Back-to-Germline (B2G) Procedure for Antibody Devolution.

Bispecific antibodies (bsAbs) with avidity-enhanced specificity can be used to address target cells with increased specificity, ideally binding efficiently to cells that express two cognate antigens, yet not to cells that express only one of those. Building blocks required to generate such bsAbs are binders that recognize the two antigens with high specificity yet with various (including very low monovalent) affinities. The herein described 'back-to-germline' (B2G) procedure defines such derivatives. It converts parent antibodies with high specificity to derivatives that retain specificity but modulate affinity. The approach defines mutations to be introduced into antibody complementarity-determining regions (CDRs) regions without requiring structures of antibody-antigen complexes. Instead, it reverses the B-cell maturation process that increases affinities, with preference on CDR residues with high antigen contact probability. Placing germline residues at those positions generates VH and VL domains and Fv-combinations thereof that retain specificities but are 'de-matured' to different degrees. De-maturation influences on-rates and off-rates, and can produce entities with extremely low affinity for which binding can only be detected in bivalent formats. A comparison with alanine replacement in CDRs (so far, the most frequently applied technology) indicates that B2G may be more reliable/predictable without introduction of stickiness or poly-reactivity. The applicability for generating sets of affinity-modulated monospecific variants is exemplarily shown for antibodies that bind CD138, Her2/neu, and EGFR.

Antibody fusion proteins with human ribonucleases 1 to 8.

ImmunoRNases combine tumor targeting by antibodies with the cytotoxic action of ribonucleases from the RNase A superfamily. This study investigated for the first time all catalytic active human RNase A family members (1 to 8) as effector components of antibody fusion proteins. ImmunoRNase fusion proteins were constructed using the CD30-specific bivalent recombinant scFv-Fc antibody SH313-B5. Production of the resulting entirely human immunoRNases 1 to 8 was done in mammalian cells by secretion of active forms. The immunoRNases mediated CD30-specific cell binding and showed ribonucleolytic activity. Interestingly, immunoRNases 1 and 2 were active in the presence of up to 5-/20-fold molar excess of the pancreatic RNase inhibitor (RI), which is supposed to efficiently inhibit all human RNase A activity. ImmunoRNases 3, 4, 6 and 7 were only inhibited by several fold molar excess of RI, whereas immunoRNases 5 and 8 were already completely inactive at equimolar RI concentrations. Compared to free RNases, activity and RI sensitivity were not significantly changed by antibody fusion or dimerisation. ImmunoRNase3 and 5 mediated tumor growth inhibition at low nanomolar concentrations. Anti-tumor activity was antigen-specific and did not show any correlation with ribonucleolytic activity or RI sensitivity.

CD30 as a therapeutic target for lymphoma.

Hodgkin's lymphoma (HL) and ALK(+) anaplastic large-cell lymphoma (ALCL) have become highly curable due to the success of modern regimens of chemotherapy and radiotherapy. However, up to one-third of the patients experience relapse or do not respond to first-line therapy, and half of them relapse again after secondary therapy with limited options for further treatment. In the last 15 years, monoclonal antibodies (mAbs) directed to surface receptors became a new and valuable therapeutic option in many hematologic malignancies. Due to its restricted expression on normal activated lymphocytes and its high expression on malignant cells, CD30 represents an attractive target molecule for HL and ALCL therapy. However, unconjugated CD30 mAbs have demonstrated a lack of objective clinical responses in patients with recurrent HL. CD30 exhibits complex signaling pathways, and binding of its natural ligand or anti-CD30 mAbs can induce apoptosis but may also promote proliferation and activation depending on the cellular context. Moreover, CD30 rapidly internalizes after crosslinking, which counteracts efficient recruitment of immunologic effectors but also provides the opportunity to transfer cytotoxic payloads coupled to CD30-specific mAbs into the tumor cells. Several tumor targeting approaches have been studied, including radio-immunoconjugates, immunotoxins, immunoRNases, immunokinases, and antibody drug conjugates (ADCs). In 2011, the ADC brentuximab-vedotin, consisting of the CD30-specific chimeric mAb cAC10 and the potent tubulin toxin monomethyl auristatin E, gained regulatory approval as a well tolerated and highly active drug in patients with refractory and relapsed HL and ALCL. SGN-35 is on the way to being incorporated in the standard management of CD30(+) lymphoma with significant therapeutic impact. This review gives a critical overview about anti-CD30 therapies with unconjugated, engineered, and conjugated mAbs and the therapeutic challenges of treatment of CD30(+) lymphoma.

Human antibodies targeting CD30(+) lymphomas.

Hodgkin- and Non-Hodgkin lymphomas are tumors of the lymphatic system, whose common therapy is chemotherapy and radiation. Immunotherapies with monoclonal antibodies are a promising strategy for treatment of malignant lymphomas and may overcome strong side effects and partial failure of and high relapse rates after common treatment. The antigen CD30 is overexpressed in Hodgkin lymphomas and some Non-Hodgkin lymphomas like anaplastic large cell lymphomas and adult T-cell lymphomas, which makes it a suitable target for antibody-based therapies. We isolated four new CD30-specific antibodies from a human naïve antibody gene library by phage display. These recombinant antibodies were produced as scFv in Escherichia coli and as bivalent immunoglobuline-like scFv-Fc antibodies in mammalian cells. They bound with high specificity to both recombinant antigen CD30 and to CD30(+) lymphoma cells. Flow cytometry and confocal laser scanning microscopy were used to show intracellular uptake by receptor-mediated endocytosis into CD30(+) lymphoma cell line Karpas299. The antibody clone SH313-B5 inhibited the proliferation of CD30(+) Karpas299 cells in a dose-dependent and effector independent manner with an IC(50) of 100 nM. Therefore, the antibody SH313-B5 is a promising candidate for further development towards treatment of CD30(+) tumors.

Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.