Acceptor Framework Research Articles

Exploring the “Goldilocks Zone” of Semiconducting Polymer Photocatalysts by Donor–Acceptor Interactions

AbstractWater splitting using polymer photocatalysts is a key technology to a truly sustainable hydrogen‐based energy economy. Synthetic chemists have intuitively tried to enhance photocatalytic activity by tuning the length of π‐conjugated domains of their semiconducting polymers, but the increasing flexibility and hydrophobicity of ever‐larger organic building blocks leads to adverse effects such as structural collapse and inaccessible catalytic sites. To reach the ideal optical band gap of about 2.3 eV, A library of eight sulfur and nitrogen containing porous polymers (SNPs) with similar geometries but with optical band gaps ranging from 2.07 to 2.60 eV was synthesized using Stille coupling. These polymers combine π‐conjugated electron‐withdrawing triazine (C3N3) and electron donating, sulfur‐containing moieties as covalently bonded donor–acceptor frameworks with permanent porosity. The remarkable optical properties of SNPs enable fluorescence on‐off sensing of volatile organic compounds and illustrate intrinsic charge‐transfer effects.

Design of a Novel Series of Donor-Acceptor Frameworks via Superalkali-Superhalogen Assemblage to Improve the Nonlinear Optical Responses.

Presently, many researches are directed toward the design of novel superatoms with high nonlinear optical responses. Inspired by a fascinating finding of superatoms which were designed by bonding superhalogen (Al13 nanocluster) with superalkalis (M3O, M = Na and K), we suggest an effective strategy to form a series of typical donor-acceptor frameworks with high nonlinear optical responses via bonding the superalkalis M3O (Li3O, Na3O, K3O, Li2NaO, Li2KO, Na2LiO, Na2KO, K2LiO, K2NaO, and LiNaKO) with low ionization potential to the superhalogen Al13 with large electron affinity. The ionization potential, electronic spatial extent, electric field gradient tensors of 17O nuclei, and natural bond orbital charge values of the superalkalis M3O were also calculated. We found that the M ligands have the remarkable effect on the ionization potential as well as 17O nuclear quadrupole resonance parameters of the superalkalis M3O. Our results also represented that the bonding superalkalis can efficiently narrow wide HOMO-LUMO gap and considerably enhance first hyperpolarizability of the pristine Al13, due to electron transfer in this type of superatom. Also, the effect of oriented external electric fields on the nonlinear optical responses of the superatoms M3O-Al13 has been systematically explored. We found that the first hyperpolarizability of the superatom compounds can be gradually increased by increasing the imposed oriented external electric field from zero to the critical external electric field along the charge transfer direction (M3O → Al13). In this respect, this work reveals an effective approach to gradually enhance the nonlinear optical responses of the superatoms through applying oriented external electric fields.

Effects of Aqueous Electrolyte, Active Layer Thickness and Bias Irradiation on Charge Transfer Rates in Solar Cells Sensitized with Top Efficient Carbazole Dyes

The photovoltaic performance and electron-transfer dynamics from femtoseconds to milliseconds are explored for very efficient carbazole sensitizers applied in solar cells, including the champion dye ADEKA-1. The photocurrent in the novel, environmentally friendly aqueous electrolyte is about 40% of that in the standard acetonitrile one, both based on cobalt complexes as a redox pair. The drop in the photocurrent is found to be correlated with increased electron recombination between sensitized titania particles and dyes, taking place with a time constant of several hundreds of picoseconds. Electron injection and recombination between titania and dye are slowed down under the presence of water (about 10 times) and with additional 1 sun bias irradiation (about 2 times). These effects are interpreted as due to reduced electronic coupling between the electron donors (dyes) and acceptor framework (titania). Moreover, a decrease in the relative photocurrent, photovoltage, and fill factor of the cells with incre...

Three birds, one stone - photo-/piezo-/chemochromism in one conjugated nanoporous ionic organic network.

A nanoporous material bearing a high ion density and inherent organic radical character was synthesized by a facile one-pot process, which exhibits photo-, piezo- and chemochromism, driven by the diverse electron transfer processes between the acceptor framework and different electron donors. The responsive behavior is useful for its sensing application, as demonstrated here for pressure, anion and gas sensing.

