Resources

  • Related Diseases
  • Related Signaling Pathways

Product Notes

This is a product of Creative Biolabs' Hi-Affi™ recombinant antibody portfolio, which has several benefits including:

• Increased sensitivity
• Confirmed specificity
• High repeatability
• Excellent batch-to-batch consistency
• Sustainable supply
• Animal-free production

See more details about Hi-Affi™ recombinant antibody benefits.

Downloads

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

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