Small Molecule Linkers Research Articles

Clickable HaloTag ligands for live cell labeling and imaging

Self-labeling protein (SLP) tags, such as HaloTag, have gained considerable interest as advanced tools for live cell labeling. However, the chloroalkane-based substrates that can be directly used for protein labeling are limited. Here, we report two bioorthogonal small molecule linkers, chloroalkane-tetrazine (CA-Tz) and chloroalkane-azide (CA-N3), which can penetrate cell membranes and facilitate click chemistry-based labeling in live cells. We compare their labeling capability using two clickable silicon rhodamine dyes (SiR-PEG3-TCO and SiR-PEG4-DBCO). Confocal imaging results demonstrate that using CA-Tz and SiR-PEG3-TCO dye exhibits superior intracellular labeling with low nonspecific signals. We subsequently compared the photostability of SiR dyes with that of green fluorescent proteins (mEmerald). Total internal reflection fluorescence (TIRF) imaging indicates that SiR dyes exhibit superior photostability under identical excitation conditions, making them suitable for long-term cell imaging. Furthermore, SiR dyes labeling also shows high structure retention for the fourth-order super-resolution optical fluctuation imaging (SOFI) compared to fluorescent proteins. This study presents clickable HaloTag linkers as effective tools for live cell labeling and imaging, highlighting the high-quality labeling of chloroalkane linkers and clickable dyes for live cell imaging.

Abstract 1764: Development of an alphalex™-auristatin low pH targeting conjugate for the treatment of solid tumors

Abstract Auristatins such as monomethyl auristatin E (MMAE) are a class of high potency microtubule targeting compounds that have an extremely narrow therapeutic window. Targeting potent auristatins to the tumor is the only feasible method of unlocking the clinical potential of such toxic molecules. While there are currently four marketed antibody-drug conjugates (ADCs) featuring auristatins, these ADCs face the same fundamental issues - tumor restriction by target antigen and the potential for off target release of payload. Alphalex࣪ is a tumor targeting technology consisting of a unique variant of a family of pH-Low Insertion Peptides (pHLIP®) that target acidic cell surfaces (references 1-2), a cleavable small molecule linker, and an anti-cancer agent warhead. Alphalex࣪ thereby allows for antigen independent targeting of the tumor and enables intracellular delivery of the warhead by leveraging the low pH microenvironment of the tumor, a universal feature common to all tumors due to the Warburg effect. Here we report the preclinical efficacy, safety, and antigen-independent tumor-targeting properties of alphalex࣪ conjugated to MMAE. We demonstrate the ability of alphalex࣪-MMAE to display potent in vitro and in vivo efficacy in colorectal, non-small cell lung, and prostate carcinoma cell lines. We further show that alphalex࣪-MMAE efficiently and safely delivers efficacious levels of MMAE selectively to tumor and demonstrates extreme plasma stability, with 0.02% warhead release over 24h in the rat. Based on the excellent preclinical safety and efficacy profile of alphalex࣪-MMAE, Cybrexa will move forward with the goal of initiating IND-enabling studies in 2022.

Efficient Human Acute Myeloid Leukemia Targeting By Universal Chimeric Antigen Receptor T-Cells Via Combinatorial Use of Linkers

