31 research outputs found
Antibody variants with improved affinity
Therapeutische Antikörper gewinnen bei der Behandlung verschiedener Krebsarten mehr und mehr an Bedeutung. Ein Zielmolekül für eine antikörperbasierte Therapie ist Mucin 1 (MUC1). In vielen Adenokarzinomen wird MUC1 basolateral überexprimiert und weist veränderte O-Glykosylierungen auf. Die aberranten Glykosylierungen machen neue Peptidepitope zugänglich und stellen selbst Neoantigene dar. Das humane single chain Fv (scFv) Antikörperfragment IIB6 wurde in einer vorhergehenden Arbeit (Toleikis, 2004) mittels Phagen-Display gegen tumorassoziiertes MUC1 selektiert. Es besitzt jedoch nur eine niedrige Affinität zu MUC1 (KD = 2,3 x 10-7 M) und bindet an ein Peptid-Epitop in der extrazellulären VNTR-Region. Durch Affinitätsreifung von IIB6 scFv wurden hochaffine Antikörperfragmente gegen MUC1 generiert und anschließend charakterisiert. Es wurden zufällige Mutationen mittels sequenzieller Error-prone-PCR in das IIB6 scFv Genfragment eingebracht und Antikörpergenbibliotheken generiert. Durch stringente Selektionsmethoden wurden Antikörperklone mit verbesserter Bindung an MUC1 isoliert. Für den scFv-Klon HT186-D11 wurde eine deutlich erhöhte Affinität zu MUC1 gemessen (KD = 5,7 x 10-10 M). Alle untersuchten Klone banden an das gleiche Peptidepitop (RPAP) in der VNTR-Region von MUC1. Die affinitätsgereiften Klone besitzen eine verringerte Aggregationsneigung und gleichzeitig eine deutlich verbesserte Langzeitstabilität verglichen mit IIB6 scFv. Mittels durchflusszytometrischer Analysen wurde eine spezifische Bindung an MUC1+ Tumorzelllinien nachgewiesen. Eine bioinformatische Analyse zeigte gehäufte Aminosäureaustausche im CDR2-Bereich von VL. Die in dieser Arbeit generierten affinitätsgereiften MUC1-spezifischen scFv-Fragmente zeigen ein großes Potential für die Diagnose und Behandlung von MUC1+ Adenokarzinomen. In weiteren Experimenten wurde die Steigerung der apparenten Affinität durch Multimerisierung der Antikörperfragmente und Ausnutzung des Aviditätseffekts untersucht.Therapeutic antibodies are gaining in importance in the treatment of different cancer diseases. One target molecule for an antibody-based therapy is Mucin 1 (MUC1). In many adenocarcinomas MUC1 is basolateral overexpressed and exhibits altered O-glycosylations. These aberrant glycosylations result in new epitopes based on the different glycosylations or the peptide backbone of MUC1. The human single chain Fv (scFv) antibody fragment IIB6 was isolated against tumor-associated MUC1 by phage display (Toleikis, 2004). This antibody fragment binds a peptide epitope within the extracellular VNTR-region of MUC1 with low affinity (KD = 2,3 x 10-7 M). High affinity binders against MUC1 were generated by an affinity maturation of IIB6 scFv. Random point-mutations were introduced into the IIB6 scFv gene by error-prone-PCR and subsequently antibody gene libraries were constructed. These libraries were screened for high affinity binders by different stringent panning strategies. For HT186-D11 a drastically increased affinity (KD = 5,7 x 10-10 M) to MUC1 was observed. All characterized clones bound to the same peptide-epitope (RPAP) in the VNTR-region of MUC1. The affinity matured clones showed a significant lower aggregation-tendency and a better long-term stability compared with IIB6. Binding to tumor-cell associated MUC1 was validated by flow cytometry experiments. Bioinformatical analysis of the amino acid sequence showed a cluster of amino acid substitutions within the CDR2 of VL. The affinity matured MUC1-specific antibody fragments show a high potential for diagnostic and antibody-based therapies for the treatment of MUC1+ adenocarcinomas. In further experiments gaining apparent affinities by antibody-multimerization utilizing the avidity effect were investigated
Study of an unusually high level of N-glycolylneuraminic acid (NGNA) sialylation on a monoclonal antibody expressed in Chinese hamster ovary cells
Sialic or neuraminic acids of recombinant therapeutic glycoproteins produced in mammalian cells, including monoclonal antibodies, have significant impact on the half-life, stability, and biological activity of these proteins (Hossler et al., 2009; Ghaderi et al., 2012). The predominant sialic acid N-acetylneuraminic acid (NANA or Neu5Ac) is added from precursor CMP-NANA to galactose residues of N-linked glycoproteins by sialytransferases. In most mammals CMP-NANA can also be modified to its hydroxylated derivative CMP-NGNA by CMP-N-acetylneuraminic acid hydroxylase (CMAH). NGNA can then be added from CMP-NGNA to galactose residues of the N-linked glycoproteins, also by sialytransferases. However, humans cannot make functional CMAH due to an inactivating exon deletion mutation in CMAH gene (Okerblom and Varki, 2017), and therefore cannot convert CMP-NANA to CMP-NGNA. Consequently, when injected into human patients, NGNA sialic acid containing mAbs or other recombinant glycoproteins may induce immune responses, which could negatively impact pharmacokinetics or efficacy. Therefore high levels of NGNA on therapeutic mAbs or other recombinant glycoproteins are an undesirable product quality attribute.