De novo design of antibody complementarity determining regions binding a FLAG tetra-peptide

Computational antibody engineering efforts to date have focused on improving binding affinities or biophysical characteristics. De novo design of antibodies binding specific epitopes could greatly accelerate discovery of therapeutics as compared to conventional immunization or synthetic library selection strategies. Here, we employed de novo complementarity determining region (CDR) design to engineer targeted antibody–antigen interactions using previously described in silico methods. CDRs predicted to bind the minimal FLAG peptide (Asp–Tyr–Lys–Asp) were grafted onto a single-chain variable fragment (scFv) acceptor framework. Fifty scFvs comprised of designed heavy and light or just heavy chain CDRs were synthesized and screened for peptide binding by phage ELISA. Roughly half of the designs resulted in detectable scFv expression. Four antibodies, designed entirely in silico, bound the minimal FLAG sequence with high specificity and sensitivity. When reformatted as soluble antigen-binding fragments (Fab), these clones expressed well, were predominantly monomeric and retained peptide specificity. In both formats, the antibodies bind the peptide only when present at the amino-terminus of a carrier protein and even conservative peptide amino acid substitutions resulted in a complete loss of binding. These results support in silico CDR design of antibody specificity as an emerging antibody engineering strategy.

Efficacy of Humanized CD19-Targeted Chimeric Antigen Receptor (CAR)-Modified T Cells in Children and Young Adults with Relapsed/Refractory Acute Lymphoblastic Leukemia

▪BackgroundTargeted immunotherapy with CTL019, CD19-specific chimeric antigen receptor (CAR)-modified T cells, can produce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). However, a subset of patients has limited persistence, which can increase the risk of relapse. Most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin; therefore, anti-mouse reactivity is one potential cause of immune-mediated rejection that may be overcome by fully human or humanized CAR designs. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19-directed CAR T cells (CTL119).DesignA pilot/phase 1 study of CAR-modified T cells containing a humanized anti-CD19 scFv domain (CTL119) enrolled children and young adults with relapsed/refractory B-ALL with or without prior exposure to a CAR T cell product. Patients previously treated with CD19-specific CAR-modified T cells were eligible if they met 1 of 3 criteria: 1) CD19+ relapse 2) no response to prior CAR T cell therapy or 3) early B cell recovery indicating poor persistence of CAR T cells. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of CD3z, 4-1BB, and humanized anti-CD19 scFv domains and activated/expanded with anti-CD3/CD28 beads. The humanized scFv domain was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Patients received lymphodepletion with cyclophosphamide and fludarabine 1 week prior to infusion with CTL119.ResultsThirty children and young adults aged 29 mo-24 yr were infused with CTL119. Eighteen patients had received prior allogeneic stem cell transplant (SCT). Eleven patients who previously received murine-derived CD19-specific CAR-modified T cells (CTL019, n=7; other, n=4) were retreated for B cell recovery (n=5), CD19+ relapse (n=5), or no response to prior CAR T cells (n=1). CNS disease or other extramedullary disease was the indication for enrollment in 6 and 3 patients, respectively.At assessment 1 month after infusion, 26/30 patients (87%) achieved a complete response (CR), defined as morphologic remission with B cell aplasia. Of 11 patients previously treated with murine CD19-specific CAR-modified T cells, 7 (64%) achieved a CR at 1 month, 4 demonstrated no response. Multiparameter flow cytometry for minimal residual disease (MRD) was negative at a detection level of 0.01% in 5/7 responding patients. Two responding patients with positive MRD progressed to CD19+ relapse at 1.6 and 3 mo.In patients with no prior exposure to a CD19 CAR T cell product, MRD-negative CR was achieved in 19/19 patients (100%). One patient relapsed with CD19+ extramedullary disease at 2.8 mo. With a median follow-up of 4.2 mo (range, 1.0-14.1 mo) for all responding patients in both cohorts, 23/26 remain in remission with 1 proceeding to SCT in remission.B cell aplasia, a functional marker of CD19-targeted CAR T cell persistence, continued for 3 months or more in 11/18 patients with adequate follow-up: 1/6 retreatment, 10/12 CAR-naïve. Cytokine release syndrome (CRS) was observed in 28/30 patients and mild in most patients (grade 1, n=6; grade 2, n=18). Three patients experienced grade 3 CRS requiring supplemental oxygen or low-dose vasopressor support and 1 experienced grade 4 CRS requiring high-dose vasopressor and ventilatory support. Severe CRS was successfully managed with the IL6R antagonist tocilizumab in 3 patients. Neurologic toxicity included encephalopathy (n=5) and seizure (n=4) and was fully reversible.ConclusionIn the first study of humanized anti-CD19 CAR T cells, CTL119 induced remissions in children and young adults with relapsed/refractory B-ALL, including 64% of patients previously treated with murine CD19-directed CAR T cells and 100% of CAR-naïve patients. Further investigation into CAR T cell persistence and anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. DisclosuresMaude:Novartis: Consultancy. Barrett:Novartis: Research Funding. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding; Vitality Institute: Research Funding. Brogdon:Novartis: Employment. Scholler:Novartis: Patents & Royalties: Royalties, Research Funding. Marcucci:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Frey:Amgen: Consultancy; Novartis: Research Funding. Porter:Novartis: Patents & Royalties, Research Funding; Genentech: Employment. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Johnson & Johnson: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership. Grupp:Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding.

