In Vitro Antibody Evolution Targeting Germline Hot Spots to Increase Activity of an Anti-CD22 Immunotoxin

126

Rational engineering methods can be applied with reasonable success to optimize physicochemical characteristics of proteins, in particular, antibodies. Here, we describe a combined CDR3 walking randomization and rational design-based approach to enhance the affinity of the human anti-gastrin TA4 scFv. The application of this methodology to TA4 scFv, displaying only a weak overall affinity for gastrin17 (K D ‫؍‬ 6 M), resulted in a set of nine affinity-matured scFv variants with near-nanomolar affinity (KD ‫؍‬ 13.2 nM for scFv TA4.112). First, CDR-H3 and CDR-L3 randomization resulted in three scFvs with an overall affinity improvement of 15-to 35-fold over the parental. Then, the modeling of two scFv constructs selected from the previous step (TA4.11 and TA4.13) was followed by a combination of manual and molecular dynamics-based docking of gastrin17 into the respective binding sites, analysis of apparent packing defects, and selection of residues for mutagenesis through phage display. Nine scFv mutants were obtained from the second maturation step. A final 454-fold improvement in affinity compared with TA4 was obtained. These scFvs showed an enhanced potency to inhibit gastrin-induced proliferation in Colo 320 WT and BxPc3 tumoral cells. In conclusion, we propose a structure-based rational method to accelerate the development of affinity-matured antibody constructs with enhanced potential for therapeutic use.antibody engineering ͉ gastrin ͉ in vitro affinity maturation ͉ pancreatic cancer

Section: Discussionmentioning

confidence: 99%

Affinity maturation of antibodies assisted by in silico modeling

Barderas

Desmet²,

Timmerman³

et al. 2008

126

“…Numerous protein engineering and mutagenesis strategies have been used for the enhancement of antibody affinity (3,5,6,11,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). For small regions (Ͻ8-10 aa), saturation mutagenesis is typically used to produce all combinations of the 20 naturally occurring amino acids.…”

Section: Discussionmentioning

confidence: 99%

“…Reduced codon complexity to limit library diversity has also been proposed (15,20,31). Alternatively, specific residues among the six CDRs are selected based on their propensity for somatic hypermutation (6,29). Although these strategies can result in significant affinity enhancement, none comprehensively interrogate all six CDR positions in a precise and informative fashion.…”

Section: Discussionmentioning

confidence: 99%

A general method for greatly improving the affinity of antibodies by using combinatorial libraries

Rajpal

Beyaz

Haber

et al. 2005

144

102

Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-␣ antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (Kd ‫؍‬ 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and͞or three CDRs for each V H and VL domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-␣. These beneficial mutations in both VH and VL were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500-and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15-to 30-fold improvement in in vitro TNF-␣ neutralization in an L929 bioassay. Thus, this LTM͞combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins.look-through mutagenesis ͉ maturation ͉ mutagenesis ͉ TNF-␣

“…A combinational engineered human V H singledomain phage display library, with an estimated size of 2.5 × 10 10 , was used for the screening and has been previously described (56). The phage library was subjected to four rounds of panning on Nunc 96-well Maxisorp plate (Thermo Scientific) as described previously (57)(58)(59). Details are provided in the SI Appendix, SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma

Hewitt

et al. 2017

Self Cite

111

126

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/ β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.glypican | neuroblastoma | single-domain antibody | chimeric antigen receptor T-cell therapy | immunotoxin