The level of total sialic acids of recombinant glycoproteins produced in Chinese hamster ovary (CHO) cells is dictated largely by the selected cell lines, upstream process, and to a lesser degree, downstream process. NGNA sialylation is generally rare in CHO cells (Könitzer et al., 2015). Hence, therapeutic glycoproteins manufactured in these cells are considered safe for human use. However, during a first-in-human (FIH) upstream process development for a novel mAb, an initially selected desirable cell line (A) was found to produce the mAb with an unexpectedly high level of the NGNA sialic acid (\u3e30%). To the best of our knowledge such high level of NGNA sialylation on a mAb produced by CHO cells has not been reported. To mitigate potential risks associated with high NGNA in human patients, a new cell line (B) that produces the mAb with very low NGNA was selected as the manufacturing cell line for this project.
In order to understand the molecular mechanism causing the high NGNA content in cell line A, we initiated comprehensive genetic gap analyses using next-generation sequencing technologies and determined the differences in genomic, transcriptomic, and miRnomic profiles of the two cell lines. The results indicate spontaneous upregulation of CMAH mRNA expression, at least 10 fold higher in cell line A compared to cell line B. In this talk we will summarize the results of our studies of this unusual sialylation behavior in CHO cells
Rise and Fall of an Anti-MUC1 Specific Antibody
So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology
A two-arm multicenter phase II trial of one cycle chemoselection split-dose docetaxel, cisplatin and 5-fluorouracil (TPF) induction chemotherapy before two cycles of split TPF followed by curative surgery combined with postoperative radiotherapy in patients with locally advanced oral and oropharyngeal squamous cell cancer (TISOC-1)
Inhestern J, Schmalenberg H, Dietz A, et al. A two-arm multicenter phase II trial of one cycle chemoselection split-dose docetaxel, cisplatin and 5-fluorouracil (TPF) induction chemotherapy before two cycles of split TPF followed by curative surgery combined with postoperative radiotherapy in patients with locally advanced oral and oropharyngeal squamous cell cancer (TISOC-1). Annals of Oncology. 2017;28(8):1917-1922
Epitope mapping.
<p><b>A</b> The epitope mapping membrane (15mer oligopeptide, 1 amino acid overlap) was stained with 30 µg scFvs. The bound scFvs were detected with mouse anti-c-myc IgG (9E10) (1∶500) and a goat anti-mouse IgG (Fab spec.) AP conjugate (1∶2000). NWS  =  detection antibodies only. <b>B</b> Sequence overview. Amino acid sequences of the single spots on the nitrocellulose membrane. Immunostained spots were marked in grey. Amino acids forming the minimal epitope are given in bold. The sequence of one complete VNTR repetitive region is underlined.</p
Germinality index and humanness (Z-score) for the variable region of the anti-MUC1 scFvs.
<p>Germinality index is calculated by aligning the amino acid sequence of the variable region to the next human germline sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015921#pone.0015921-Pelat3" target="_blank">[62]</a>. Z-scores were calculated using the SHAB web interface (<a href="http://www.bioinf.org.uk/abs/shab/" target="_blank">http://www.bioinf.org.uk/abs/shab/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015921#pone.0015921-Abhinandan1" target="_blank">[61]</a>.</p
Analysis of the IHC staining with non tumour tissues.
<p>Eight slides of each tissue type were analysed.</p><p>*suspected reaction of the detection system with endogenous biotin.</p
Flow cytometry analysis of anti-MUC1 IgG and scFv-Fc antibodies on different tumour cell lines HEK293T (A), SKOV3 (B) and MCF-7 (C).
<p>Different anti-MUC1 antibody concentrations were incubated on two different MUC1 positive cell lines and one MUC1 negative cell line. Detection was performed as given for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015921#pone-0015921-g008" target="_blank">figure 8B</a>.</p
Internalisation of anti-MUC1 HT186-D11 and huHMFG1 IgGs.
<p>Internalisation was analysed by incubation of MCF-7 cells with anti-MUC1 antibodies at 37°C or on ice (on ice or 4°C, should be consistent throughout the paper) for 1h. Remaining cell surface-associated mAbs were detected by staining with PE-conjugated mouse anti-human IgG mab.</p