Integration of aggregation-induced emission and delayed fluorescence into electronic donor–acceptor conjugates

Aggregation-induced emission and delayed fluorescence are observed simultaneously for the luminogens with an electron-donor and acceptor framework.

Computational study on thermally activated delayed fluorescence of donor–linker–acceptor network molecules

Using density functional theory we have investigated the structure–property relationships of organic molecules with a donor–linker–acceptor (DLA) framework, which can be used as precursors of OLED materials.

Tuning of the HOMO–LUMO Gap of Symmetrical and Unsymmetrical Ferrocenyl‐Substituted Diketopyrrolopyrroles

AbstractFerrocenyl‐substituted diketopyrrolopyrroles (DPPs) with the symmetrical π‐bridged donor–acceptor (D–π–A) frameworks D′–π–D–A–D–π–D′ and D′–A′–D–A–D–A′–D′ as well as unsymmetrical D–A–D–π–D′ and D–A–D–A′–D′ frameworks were designed and synthesized by a Pd‐catalyzed Sonogashira cross‐coupling reaction followed by a [2+2] cycloaddition–retroelectrocyclization reaction. The tetracyanobutadiene (TCBD) DPPs 7 and 8 have redshifted absorption bands and lower HOMO–LUMO gap values relative to those of ferrocenyl‐DPPs 5 and 6. The electronic absorption spectra show a systematic redshift in the intramolecular charge transfer (ICT) band from the ferrocene to the diketopyrrolopyrrole core. The photophysical, computational, and electrochemical properties were also investigated and show substantial donor–acceptor interactions between the ferrocene and DPP moieties. The symmetrical ferrocenyl‐DPPs have more thermal stability than the unsymmetrical derivatives.

Efficacy and Safety of Humanized Chimeric Antigen Receptor (CAR)-Modified T Cells Targeting CD19 in Children with Relapsed/Refractory ALL

