Nikolaus Krall

Site-selective protein-modification chemistry for basic biology and drug development

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

A Small‐Molecule Drug Conjugate for the Treatment of Carbonic Anhydrase IX Expressing Tumors

Antibody-drug conjugates are a very promising class of new anticancer agents, but the use of small-molecule ligands for the targeted delivery of cytotoxic drugs into solid tumors is less well established. Here, we describe the first small-molecule drug conjugates for the treatment of carbonic anhydrase IX expressing solid tumors. Using ligand-dye conjugates we demonstrate that such molecules can preferentially accumulate inside antigen-positive lesions, have fast targeting kinetics and good tumor-penetrating properties, and are easily accessible by total synthesis. A disulfide-linked drug conjugate with the maytansinoid DM1 as the cytotoxic payload and a derivative of acetazolamide as the targeting ligand exhibited a potent antitumor effect in SKRC52 renal cell carcinoma in vivo. It was furthermore superior to sunitinib and sorafenib, both small-molecule standard-of-care drugs for the treatment of kidney cancer.

Small Targeted Cytotoxics: Current State and Promises from DNA‐Encoded Chemical Libraries

The targeted delivery of potent cytotoxic agents has emerged as a promising strategy for the treatment of cancer and other serious conditions. Traditionally, antibodies against markers of disease have been used as drug-delivery vehicles. More recently, lower molecular weight ligands have been proposed for the generation of a novel class of targeted cytotoxics with improved properties. Advances in this field crucially rely on efficient methods for the identification and optimization of organic molecules capable of high-affinity binding and selective recognition of target proteins. The advent of DNA-encoded chemical libraries allows the construction and screening of compound collections of unprecedented size. In this Review, we survey developments in the field of small ligand-based targeted cytotoxics and show how innovative library technologies will help develop the drugs of the future.

Curative Properties of Noninternalizing Antibody–Drug Conjugates Based on Maytansinoids

It is generally thought that the anticancer efficacy of antibody-drug conjugates (ADC) relies on their internalization by cancer cells. However, recent work on an ADC that targets fibronectin in the tumor microenvironment suggests this may not be necessary. The alternatively spliced extra domains A and B (EDA and EDB) of fibronectin offer appealing targets for ADC development, because the antigen is strongly expressed in many solid human tumors and nearly undetectable in normal tissues except for the female reproductive system. In this study, we describe the properties of a set of ADCs based on an antibody targeting the alternatively spliced EDA of fibronectin coupled to one of a set of potent cytotoxic drugs (DM1 or one of two duocarmycin derivatives). The DM1 conjugate SIP(F8)-SS-DM1 mediated potent antitumor activity in mice bearing DM1-sensitive F9 tumors but not DM1-insensitive CT26 tumors. Quantitative biodistribution studies and microscopic analyses confirmed a preferential accumulation of SIP(F8)-SS-DM1 in the subendothelial extracellular matrix of tumors, similar to the pattern observed for unmodified antibody. Notably, we found that treatments were well tolerated at efficacious doses that were fully curative and compatible with pharmaceutical development. Our findings offer a preclinical proof-of-concept for curative ADC targeting the tumor microenvironment that do not rely upon antigen internalization.

A bivalent small molecule-drug conjugate directed against carbonic anhydrase IX can elicit complete tumour regression in mice

There is a pressing need for the development of innovative chemical drug delivery strategies in oncology, since conventional chemotherapeutic agents typically do not localise to solid tumours in vivo. It is widely accepted that bivalent antibody formats accumulate in tumours more strongly than monovalent ones and that they should thus be preferred for antibody-based pharmacodelivery approaches. For small molecule-drug conjugates this is less clear. Here, we show that a bivalent ligand against the tumour marker carbonic anhydrase IX leads to an improved tumour-targeting performance compared with the corresponding monovalent counterpart in the SKRC52 model of constitutively CAIX-positive renal cell carcinoma. A bivalent disulfide-linked small drug conjugate with the potent cytotoxic maytansinoid DM1 as the payload can mediate complete eradication of the same tumours, which are resistant to standard-of-care therapeutics, in a proportion of treated mice. In the A375 melanoma model, which preferentially expresses CAIX at sites distant to blood vessels, no measurable tumour accumulation could be observed. Our results suggest that the use of bivalent small molecule ligand-drug conjugates against CAIX may represent an attractive chemical strategy for the treatment of constitutively CAIX-positive kidney cancer.

Tumor-targeting antibody-anticalin fusion proteins for in vivo pretargeting applications.

Pretargeting approaches rely on the injection of bispecific antibodies capable of recognizing both an accessible disease marker and a small ligand, which is typically administered at a later stage and which serves as delivery vehicle for a payload for imaging or therapy applications. In the oncology field, pretargeting strategies have exhibited extremely promising biodistribution results and in vivo selectivity, but have often relied on the cumbersome preparation of multispecific antibodies by chemical conjugation techniques. Here, we describe the design, production, and characterization of a novel class of bispecific multivalent antibody products, which contain both antibody fragments and an anticalin moiety for the simultaneous recognition of tumor-associated antigens and a small organic molecule. Anticalins are derivatives of the naturally occurring binding proteins lipocalins, which have been engineered to recognize a target molecule with high affinity. In particular, we produced and compared in vitro and in vivo different fusion proteins, which contained the anticalin FluA that selectively recognizes various different fluorescein derivatives and the F8 antibody specific to the alternatively spliced EDA domain of fibronectin (a marker of tumor angiogenesis). The selective accumulation of the most promising fusion-protein scFv(F8)-FluA-scFv(F8) on solid tumors and simultaneous binding of fluorescein derivatives could be visualized in vivo using a fluorescein-near-infrared fluorescent dye conjugate, confirming the potential of antibody-anticalin fusion proteins for pretargeting applications.