Chimeric antigen receptor (CAR) T-cells are genetically engineered T-cells with potent biocidal activity against respective target-expressing cells. Recently, CAR T-cells have been successfully used clinically to eradicate B cell-derived malignancies by targeting B-cell lineage specific surface antigens (e.g. CD19, BCMA). However, several limitations of current single-antigen targeting CAR T-cell therapies are becoming evident: a) low target tumor-antigen expression might lead to low CAR T-cell targeting efficacy; b) single antigen-targeting might lead to rapid selection of tumor cells with low or loss of antigen expression; c) single antigen-targeting does frequently not generate a high tumor-selectivity as tumor-antigens are frequently also expressed on healthy tissues; d) production of single-antigen CAR T-cells is time- and resource-consuming but results in effectors that target only one antigen; e) on-target off-tumor as well as off-target side-effects are difficult to control without terminally eliminating CAR T-cells in the recipient. While a-c) might be overcome by combinatorial tumor-antigen targeting, d-e) might be addressed by production of Universal CAR T-cells that recognize a specific tag on selective bridging molecules with short half-life.To address some of these limitations in principle, we have here developed universal CAR T-cells targeting fluorescein, as well as fluorescein-labeled antibody-constructs directed against several cell surface antigens, that would serve as versatile, combinatorial selective linkers and, upon target binding, also CAR T-cells activators. We then tested the system on human acute myeloid leukemia (AML) cell lines and primary patient AML blasts. Specifically, we engineered CAR T-cells, termed FluA-CAR T-cells, to display the anti-fluorescein engineered lipocalin FluA, which mediated recognition of randomly or site-specifically fluorescein-labeled antibodies in IgG or short half-life diabody (Db) format, directed against the frequently AML expressed antigens CD33, CD117 and CD371. Site-specific chemical modification methods and cysteine-tagged Db mediated the strongest AML killing results in vitro over a broad range of antibody concentrations.We then hypothesized that FluA-CAR T-cells, targeting AML cells via combinatorial use of linker molecules adapted to specific AML antigen-expression profiles, would allow to most efficiently eliminate AML cells in a dose- and timely-regulated fashion. To this end, we tested single and combinatorial use of fluoresceinated anti-CD117 Db and anti-CD371 Db on MOLM13 AML cell lines, engineered to be either CD117 highCD371 neg, CD117 negCD371 high, or CD117 highCD371 high. Indeed, combination of anti-CD117 and anti-CD371 Db-linkers, resulted in significantly improved MOLM13 cell lysis compared to equimolar concentrations of single agents in vitro (example figure). We then tested the same approach, targeting CD117 +CD371 + primary AML cells from two different patients, using either patient-derived or allogeneic FluA CAR T-cells. Again, the combinatorial use of linkers generated a significantly higher AML cell lysis than the use of single Db linkers.We thus here provide proof-of-concept for the generation of highly potent universal targeting FluA CAR T-cells from healthy donors and AML patients. By choosing suitable CAR-adaptors with respect to their conjugation chemistry and size, it is possible to tightly regulate CAR-T cell activity against CD33, CD117 and CD371 expressing AML cells and likely any tumor cell expressed antigen. Short half-life small molecule linkers will allow to control FluA CAR T-cell on-off activity and combinatorial use of linkers will allow to maximize anti-tumor activity and to minimize on-target off-tumor toxicity. [Display omitted] DisclosuresMyburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

New Bifunctional Chelators Incorporating Dibromomaleimide Groups for Radiolabeling of Antibodies with Positron Emission Tomography Imaging Radioisotopes.

Positron Emission Tomography (PET) imaging with antibody-based contrast agents frequently uses the radioisotopes [64Cu]Cu2+ and [89Zr]Zr4+. The macrobicyclic chelator commonly known as sarcophagine (sar) is ideal for labeling receptor-targeted biomolecules with [64Cu]Cu2+. The siderophore chelator, desferrioxamine-B (dfo), has been widely used to incorporate [89Zr]Zr4+ into antibodies. Here, we describe new bifunctional chelators of sar and dfo: these chelators have been functionalized with dibromomaleimides (dbm), that enable site-specific and highly stable attachment of molecular cargoes to reduced, solvent-accessible, interstrand native disulfide groups. The new sar–dbm and dfo–dbm derivatives can be easily conjugated with the IgG antibody trastuzumab via reaction with reduced interstrand disulfide groups to give site-specifically modified dithiomaleamic acid (dtm) conjugates, sar–dtm–trastuzumab and dfo–dtm–trastuzumab, in which interstrand disulfides are rebridged covalently with a small molecule linker. Both sar– and dfo–dtm–trastuzumab conjugates have been radiolabeled with [64Cu]Cu2+ and [89Zr]Zr4+, respectively, in near quantitative radiochemical yield (>99%). Serum stability studies, in vivo PET imaging, and biodistribution analyses using these radiolabeled immunoconjugates demonstrate that both [64Cu]Cu-sar–dtm–trastuzumab and [89Zr]Zr-dfo–dtm–trastuzumab possess high stability in biological milieu. Dibromomaleimide technology can be easily applied to enable stable, site-specific attachment of radiolabeled chelators, such as sar and dfo, to native interstrand disulfide regions of antibodies, enabling tracking of antibodies with PET imaging.