Background Targeted immunotherapy with T cells expressing a chimeric antigen receptor (CAR) can produce dramatic anti-tumor responses. We previously demonstrated complete remissions (CRs), prolonged persistence, and sustained responses in children and adults with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL) treated with CD19-specific CAR-modified T cells (CTL019). However, a subset (~25%) of patients has limited persistence of CTL019, which can increase the risk of relapse. We hypothesized that repeat infusion may prolong persistence in some patients but would be ineffective in patients with immune-mediated rejection. As most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin, anti-mouse reactivity is one potential cause of immunogenicity that may be overcome by fully human or humanized CAR design. We now report on retreatment with murine or humanized CD19-directed CAR T cells. Aims We sought to 1) prolong persistence by reinfusion of murine CTL019 in children with r/r CD19+ ALL and evidence of poor persistence and 2) determine the safety and efficacy of CD19-directed CAR T cells with a humanized scFv domain in children with B cell recovery or CD19+ relapse after prior CAR T cell therapy. Method T cells collected from patients were transduced with a lentiviral vector encoding a CAR composed of murine or humanized anti-CD19 scFv, CD3z, and 4-1BB domains, activated/expanded ex vivo with anti-CD3/anti-CD28 beads, cryopreserved, and then infused. The humanized scFv was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Results Of 50 patients in CR at 1 month after CTL019 infusion (50/53 CR), 14 patients received a repeat infusion of murine CTL019 at 3 and/or 6 months after initial infusion and 11 are evaluable for response. Indications were B cell recovery (n=4), CD19+ minimal residual disease (MRD, n=2), or undetectable CTL019 in peripheral blood by flow cytometry (≤0.1% CTL019+ of CD3+, n=5). Minimal toxicities were observed and included fever and fatigue. CTL019 reinfusion induced B cell aplasia for a second time in 1 of 4 children treated for B cell recovery. Two of these patients later relapsed, 1 with CD19+ and 1 with CD19- disease. Of 2 patients with CD19+ MRD, 1 had no response and subsequently experienced a CD19+ relapse and the second became MRD-negative but had B cell recovery. All 5 children reinfused for poor CTL019 persistence demonstrated continued B cell aplasia 3-15 months after repeat infusion with detectable CTL019 in peripheral blood by flow cytometry (0.2-2.8% CTL019+ of CD3+) in 4 patients. Of this group, 4 patients remain in remission 12-21 months after initial infusion, and 1 experienced a CD19- relapse. To overcome the potential for anti-murine immune-mediated rejection, patients previously treated with CAR-modified T cells were eligible for a phase 1 study of humanized CD19-directed CAR T cells (CTL119). Five children were infused for B cell recovery (n=3) or CD19+ relapse (n=2). Cytokine release syndrome (CRS), seen in 2 patients, was limited to fever, headache, and nausea and did not require vasopressor or respiratory support. Three patients showed no response and progressed (n=1) or relapsed with CD19+ (n=1) or CD19- (n=1) ALL. Responses were demonstrated in 2 patients: 1) MRD-negative CR in a patient treated for CD19+ relapse and 2) reduction in MRD and return of B cell aplasia in a patient treated for CD19+ MRD and B cell recovery, with subsequent progression to CD19+ relapse. Both responding patients were previously resistant to murine CTL019 reinfusion, suggesting a possible immune-mediated rejection mechanism. Ongoing studies are investigating anti-CAR responses. Conclusion Repeat infusion of murine CTL019 may prolong B cell aplasia in patients with early evidence of poor persistence and in a fraction of patients with B cell recovery. In the first demonstration of humanized anti-CD19 CAR efficacy, retreatment with CTL119 expressing a humanized scFv induced remission of ALL refractory to prior CD19-directed murine CAR T cell therapy, suggesting that immune-mediated rejection may be an important mechanism of resistance in the subset of patients with rapid CAR cell loss and loss of B cell aplasia. Further investigation into anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. Disclosures Maude: Novartis: Consultancy, Research Funding. Off Label Use: CTL019 and CTL119 for relapsed/refractory ALL. Rheingold:Endo: Other: Husband's employer, has equity interest; Novartis: Consultancy. Aplenc:Sigma Tau: Consultancy. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding. Brogdon:Novartis: Employment. Loew:Novartis: Employment. Zheng:Novartis: Patents & Royalties. Levine:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Grupp:Novartis: Consultancy, Research Funding.

Synthesis, crystal structures of a series of novel 2, 2′:6′, 2″-terpyridine derivatives: The influences of substituents on their photophysical properties and intracellular acid organelle targeting

Five novel 2, 2′:6′, 2″-terpyridine derivatives (L1-L5) with subtle substitute changes have been designed and synthesized. Their structures were determined by the single crystal X-ray diffraction analysis. It was observed that L1 and L2 containing an acceptor (–NO2) group exhibited three strong absorption bands, while L3, L4 and L5 containing donors (–OCH3/NH2) exhibited two absorption bands similar to that of ligand L0. Two-photon absorption cross sections were measured by two-photon excited fluorescence and open aperture Z-scan methods, respectively. Further, the two-photon absorption properties were explained by the hybrid density functional theory (DFT/B3LYP). The results indicated that L5 with amino group possessed the largest two-photon absorption cross-section values. The systematic investigations revealed that the linear/nonlinear optical properties could be tuned by subtle terminal unit changes for the Donor–Acceptor framework. Due to the existed –NH2 moiety, the subcellular acidic organelle targeting in living cells finally observed using such compounds under two-photon confocal microscopy.

