Anti-epidermal Growth Factor Receptor mAb Research Articles

Meditopia: Meditope‐Enabled Bonds and their Affinity with Cetuximab

A meditope is a peptide found in the fragment antigen‐binding (Fab) region of an antibody, and can be used to bind substances, such as monoclonal antibodies (mAbs) and pharmaceuticals. We centered on four mutations that can increase the affinity between the meditope and the mAb, allowing for a stronger bond. Our focus was on the anti‐epidermal growth factor receptor mAb cetuximab, which tracks the overexpression of the epidermal growth factor receptor (EGFR) by cancer cells. Utilizing a meditope, cetuximab can be linked to chemotherapy, creating a more effective treatment that only attacks cancer cells, significantly reducing possible long‐term side effects. In this study, yeast display and deep mutational scanning tracked, enhanced, and joined four mutations within the meditope, increasing affinity, or bond strength, for cetuximab 10‐fold, from Kd 130 nm to 15 nm. Those mutations led to heightened cetuximab recruitment to EGFR‐overexpressing cancer cells, and therefore more efficient targeting of cancer. The methods used in this study can develop other effective meditope‐linked monoclonal antibodies and chemotherapy treatments. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team will use 3‐D modeling and printing technology to examine structure‐function relationships of meditope‐mAb binding within cetuximab. The visual model will be a valuable tool in developing our story.

Significant antitumor effect of an antibody against TMEM180, a new colorectal cancer-specific molecule.

The present state of therapy for colorectal cancer (CRC) is far from satisfactory, highlighting the need for new targets for this disease. We identified a new CRC‐specific molecule, TMEM180, a predicted 11‐pass transmembrane protein that apparently functions as a cation symporter. We developed an anti‐TMEM180 mAb and then succeeded in humanizing the mAb. Immunohistochemistry (IHC) in CRC with the mAb showed a similar positivity rate as compared with anti‐epidermal growth factor receptor mAb, and IHC with anti‐TMEM180 mAb did not show staining in major organs used in this study. Immune electron microscopy clearly indicated that TMEM180 was present on the tumor exosome. The TMEM180 promoter region contains 10 hypoxia‐responsive element consensus sequences; accordingly, SW480 cells upregulated TMEM180 under low‐oxygen conditions. Anti‐TMEM180 mAb has in vitro antibody‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity activity, and SW480 CRC xenografts were eradicated by the mAb. These data indicate that TMEM180 may be a new CRC marker and that a mAb against this protein could be used as antibody‐based therapy against CRC.

Efficacy of Patritumab (U3-1287), a Fully Human Anti–Human Epidermal Growth Factor Receptor 3 (Her3) Monoclonal Antibody (Mab), in Head and Neck (H&N) Cancer Models

ABSTRACT Aim: Activation of the receptor tyrosine kinase HER3 and its downstream signaling pathways promote the development of various cancers. HER3 facilitates phosphatidylinositol-3-kinase/AKT signaling via heterodimeric interaction with other HER family members, mediated by binding of the HER3 ligand heregulin (HRG). Patritumab (P) competes with HRG for receptor binding and mediates internalization of HER3. We examined expression of HRG in H&N cancer models and patient samples and evaluated the in vitro and in vivo efficacy of P alone or with cetuximab (C). Methods: Selection of H&N cell lines for reverse transcription polymerase chain reaction was based on microarray data. Tumor cells were treated with 10 µg/mL of P, C, or a combination of both, and cell lysates were analyzed for HER3/pHER3-, AKT/pAKT- and extracellular regulated kinase (ERK)/pERK-levels by Western blotting. The effect of P and C on proliferation and apoptosis was evaluated by bromodeoxyuridine- and caspase-3/7 assays. To address efficacy in vivo, mice bearing tumor xenografts were treated with P and/or C twice weekly, and tumor volumes were assessed. HRG expression was also evaluated in patient samples. Results: HRG expression was frequent in H&N cell lines. HRG mRNA expression was elevated in 67% of patient tumor samples compared with controls. In H&N cell lines, basal HER3 and AKT activation were inhibited by P, with additional blockage of the ERK pathway by co-treatment with C. In vitro combination effects were also observed on the inhibition of proliferation and caspase-3/7 activation. Co-administration of P + C enhanced tumor growth inhibition (TGI) in vivo. Conclusions: High expression frequencies of HRG were observed in H&N tumor cell lines and patient samples, and were associated with single-agent efficacy of P on cell signaling in vitro and on TGI in vivo. The combination of P + C extended the inhibition of signaling pathways, induced apoptosis and inhibited proliferation in vitro, and enforced TGI in vivo. Thus, these data strongly support the combination of P with an anti-epidermal growth factor receptor mAb as a promising strategy for the treatment of H&N cancer. Disclosure: All authors have declared no conflicts of interest.

Phase II trial of preoperative radiotherapy (RT) and panitumumab (PAN) in KRAS wild type (wt) rectal cancer (RC).

