Generation and in vivo characterization of a chimeric αvβ5-targeting antibody 14C5 and its derivatives

Background: Previous studies showed that radiolabeled murine monoclonal antibody (mAb) 14C5 and its Fab and F(ab')2 fragments, targeting αvβ5 integrin, have promising properties for diagnostic and therapeutic applications in cancer. To diminish the risk of generating a human anti-mouse antibody response in patients, chimeric variants were created. The purpose of this study was to recombinantly produce chimeric antibody (chAb) derivatives of the murine mAb 14C5 and to evaluate the in vitro and in vivo characteristics. Methods: In vitro stability, specificity, and affinity of radioiodinated chAb and fragments (Iodo-Gen method) were examined on high-expressing αvβ5 A549 lung tumor cells. In vivo biodistribution and pharmacokinetic characteristics were studied in A549 lung tumor-bearing Swiss Nu/Nu mice. Results: Saturation binding experiments revealed high in vitro affinity of radioiodinated chAb, F(ab')2, and Fab, with dissociation constants (KD) of 1.19 ± 0.19, 0.68 ± 0.10, and 2.11 ± 0.58 nM, respectively. ChAb 14C5 showed highest tumor uptake (approximately 10%ID/g) at 24 h post injection, corresponding with other high-affinity Abs. ChF(ab')2 and chFab fragments showed faster clearance from the blood compared to the intact Ab. Conclusions: The chimerization of mAb 14C5 and its fragments has no or negligible effect on the properties of the antibody. In vitro and in vivo properties show that the chAb 14C5 is promising for radioimmunotherapy, due to its high maximum tumor uptake and its long retention in the tumor. The chF(ab')2 fragment shows a similar receptor affinity and a faster blood clearance, causing less non-specific retention than the chAb. Due to their fast blood clearance, the fragments show high potential for radioimmunodiagnosis

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic <it>P. pastoris </it>yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs.</p> <p>Results</p> <p>Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and <it>P. pastoris </it>cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from <it>Pichia </it>supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in <it>Pichia </it>more efficient than most other previously described trispecific or trivalent molecules produced in <it>E. coli</it>.</p> <p>Conclusion</p> <p>Bi- and tribody molecules are efficiently produced in <it>P. pastoris</it>. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of <it>P. pastoris </it>as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.</p

Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells

Self-renewing tissue-resident macrophages are thought to be exclusively derived from embryonic progenitors. However, whether circulating monocytes can also give rise to such macrophages has not been formally investigated. Here we use a new model of diphtheria toxin-mediated depletion of liver-resident Kupffer cells to generate niche availability and show that circulating monocytes engraft in the liver, gradually adopt the transcriptional profile of their depleted counterparts and become long-lived self-renewing cells. Underlining the physiological relevance of our findings, circulating monocytes also contribute to the expanding pool of macrophages in the liver shortly after birth, when macrophage niches become available during normal organ growth. Thus, like embryonic precursors, monocytes can and do give rise to self-renewing tissue-resident macrophages if the niche is available to them

Production of antibody derivatives in the methylotrophic yeast Pichia pastoris

New antibody derivatives are continuously being generated to interact with a range of therapeutic targets. The cost-effective and efficient production of these and other antibody derivatives is crucial for their further success. Here, we describe the construction of the expression vectors needed for heterologous expression of a Fab fragment in the yeast Pichia pastoris. The experimental conditions for lab-scale expressions are discussed, and an overview of an efficient purification strategy is presented

New recombinant bi- and trispecific antibody derivatives

Bispecific antibodies (BsAb) are promising therapeutic tools in tomorrow's medicine. When constructing BsAbs, the final molecular size should be large enough to avoid rapid renal clearing, but small enough to allow efficient tissue distribution. In order to produce such intermediate sized BsAb, a good heterodimerisation technique will improve existing production methods. When considering recombinant expression of BsAbs, the heterodimerisation motif can be incorporated into the molecule. Recombinant BsAb can e.g. be made by fusing single chain variable fragments (scFv) to a heterodimerisation domain. We compared the efficiency of the isolated CL and CH1 constant domains with complete Fab chains to drive heterodimerisation of BsAbs in mammalian cells. We found that the isolated CL:CHI domain interaction was inefficient for secretion of heterodimers. However, when the complete Fab chains were used, secretion of a heterodimerised bispecific antibody was successful. By C-terminal fusion of scFv molecules to the Fd- and the L-chains efficient heterodimerisation in mammalian cells was obtained and a novel intermediate sized, disulfide stabilised BsAb could be efficiently produced. Since the Fab chain encodes a binding specificity on its own, bispecific (BsAb) or trispecific (TsAb) antibodies can be made. This gave rise to disulphide stabilised Fab-scFv BsAb (Bibody) or Fab-(scFv)2 TsAb (Tribody) of intermediate molecular size. Heterodimerisation of the L and Fd-containing fusion proteins was very efficient, and up to 90% of all secreted antibody fragments was in the desired heterodimerised format. All building blocks remained functional in the fusion product, and the bispecific character of the molecules as well as the functionality was demonstrated. Due to the high heterodimerisation efficiency, the ease of purification of the desired product from by-products and the lack of post-production processing, this method for producing bi- or trispecific antibodies in mammalian cells could become a method of choice for the production of intermediate sized trispecific antibodies, BsAb with monovalent or bivalent binding for one antigen, or immunoconjugates

PH1-derived bivalent bibodies and trivalent tribodies bind differentially to shed and tumour cell-associated MUC1

Most adenocarcinomas express altered MUC1 as a tumour-associated antigen. Due to suboptimal glycosylation in tumour-associated MUC1, the apomucin core is exposed, revealing new epitopes for antibody-directed immunotherapy. The human PH1 Fab binds specifically to this MUC1 apomucin. We describe the engineering and functional characterization of bi- and trivalent recombinant antibody derivatives from the PH1 Fab. Bi- and tribodies were made using the disulfide-stabilized Fab fragment as a heterodimerization scaffold with PH1 single-chain variable fragments fused to either one or both Fab-chain C-termini. Immunoassays revealed 27- and 165-fold improved dissociation constants (K(D) = 30 and 5 nM) of the PH1 bi- and tribodies compared with the parental Fab (K(D) = 820 nM). Unexpectedly, major differences were seen in the ability of the antibody constructs to bind shed and tumour cell-tethered MUC1. While the tribody did not discriminate between both MUC1 forms, the bibody demonstrated preferential interaction with membrane-bound MUC1 compared with shed MUC1. This preferential recognition of membrane-bound MUC1, along with the high serum stability of the bibody, its intermediate size and efficient internalization by MUC1(+) cells, makes the human PH1-derived bibody a valuable candidate as a cancer-targeting therapeutic