Aluminum textile-based binder-free nanostructured battery cathodes using a layer-by-layer assembly of metal/metal oxide nanoparticles

Despite considerable interest in textile-based battery electrodes with large surface areas and mechanical flexibility, issues have restricted further advances in the energy performance of textile electrodes. These issues include the ineffective incorporation of conductive and/or active components into textile frameworks, the poor charge transfer between energy materials, and the formation of numerous unstable interfaces within textile electrodes. Herein, we introduce an aluminum textile-based lithium-ion battery cathode with remarkable areal capacity, high rate performance, and good cycling stability. Ligand exchange reaction-induced layer-by-layer (LbL) assembly of metal nanoparticles and small molecule linkers, with subsequent metal electroplating, perfectly converted polyester textiles to 3D-porous aluminum textiles that can be used as current collectors and high-energy reservoirs. The consecutive LbL assembly of high-energy LiFePO4 and conductive indium tin oxide nanoparticles onto the aluminum textiles using small organic linkers significantly increased the areal capacity and cycling stability (at least 580 cycles) of the resultant cathode, allowing facile charge transfer within the textile electrodes. Furthermore, the areal capacity of these textile electrodes increased from 1.07 to 3.28 mA h cm−2, with an increase in the folding number from 0 to 2.

Abstract 6242: Development of alphalex™-toxin low pH targeting conjugates for the treatment of solid tumors

Abstract Maytansines and their derived maytansinoid DMx compounds are high potency microtubule targeting compounds that have an extremely narrow therapeutic window. Unacceptable dose limiting systemic toxicity has limited the therapeutic potential of these potent anti-oncogenic compounds. Targeting maytansinoids to the tumor is the only feasible method of unlocking the clinical potential of such toxic molecules. To date Trastuzumab-DM1 (Kadcyla®) remains the only approved antibody-maytansinoid conjugate on the market. Most preclinical maytansinoid conjugates to date face the same issues encountered by Kadcyla® - tumor restriction by target antigen and the potential for off target release of payload. alphalexTM is a tumor targeting technology consisting of a unique variant of pH-Low Insertion Peptide (pHLIP®; references 1-3), cleavable small molecule linker and anti-cancer agent warhead. alphalexTM thereby allows for antigen independent targeting of the tumor and enables intracellular delivery of the warhead by leveraging the low pH microenvironment of the tumor, a universal feature common to all tumors due to the Warburg effect. Here we demonstrate the ability to conjugate the maytansinoids DM1 and DM4 to alphalexTM via both direct and linker-mediated conjugation. We have demonstrated the ability of our alphalexTM-DM4 conjugate candidates (CBX-13) to have single digit nanomolar potency in vitro as well as exquisitely potent and long-lasting anti-tumor activity in a HER2-negative xenograft model that is un-targetable by competing therapies. In particular we have demonstrated that CBX-13 safely delivers amounts of maytansinoid in vivo that otherwise result in systemic toxicity and death when dosed as free warhead. Based on the SAR of this first generation of maytansinoid conjugates we are further optimizing our alphalexTM - maytansinoid conjugation strategy with the goal of moving forward with IND-enabling studies in the near future.

A Metal-Like Conductive Elastomer with a Hierarchical Wrinkled Structure.

For the development of wearable electronics, the replacement of rigid, metallic components with fully elastomeric materials is crucial. However, current elastomeric electrodes suffer from low electrical conductivity and poor electrical stability. Herein, a metal-like conductive elastomer with exceptional electrical performance and stability is presented, which is used to fabricate fully elastomeric electronics. The key feature of this material is its wrinkled structure, which is induced by in situ cooperation of solvent swelling and densely packed nanoparticle assembly. Specifically, layer-by-layer assembly of metal nanoparticles and small-molecule linkers on elastomers generates the hierarchical wrinkled elastomer. The elastomer demonstrates remarkable electrical conductivity (170 000 and 11 000 S cm-1 at 0% and 100% strain, respectively), outperforming previously reported elastomeric electrodes based on nanomaterials. Furthermore, a fully elastomeric triboelectric nanogenerator based on wrinkled elastomeric electrode exhibits excellent electric power generation performance due to the compressible, large contact area of the wrinkled surface during periodic contact and separation.

Regulation of Protein Structural Changes by Incorporation of a Small-Molecule Linker.