Luminescent metallomesogens based on platinum complex containing triphenylene unit

To explore the influence of mesogenic unit on the liquid crystals and luminescence properties of metal complex, two novel triphenylene-based platinum complexes of TppyPtacac and TppyPtPhacac featuring donor–acceptor framework have been designed and synthesized, in which Tppy is triphenylene–phenylpyridine skeleton and acac/Phacac is pentane-2,4-dione derivative. Differential scanning calorimetry (DSC), polarized optical microscopy (POM) and X-ray diffraction (XRD) techniques demonstrate the complex TppyPtPhacac shows a column mesophase in the region of 55–110 °C. Both platinum complexes exhibit intense emission at about 550 nm in CH2Cl2 solution. Compared to the analogous platinum complexes without triphenylene unit, TppyPtacac and TppyPtPhacac display a remarkable red-shifted emission profiles (ca. 50 nm) due to strong intramolecular charge transfer. Furthermore, the hole mobilities up to 2.5×10−4 cm2 V−1 s−1 and 2.99×10−5 cm2 V−1 s−1 are achieved for the annealed film of TppyPtacac and TppyPtPhacac, respectively, which is one of the highest hole mobility with respect to cyclometalated platinum complexes.

Influence of Spiral Framework on Nonlinear Optical Materials

A series of spiral donor-π-acceptor frameworks (i.e. 2-2, 3-3, 4-4, and 5-5) based on 4-nitrophenyldiphenylamine with π-conjugated linear acenes (naphthalenes, anthracenes, tetracenes, and pentacenes) serving as the electron donor and nitro (NO2 ) groups serving as the electron acceptor were designed to investigate the relationships between the nonlinear optical (NLO) responses and the spirality in the frameworks. A parameter denoted as D was defined to describe the extent of the spiral framework. The D value reached its maximum if the number of NO2 groups was equal to the number of fused benzene rings contained in the linear acene. A longer 4-nitrophenyldiphenylamine chain led to a larger D value and, further, to a larger first hyperpolarizability. Different from traditional NLO materials with charge transfer occurring in the one-dimensional direction, charge transfer in 2-2, 3-3, 4-4, and 5-5 occur in three-dimensional directions due to the attractive spiral frameworks, and this is of great importance in the design of NLO materials. The origin of such an enhancement in the NLO properties of these spiral frameworks was explained with the aid of molecular orbital analysis.

Theoretical investigation on nonlinear optical properties of carbon nanotubes with Stone–Wales defect rings

Based on (6, 0) zigzag carbon nanotubes (ZCNTs), a Stone–Wales defect ring ([5.7]3) is constructed at one end of the ZCNTs, forming a novel nanostructure [5.7]3ZCNT. The introduction of [5.7]3 breaks the centrosymmetry of the nanotube and remarkably changes electronic and magnetic properties of the nanotube. Unlike the (6, 0)ZCNT which has an open-shell singlet ground state, the [5.7]3ZCNT has a closed-shell singlet ground state with a large dipole moment, polarizability, and first hyperpolarizability. Interestingly, the [5.7]3ZCNT itself has a donor–π–acceptor framework, in which the Stone–Wales defect ring serves as an electron donor (D) while the zigzag nanotube works as an electron acceptor (A) and a conjugated bridge (π). Moreover, increasing both the tube diameter and tube length could enhance second-order nonlinear optical (NLO) responses, with the former being more effective than the latter.