e14060 Background: The addition of anti Epidermal Growth Factor Receptor (EGFR) monoclonal antibodies (mabs) to preoperative chemoradiation in RC has produced low pathological complete response (pCR) rate. The postulated reasons for the poor outcome are: lack of patient selection, suboptimal treatment sequencing, and potential negative interaction between anti EGFR mabs and chemotherapy (CT) when used concurrently as radiosensitizers. Given these issues, we conducted a phase II trial of PAN in combination with preoperative RT in Kras wt RC. Methods: We studied several treatment sequences in murine models of Kras wt human colorectal cancer. Next, we used our preclinical results to design the treatment schedule of the phase II trial. PAN (6mg/kg/q2w) was combined with RT (45 Gy) to treat T3-4, Kras wt RC. Surgery was done 6-8 weeks after the end of RT. The primary endpoint, the pCR rate on surgical specimen, was assessed centrally (Simon: H0=5%, H1=17%, α=0.05, β=0.2). Results: In our murine models, the addition of anti EGFR mabs to RT significantly improved response when anti EGFR mabs were started during RT and extended after the end of RT (p<0.05). No benefit was found when anti EGFR mabs were given before the start of RT. In the phase II trial, we therefore started RT on day 1 and introduced PAN 7 days later (day 8). After RT ended (day 36), PAN treatment was continued to day 50. To fully explore PAN’s potential as radiosensitizer, CT was not given during RT but postoperatively. 19 patients were evaluable for pCR. 47% of tumours were cT3N+. Hypomagnesemia (16%) and hypophosphatemia (16%) were most common grade ≥3 toxicities. As no pCR was found, the study was closed prematurely for lack of efficacy according to the statistical rule. 7 patients (41%) had >50% pathological tumour regression (grade 3 Dworak) and 12 (70%) showed >2mm of tumour-free circumferential resection margin. Conclusions: Preclinical data confirm the need of optimal treatment sequencing to potentiate synergism between anti EGFR mabs and RT. However, the pCR rate in our phase II trial remains low despite adequate patient selection and optimized study design. Thus, we do not recommend the concurrent use of anti EGFR mabs and RT in the neoadjuvant treatment of RC.

Synthesis and Preliminary Biological Evaluation of High-Drug-Load Paclitaxel-Antibody Conjugates for Tumor-Targeted Chemotherapy

The goal of this study was to design paclitaxel (PTX)-monoclonal antibody (mAb) prodrug conjugates (PTXMAbs) with the ability to deliver therapeutically significant doses of the drug to the tumor while avoiding the previously observed solubility limitations of conjugates with PTX:mAb molar ratios of >3. New PTX conjugates were synthesized using the discrete poly(ethylene glycol) (dPEG) as linkers. These compounds, PTX-L-Lys[(dPEG12)(3)-dPEG4]-dPEG6-NHS (9a and 9b, for L = GL or SX, respectively), were then conjugated to the antiepidermal growth factor receptor mAb, C225 at increasing PTX:C225 ratios, producing completely soluble conjugates. Unlike the earlier PTXMAbs, buffered solutions of these conjugates remained homogeneous for extended periods of time. Fluorescence-activated cell sorting (FACS) analysis indicated preserved immunogenicity of the conjugates at all four substitution ratios, while cytotoxicity studies in MDA-MB-468 breast cancer cells indicated preservation of drug cytotoxicity. These conjugates may have potential in the development of high-drug-load tumor-targeting taxanes.

Comparing ELISA and Surface Plasmon Resonance for Assessing Clinical Immunogenicity of Panitumumab

Evaluation of the immunogenicity of panitumumab, a fully human anti-epidermal growth factor receptor mAb approved for use in colorectal cancer patients, led to the development of two separate immunoassays for the detection of anti-panitumumab Abs. The first immunoassay used a bridging ELISA capable of detecting 10 ng/ml positive control anti-panitumumab Ab. The ELISA incorporated an acid dissociation step to reduce drug interference and tolerated the presence of approximately 100-fold molar excess of drug. During eight clinical trials, the ELISA detected developing Ab responses in 2 of 612 (0.3%) subjects. In one of the ELISA positive subjects, neutralizing Abs were detected using an epidermal growth factor receptor phosphorylation bioassay. The second immunoassay used a Biacore biosensor immunoassay format capable of detecting 1 mug/ml positive control Ab while tolerating the presence of equal molar amounts of drug. Although less sensitive and less tolerant to competing drug in the assay, the Biacore assay detected developing Ab responses in 25 of the 604 (4.1%) subjects. Additionally, the Biacore assay identified eight subjects who developed neutralizing Abs. Mouse mAbs with affinities ranging from 1.1 x 10(-6) to 8.4 x 10(-10) M were used to characterize both assay types. The ELISA was more sensitive for the detection of higher affinity mAbs and detected high-affinity mAbs in the presence of higher molar ratio of drug to mAb. The Biacore assay was more sensitive for detection of lower affinity mAbs and detected low affinity Abs in the presence of higher molar ratios of drug to mAb.

Radiotoxicity of h-R3 monoclonal antibody labeled with 188Re administered intracerebrally in rats

Brain tumors are often incurable despite current aggressive treatment modalities. Regional intracerebral administration of labeled monoclonal antibodies (Mabs) can maximize the radioisotope and Mab concentration to tumor sites while reducing systemic toxicity. h-R3 is a humanized antiepidermal growth factor receptor Mab that successfully targets the epidermal growth factor receptor, which is overexpressed in glioblastomas. We studied the acute local and systemic toxicity effects of intraventricular 188Re-h-R3 in rats. Forty rats were distributed into four groups with five animals of each sex in each group. A single 5 -microl dose (2.5 microl into the left and 2.5 microl into the right lateral ventricles) of neutral solution containing 50 microg of h- R3 labeled with 49.5 +/- 1.7,284 +/- 13.7 or 579 +/- 23.7 muCi of 188Re were stereotactically administered to each animal. Control animals received vehicle alone. Each animal was observed twice daily for detection of toxicity signs. Body weights were recorded on days 0, 7 and 14. Blood samples for analysis of hematological and clinical chemistry parameters were taken on days 0 and 14. Necropsy and histopathological studies were carried out after completion of the study. All animals, but one, remained clinically stable. Toxicities included local radionecrosis, discrete increase in ALAT and creatinine blood values at higher dose level. We concluded that a single intraventricular administration of relatively large doses of 188Re-h-R3 is tolerable and causes minimal local and systemic toxicity effects in rats. Nevertheless, further studies are necessary to discard learning and behavioral problems.