Proteins have the potential to serve as nanomachines with well-controlled structural movements, and artificial control of their conformational changes is highly desirable for successful applications exploiting their dynamic structural characteristics. Here, we demonstrate an experimental approach for regulating the degree of conformational change in proteins by incorporating a small-molecule linker into a well-known photosensitive protein, photoactive yellow protein (PYP), which is sensitized by blue light and undergoes a photo-induced N-terminal protrusion coupled with chromophore-isomerization-triggered conformational changes. Specifically, we introduced thiol groups into specific sites of PYP through site-directed mutagenesis and then covalently conjugated a small-molecule linker into these sites, with the expectation that the linker is likely to constrain the structural changes associated with the attached positions. To investigate the structural dynamics of PYP incorporated with the small-molecule linker (SML-PYP), we employed the combination of small-angle X-ray scattering (SAXS), transient absorption (TA) spectroscopy and experiment-restrained rigid-body molecular dynamics (MD) simulation. Our results show that SML-PYP exhibits much reduced structural changes during photo-induced signaling as compared to wild-type PYP. This demonstrates that incorporating an external molecular linker can limit photo-induced structural dynamics of the protein and may be used as a strategy for fine control of protein structural dynamics in nanomachines.

Synthesis of Cyclic Megamolecules.

This paper describes the synthesis of giant cyclic molecules having diameters of 10-20 nm. The molecules are prepared through the reactions of a fusion protein building block with small molecule linkers that are terminated in irreversible inhibitors of enzyme domains present in the fusion. This building block has N-terminal cutinase and C-terminal SnapTag domains that react irreversibly with p-nitrophenyl phosphonate (pNPP) and benzylguanine (BG) groups, respectively. We use a bis-BG and a BG-pNPP linker to join these fusion proteins into linear structures that can then react with a bis-pNPP linker that joins the ends into a cyclic product. The last step can occur intramolecularly, to give the macrocycle, or intermolecularly with another equivalent of linker, to give a linear product. Because these are coupled first- and second-order processes, an analysis of product yields from reactions performed at a range of linker concentrations gives rate constants for cyclization. We determined these to be 9.7 × 10-3 s-1, 2.3 × 10-3 s-1, and 8.1 × 10-4 s-1 for the dimer, tetramer, and hexamer, respectively. This work demonstrates an efficient route to cyclic macromolecules having nanoscale dimensions and provides new scaffolds that can be generated using the megamolecule approach.

A General Strategy for the Preparation of Thalidomide-Conjugate Linkers

The synthesis of small-molecule linkers for installation of thalidomide-based conjugates is described. Linker properties have been recognized as vital to conjugate success in drug discovery and delivery systems. These small-molecule tethers act as linkages between molecules, can also aid in cell permeability, and act as solubilizing agents. This work shows our progress in synthesizing conjugates with a variety of linker characteristics. The adaptability and manipulation of these and other linkers holds potential in improving synthetic control of chemical connectivities toward therapeutic development.

Self-Accelerating Click Reaction for Cyclic Polymer

As the most straightforward synthetic strategy for cyclic polymers in theory, the traditional homodifunctional bimolecular ring-closure methods showed limited success for preparing pure cyclic polymers in practice even after several decades of development. A breakthrough was achieved in this paper to develop a successful homodifunctional bimolecular ring-closure method using a self-accelerating double strain-promoted azide–alkyne click reaction as the intermolecular and subsequent intramolecular coupling reactions. Because of the self-accelerating property of coupling reaction, this novel approach eliminated the usage of equimolar quantities between telechelic polymers and small molecule linkers, which was the prerequisite of traditional homodifunctional bimolecular ring-closure methods for pure cyclic polymers. More importantly, this approach could use an excess amount of small linkers to increase the intermolecular coupling reaction rate, further resulting in a significantly enhanced preparation efficie...

Surface modification of PLGA nanoparticles to deliver nitric oxide to inhibit Escherichia coli growth

Polymer nanoparticles consisting of poly (DL-lactic-co-glycolic acid) were surface functionalized to deliver nitric oxide. These biodegradable and biocompatible nanoparticles were modified with an S-nitrosothiol molecule, S-nitrosocysteamine, as the nitric oxide delivery molecule. S-nitrosocysteamine was covalently immobilized on the nanoparticle surface using small organic molecule linkers and carbodiimide coupling. Nanoparticle size, zeta potential, and morphology were determined using dynamic light scattering and scanning electron microscopy, respectively. Subsequent attachment of the S-nitrosothiol resulted in a nitric oxide release of 37.1±1.1nmol per milligram of nanoparticles under physiological conditions. This low concentration of nitric oxide reduced Escherichia coli culture growth by 31.8%, indicating that the nitric oxide donor was effective at releasing nitric oxide even after attachment to the nanoparticle surface. Combining the nitric oxide modified nanoparticles with tetracycline, a commonly prescribed antibiotic for E. coli infections, increased the effectiveness of the antibiotic by 87.8%, which allows for lower doses of antibiotics to be used in order to achieve the same effect. The functionalized nanoparticles were not cytotoxic to mouse fibroblasts.