A comprehensive surface proteome analysis of myeloid leukemia cell lines for therapeutic antibody development

UNLABELLED A detailed characterization of the cell surface proteome facilitates the identification of target antigens, which can be used for the development of antibody-based therapeutics for the treatment of hematological malignancies. We have performed cell surface biotinylation of five human myeloid leukemia cell lines and normal human granulocytes, which was used for mass spectrometric analysis and allowed the identification and label-free, relative quantification of 320 membrane proteins. Several proteins exhibited a pronounced difference in expression between leukemia cell lines and granulocytes. We focused our attention on CD166/ALCAM, as this protein was strongly up-regulated on all AML cell lines and AML blasts of some patients. A human monoclonal antibody specific to CD166 (named H8) was generated using phage display technology. H8 specifically recognized AML cells in FACS analysis while demonstrating tumor targeting properties in vivo. After in vitro screening of five potent cytotoxic agents, a duocarmycin derivative was used for the preparation of an antibody-drug conjugate, which was able to kill AML cells in vitro with an IC50 of 8nM. The presented atlas of surface proteins in myeloid leukemia provides an experimental basis for the choice of target antigens, which may be used for the development of anti-AML therapeutic antibodies. BIOLOGICAL SIGNIFICANCE The ability to discriminate between malignant and healthy, essential cells represents an important requirement for the development of armed antibodies for the therapy of hematological malignancies. Our proteomic study is, to our knowledge, the first large scale comparison of the accessible cell surface proteome of leukemia cells and normal blood cells, facilitating the choice of a suitable target for the treatment of acute myeloid leukemia (AML). An antibody drug conjugate was generated recognizing the CD166 antigen which was found to be strongly up-regulated in all AML cell lines and AML blasts of some patients. This antibody drug conjugate SIP(H8)-Duo might be further characterized in therapy experiments and might lead to a new targeted treatment option for AML.

Targeting carbonic anhydrase IX with small organic ligands.

Carbonic anhydrase IX (CAIX) is expressed in many solid tumors in response to hypoxia and plays an important role in tumor acid-base homeostasis under these conditions. It is also constitutively expressed in the majority of renal cell carcinoma. Its functional inhibition with small molecules has recently been shown to retard tumor growth in murine models of cancer, reduce metastasis and tumor stem cell expansion. Additionally, CAIX is a promising antigen for targeted drug delivery approaches. Initially validated with anti-CAIX antibodies, the tumor-homing capacity of high-affinity small-molecule ligands of CAIX has recently been demonstrated. Indeed, conjugates formed of CAIX ligands and potent cytotoxic drugs could eradicate CAIX-expressing solid tumors in mice. These results suggest that CAIX is a promising target for the development of novel therapies for the treatment of solid tumors.

A 99mTc-Labeled Ligand of Carbonic Anhydrase IX Selectively Targets Renal Cell Carcinoma In Vivo

Small organic ligands, selective for tumor-associated antigens, are increasingly being considered as alternatives to monoclonal antibodies for the targeted delivery of diagnostic and therapeutic payloads such as radionuclides and drugs into neoplastic masses. We have previously described a novel acetazolamide derivative, a carbonic anhydrase ligand with high affinity for the tumor-associated isoform IX (CAIX), which can transport highly potent cytotoxic drugs into CAIX-expressing solid tumors. The aim of the present study was to quantitatively investigate the biodistribution properties of said ligand and understand whether acetazolamide conjugates merit further development as drug carriers and radioimaging agents. Methods: The conjugate described in this study, consisting of a derivative of acetazolamide, a spacer, and a peptidic 99mTc chelator, was labeled using sodium pertechnetate under reducing conditions and injected intravenously into CAIX-expressing SKRC-52 xenograft–bearing mice. Animals were sacrificed, and organ uptake as percentage injected activity of radiolabeled ligand per gram of tissues (%IA/g) was evaluated between 10 min and 24 h. Additionally, postmortem imaging by SPECT was performed. Results: The acetazolamide conjugate described in this study could be labeled to high radiochemical purity (>95%, 2.2–4.5 MBq/nmol). Analysis of organ uptake at various time points revealed that the ligand displayed a maximal tumor accumulation 3 h after intravenous injection (22 %IA/g), with an excellent tumor-to-blood ratio of 70:1 at the same time point. The ligand accumulation in the tumor was more efficient than in any other organ, but a residual uptake in the kidney, lung, and stomach (9, 16, and 10 %IA/g, respectively) was also observed, in line with patterns of carbonic anhydrase isoform expression in those tissues. Interestingly, tumor-to-organ ratios improved on administration of higher doses of radiolabeled ligand, suggesting that certain binding sites in normal organs can be saturated in vivo. Conclusion: The 99mTc-labeled acetazolamide conjugate exhibits high tumor uptake and favorable tumor-to-kidney ratios of up to 3 that may allow imaging of tumors in the kidney and distant sites at earlier time points than commonly possible with antibody-based products. These data suggest that the described molecule merit further development as a radioimaging agent for CAIX-expressing renal cell carcinoma.