Quantifying the Relationship between the Maximum Achievable Voltage and Current Levels in Low-Bandgap Polymer Photovoltaics

A critical problem in the design of materials for organic photovoltaics is quantifying the driving force needed for efficient charge separation without losses associated with a large overpotential. Here, we directly measured the effect of the molecular driving force on the charge transfer rate in films of low-bandgap push–pull type polymers mixed with a series of fullerene-based molecular acceptors using broadband near-infrared transient absorption spectroscopy. By systematically tuning the absolute energy levels of the donor and acceptor, as well as the relative offset between them, we determine the minimum voltage loss required to achieve a high short circuit current. A molecular donor–acceptor framework provides a quantitative description of the charge transfer rate constants in our system and describes the scaling of the photogenerated current with S1–LUMO energy offset. These results point to potential efficiency gains for high performing polymer devices through recovery of additional voltage without sacrificing current output.

Antibody humanization by redesign of complementarity-determining region residues proximate to the acceptor framework

Antibodies are key components of the adaptive immune system and are well-established protein therapeutic agents. Typically high-affinity antibodies are obtained by immunization of rodent species that need to be humanized to reduce their immunogenicity. The complementarity-determining regions (CDRs) contain the residues in a defined loop structure that confer antigen binding, which must be retained in the humanized antibody. To design a humanized antibody, we graft the mature murine CDRs onto a germline human acceptor framework. Structural defects due to mismatches at the graft interface can be fixed by mutating some framework residues to murine, or by mutating some residues on the CDRs’ backside to human or to a de novo designed sequence. The first approach, framework redesign, can yield an antibody with binding better than the CDR graft and one equivalent to the mature murine, and reduced immunogenicity. The second approach, CDR redesign, is presented here as a new approach, yielding an antibody with binding better than the CDR graft, and immunogenicity potentially less than that from framework redesign. Application of both approaches to the humanization of anti-α4 integrin antibody HP1/2 is presented and the concept of the hybrid humanization approach that retains “difficult to match” murine framework amino acids and uses de novo CDR design to minimize murine amino acid content and reduce cell-mediated cytotoxicity liabilities is discussed.

Theoretical investigation of second-order nonlinear optical response by linking hexamolybdate with graphene in the donor–acceptor (D–A) framework

The graphenes and polyoxometalates (POMs) have been investigated individually for finding a material with intrinsic nonlinear optical (NLO) behaviours. In this study, we designed a novel molecular hybrid containing POM cluster linked with nanographene. We could be able to design a material with very large second-order NLO response. The Coulomb-attenuating method (CAM–B3LYP) is used with the LANL2DZ basis set for molybdenum atoms and both 6–31G (d) and 6–31+G (d) basis sets for main group elements to calculate polarisability and first hyperpolarisability. The second-order polarisability of the system is significantly enhanced by replacing NH2 or NO2 with hexamolybdate in [NO2–HBC(derivative of hexabenzocoronene)–NH2] from 33.33 × 10− 30 esu (system 1) to 57.71 × 10− 30 esu (system 2) and 231.10 × 10− 30 esu (system 3), respectively. The time-dependent density functional theory (TDDFT) calculations show that the charge transfer from polyanion to graphene in systems 2 and 3 is responsible for the NLO properties of this kind of compound. We believe that the novel designed link between graphene and POMs is a door for further studies to find materials with an excellent NLO response.

Anti-idiotypic antibody Ab2/3H6 mimicking gp41: a potential HIV-1 vaccine?