Nanoscale imaging of RNA with expansion microscopy.

The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy (ExM) of RNA. We developed a small molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, post-expansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity, with single molecule precision, in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) de-crowds RNAs and supports amplification of single molecule signals (i.e., via hybridization chain reaction (HCR)) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.

Dynamic covalent side‐chain cross‐links via intermolecular oxime or hydrazone formation from bifunctional peptides and simple organic linkers

Peptide cyclization via chemoselective reactions between side chains has proven a useful strategy to control folded structure. We report here a method for the synthesis of side-chain to side-chain cyclic peptides based on the intermolecular reaction between a linear peptide functionalized with two aminooxy or hydrazide side chains and an organic dialdehyde linker. A family of oxime-based and hydrazone-based cyclic products is prepared in a modular and convergent fashion by combination of unprotected linear peptide precursors and various small molecule linkers in neutral aqueous buffer. The side-chain to side-chain linkages that result can alter peptide folding behavior. The dynamic covalent nature of the Schiff bases in the cyclic products can be utilized to create mixtures where product composition changes in response to experimental conditions. Thus, a linear peptide precursor can select one organic linker from a mixture, and a cyclic product can dynamically exchange the small molecule component of the macrocycle.

Convergent diversity-oriented side-chain macrocyclization scan for unprotected polypeptides.

Here we describe a general synthetic platform for side-chain macrocyclization of an unprotected peptide library based on the SNAr reaction between cysteine thiolates and a new generation of highly reactive perfluoroaromatic small molecule linkers. This strategy enabled us to simultaneously "scan" two cysteine residues positioned from i, i + 1 to i, i + 14 sites in a polypeptide, producing 98 macrocyclic products from reactions of 14 peptides with 7 linkers. A complementary reverse strategy was developed; cysteine residues within the polypeptide were first modified with non-bridging perfluoroaryl moieties and then commercially available dithiol linkers were used for macrocyclization. The highly convergent, site-independent, and modular nature of these two strategies coupled with the unique chemoselectivity of a SNAr transformation allows for the rapid diversity-oriented synthesis of hybrid macrocyclic peptide libraries with varied chemical and structural complexities.

Modular assembly of protein building blocks to create precisely defined megamolecules.

Enzyme-promoted assembly: The construction of a hetero-bifunctional protein building block, HaloTag-cutinase, that reacts rapidly and selectively with a small-molecule linker is described. The step-wise combination of these building blocks generates a 300 kDa "megamolecule" with precisely defined domain orientation, connectivity, and composition.

A virus-like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose

We show that a model antigen, ovalbumin (OVA), can be chemically conjugated to the exterior of a small heat shock protein (sHsp) cage that has structural similarities to virus-like particles (VLPs). OVA–sHsp conjugation efficiency was dependent upon the stoichiometry and the length of the small molecule linker utilized, and the attachment position on the sHsp cage. When conjugated OVA–sHsp was delivered intranasally to naïve mice, the resulting immune response to OVA was accelerated and intensified, and OVA-specific IgG1 responses were apparent within 5 days after a single immunizing dose, illustrating its utility for vaccine development. If animals were pretreated with a disparate VLP, P22 (a non-replicative bacteriophage capsid), before OVA–sHsp conjugate immunization, OVA-specific IgG1 responses were apparent already by 4 days after a single immunizing dose of conjugate in OVA-naïve mice. Additionally, the mice pretreated with P22 produced high titer mucosal IgA, and isotype-switched OVA-specific serum IgG. Similarly, sHsp pretreatment enhanced the accumulation of lung germinal center B cells, T follicular helper cells, and increased polymeric Ig receptor expression, priming the lungs for subsequent IgG and IgA responses to influenza virus challenge. Thus, sHsp nanoparticles elicited quick and intense antibody responses and these accelerated responses could similarly be induced to antigen chemically conjugated to the sHsp. Pretreatment of mice with P22 further accelerated the onset of the antibody response to OVA–sHsp, demonstrating the utility of conjugating antigens to VLPs for pre-, or possibly post-exposure prophylaxis of lung, all without the need for adjuvant.