Background and aims Anti-idiotypic antibodies (Abs) represent an alternative vaccination approach in human therapy. This approach is based on the idiotype (Id) network theory postulated by Jerne describing the Ab (Ab1) – anti-idiotypic Ab (Ab2) – anti-anti-idiotypic Ab (Ab3) cascade stimulation. Specific anti-Id Abs serve as an “internal image” of the target antigen and can be used to induce Abs able to bind to the cognate antigen [1]. The anti-Id Ab Ab2/ 3H6 [2], developed at our Institute was generated in mouse and is directed against the human monoclonal Ab (mAb) 2F5, which broadly and potently neutralizes primary HIV-1 isolates [3]. Ab2/3H6 which has been characterized previously [4,5] is able to mimic the antigen recognition site of 2F5 and therefore it is suggested as a putative candidate for an HIV-1 vaccine. We investigated the potential of Ab2/3H6 by immunization of Fab fragments and fusion proteins with interleukin 15 (IL15) and tetanus toxin (TT) tags as immune modulators. After three prime/boost administrations rabbit sera were purified and analyzed for 2F5-like specific Abs. Further, the 2F5-like Abs from the sera were enriched by affinity purification and characterized for their binding affinity to 2F5. In an additional approach we applied different humanization methods to reduce the immunogenicity of the originally mouse derived Ab2/3H6. The mouse variable regions of Ab2/3H6 were subjected to three different humanization methods, namely resurfacing, CDR-grafting and superhumanization. Four differently humanized Ab2/3H6 variants were characterized for their binding affinity to 2F5 in comparison to the original Ab2/3H6. Results To evaluate the humoral immune response of Ab2/3H6 we designed Ab2/3H6 fusion proteins with IL15 and TT. Recombinant CHO cell lines were established and after protein purification New Zealand white rabbits were immunized with the Ab2/3H6 variants. Ten days after the final boost sera were collected and analyzed for total rabbit IgG levels. After proteinA affinity purification of the sera the isolated rabbit IgGs were tested for Ab2/3H6 and recombinant gp140 (UG37) specificity (Figure 1A). Further an affinity enrichment step using a UG37/ELDKWA column was performed and the obtained Ab3 fraction was tested on UG37 (Figure 1B) and additionally on the original 2F5 epitope ELDKWA (Figure 1C). Finally the Ab3 fraction was tested for binding affinity to the UG37 in a bio-layer interferometry assay which showed that the Ab3 fraction has a 6.6 fold reduced affinity towards UG37 compared to the mAb 2F5 (Table 1). For the humanization approach three different methods were chosen. The “resurfaced” variant (RS3H6) was developed by a computer model and surface exposed amino acids in the murine framework (FR) were substituted by residues usually found at equivalent positions in human Abs. The “superhumanized” form (SH3H6) was designed by structural homologies between the murine Ab2/3H6 CDRs and human germline CDRs. The most homologous human germline Ab was then used as acceptor FR. For the “CDR-grafted” variant two versions were expressed. An “aggressive” graft (GA3H6) harbouring less backmutations, making the grafted Ab more human-like and a “conservative” graft (GC3H6) with more backmutations. The different “reshaped” variable regions of Ab2/3H6 were expressed in CHO-cells as IgG1 molecules. The obtained “humanized” Ab variants were further characterized by competition ELISA (Figure 1D). * Correspondence: renate.kunert@boku.ac.at Department of Biotechnology, Institute for Applied Microbiology, BOKU – University of Natural Resources and Life Sciences, A-1190 Vienna, Austria Kunert and Mader BMC Proceedings 2011, 5(Suppl 8):P64 http://www.biomedcentral.com/1753-6561/5/S8/P64

Stabilization and humanization of a single-chain Fv antibody fragment specific for human lymphocyte antigen CD19 by designed point mutations and CDR-grafting onto a human framework

A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut. In the second variant, the murine CDRs were grafted to the human acceptor framework huVkappa3-huV(H)3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic applications.

Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering

By combining the knowledge gained from an analysis of the biophysical properties of natural antibody variable domains, the effects of mutations obtained in directed evolution experiments, and the detailed structural comparison of antibodies, it has now become possible to engineer antibodies for higher thermodynamic stability and more efficient folding. This is particularly important when antibodies are to be used under conditions where the disulfide bonds cannot form, i.e., in intracellular applications (as “intrabodies”). We describe in detail two methods for the knowledge-based improvement of antibody stability and folding efficiency. While CDR grafting from a non-human to the most closely related human antibody framework is an established technique to reduce the immunogenicity of a therapeutic antibody, CDR grafting for stabilization implies the use of a more distantly related acceptor framework with superior biophysical characteristics. The use of such dissimilar frameworks requires particular attention to antigen contact residues outside the classical CDR definition and to residues capable of indirectly affecting the conformation of the antigen binding site. As a second alternative, the stability of a suboptimal framework can be improved by the introduction of point mutations designed to optimize key residue interactions. We describe the analysis methods used to identify such point mutations, which can be introduced all at once, while maintaining the framework features necessary for antigen binding. These rational approaches render the continued “rediscovery” of certain mutations by directed evolution unnecessary, but they can also be used in conjunction with such methods to discover even better molecules.