Self‐assembly of well‐defined amphiphilic polymeric miktoarm stars, dendrons, and dendrimers in water: The effect of architecture

AbstractFive polymeric architectures with a systematic increase in architectural complexity were synthesized by “click” reactions from a toolbox of functional linear polymers and small molecule linkers. The amphiphilic architectures ranged from a simple 3‐miktoarm star block copolymer to the more complex third generation dendrimer‐like block copolymer, consisting of polystyrene (PSTY) and polyacrylic acid (PAA). Micellization of these architectures in water at a pH of 7 under identical ionic strength gave spherical micelles ranging in size from 9 to 30 nm. Subsequent calculations of the PSTY core density, average surface area per PAA arm on the corona‐core interface, and the relative stretching of the PAA arms provided insights into the effect of architecture on the self‐assembly processes. A particular trend was observed that with increased architectural complexity the hydrodynamic diameter, radius of the core in the dry state and the aggregation number also increased with the exception of the third generation dendrimer. On the basis of these observations, we postulate that thermodynamic factors controlling self‐assembly were the entropic penalty of forming PSTY loops in the core counterbalanced by the reduction in repulsive forces through chain stretching. This results in a greater number of aggregating unimers and consequently larger micelle sizes. The junction points within the architecture also play an important role in controlling the self‐assembly process. The G3 dendrimer showed results contradictory to the aforementioned trend. We believe that the self‐assembly process of this architecture was dominated by the increased attractive forces due to stretching of the PSTY core chains to form a more compact core. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6292–6303, 2009

Peptide-Targeted Diagnostics and Radiotherapeutics

Radiotherapeutic drugs and medical imaging agents, although used for different purposes, both benefit from precise targeting. When systemically administered, either would be most useful if designed to find and bind only a tumor, single type of cell, or unique molecular assembly thereon. In this Account, we examine the use of small peptides, natural and synthetic, to create biochemically specific molecular imaging agents and radiotherapeutic pharmaceuticals, discussing three distinct examples. In one project, a small natural peptide known to target members of the bombesin family of receptors was chemically attached to a strong, versatile metal chelator, DO3A, through a series of small-molecule linkers. The linkers powerfully affected not only binding strength for the bombesin receptors, tissue distribution, and tumor uptake in vivo but also receptor subtype specificity. When the assembly is combined with an active metal ion for human trials, the versatility of the DO3A (dodecanetriacetate) chelate affords choices in selecting the metal ion for different purposes: lutetium for a combination radiotherapeutic and diagnostic agent, (177)Lu-AMBA, and gallium for a positron emission tomography (PET) imaging agent, (68)Ga-AMBA. We also created small (approximately 5-kDa) bivalent peptides, each composed of different chemically linked peptides derived from phage display. The monomer peptides bound to the same target protein, VEGF-R2, a primary target of vascular endothelial growth factor (VEGF), the angiogenesis-stimulating protein. Several families of the monomer peptides did not compete with one another for the binding site on VEGF-R2. Their combination into fully synthetic hetero-bivalent molecules yielded subnanomolar K(d) values and greater than 100-fold improvements over homo-bivalent molecules. Biological activity was evident in the hetero-bivalents, whereas none or very little existed in homo-bivalents, monomers, and monomer mixtures. In ultrasound imaging, tiny bubbles (2 microm in diameter) filled with inert gas can be used as effective contrast agents. By coating the shell of such bubbles with the peptide TKPPR (a tuftsin antagonist), we created contrast agents that bound unexpectedly to cultured endothelial cells expressing angiogenesis targets; the binding was attributable to a previously unnoticed and powerful multivalency effect. TKPPR binds specifically to neuropilin-1 (NP-1), a VEGF co-receptor, but only when multimerized is it avid. Tuftsin, a small peptide derived from immunoglobulin G (IgG) that binds to macrophages during inflammation, has been studied for over 30 years; the receptor has never been cloned. Our results led to new conclusions about tuftsin, NP-1, and the purpose, heretofore unknown, of exon 8 in VEGF, which appears to be involved in NP-1 binding. Our disparate projects demonstrate that small-peptide targeted molecules can be very versatile in drug discovery in combination with classical medicinal chemistry. In particular, multivalent interactions can lead to unpredictable and useful biochemical information, as well as new drug candidates.

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.