Most chemical techniques used to produce antibody−drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, onestep attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide−alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC 50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.

Small phosphorylated metabolites from mycobacteria stimulate human ␥␦ T lymphocytes. Although such phosphoantigens could prove useful in the composition of vaccines involving ␥␦ T cell-mediated immunity, their very low abundance in natural sources limits such applications. Here, we describe the chemical production, purification, and bioactivity of a phosphorylated bromohydrin (BrHPP) analogue that mimics the biological properties of natural phosphoantigens. This compound can be obtained in gram amounts, is easy to detect, and is of high stability in aqueous solutions. Whereas unspecific binding of BrHPP to a wide panel of cell surface receptors is not detected even at micromolar concentrations, nanomolar concentrations specifically trigger effector responses of human ␥␦ T lymphocytes. Thus, BrHPP is a novel molecule enabling potent immunostimulation of human ␥␦ T lymphocytes.Stimulating ligands for ␣␤ T lymphocytes are usually composed of single peptides complexed at the surface of major histocompatibility complex molecules. Some small non-peptidic structures, however, may also constitute specific agonist ligands for T cells, particularly ␥␦ T lymphocytes. In human blood, about 3% of T cells initiate their physiological function upon recognition of small phosphorylated non-peptide antigens (phosphoantigens). This cognate interaction involves on the one hand phosphoantigens in the absence of major histocompatibility complex-presenting molecules, and on the other hand, highly selective receptors (TCR) 1 of ␥␦ subtype. In nature, phosphoantigens that can activate human ␥␦ T cells at nanomolar concentrations are produced by Gram-positive and Gram-negative bacteria and also by some eukaryotic parasites and plants. Synthetic analogues of natural phosphoantigens are also known, but their stimulating concentrations for the reactive cells never go below the micromolar range. Mycobacterium tuberculosis, the agent of human tuberculosis, produces four distinct phosphoantigens. These molecules share a moiety that is responsible for the potent stimulation of ␥␦ cells seen in tuberculosis patients (1). The structure of this common core is 3-formyl-1-butyl-pyrophosphate, a recently described phosphoester (2). Its metabolic production might be related to the non-mevalonate (or so-called Rohmer's) pathway for isoprenoid precursor biosynthesis (3). 3-formyl-1-butyl-pyrophosphate is produced in very small amounts in slow-growing mycobacteria such as Mycobacterium tuberculosis and only accumulates to submicromolar concentrations in culture media from fast-growing mycobacterial species (4). Getting large amounts of highly bioactive phosphoantigens by purification routes from such natural sources is therefore hard to conceive.Such molecules could prove therapeutically useful for immunotherapeutic approaches involving ␥␦ T cell-mediated immunity, such as elicitation of anti-infectious protection or antitumor immunity (5, 6). To address the need for readily available highly bioactive phosphoantigens, we have developed a synthetic reagen...

Biochemistry. In the article "Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays" by Ingrid Remy and Stephen W. Michnick, which appeared in number 10, May 11, 1999, of Proc. Natl. Acad. Sci. USA (96, 5394-5399) Immunology. In the article entitled "Murine natural killer cells contribute to the granulomatous reaction caused by mycobacterial cell walls" by I. Apostolou, Y. Takahama, C. Belmant, T. Kawano, M. Huerre, G. Marchal, J. Cui, M. Taniguchi, H. Nakauchi, J.-J. Fournié, P. Kourilsky, and G. Gachelin, which appeared in number 9, April 27, 1999 of Proc. Natl. Acad. Sci. USA (96,(5141)(5142)(5143)(5144)(5145)(5146), the authors request that the following correction be noted: the title should be "Murine natural killer T (NKT) cells contribute to the granulomatous reaction caused by mycobacterial cell walls." Neurobiology. In the articles "An empirical basis for Mach bands" by R. Beau Lotto, S. Mark Williams, and Dale Purves, which appeared in number 9, April 27, 1999, of Proc. Natl. Acad. Sci. USA (96,(5239)(5240)(5241)(5242)(5243)(5244), and "Mach bands as empirically derived associations" by R. Beau Lotto, S. Mark Williams, and Dale Purves, which appeared in number 9, April 27, 1999, of Proc. Natl. Acad. Sci. USA (96,(5245)(5246)(5247)(5248)(5249)(5250), the following correction should be noted. The reproduction of some of the figures in these papers was unsatisfactory due to the presence of moiré patterns and other deficiencies in the published versions. Given the difficulty in faithfully reproducing gradients in print, readers may wish to view the electronic versions of the figures at purveslab.neuro.duke.edu.Psychology. In the article "Spatial attention affects brain activity in human primary visual cortex" by Sunil P. Gandhi, David J. Heeger, and Geoffrey M. Boynton, which appeared in number 6, March 16, 1999, of Proc. Natl. Acad. Sci. USA (96, 3314-3319), due to an error in the PNAS office, a sentence was omitted. The sentence is shown in bold type in context in the complete paragraph below.On the other hand, it is certainly possible that the V1 modulation we observed might have nothing to do with the improved behavioral performance. For example, the memory load differs between the tasks in the main experiment and the spatial uncertainty experiment. In the spatial uncertainty experiment, subjects must remember two speeds instead of one during the 250 msec inter-stimulus interval. This difference in memory load might be causing the improved behavioral performance. Or the improved performance may result from subjects simply ignoring information from the uncued side, and thus may not be causally related to V1 modulation. It is difficult, however, to imagine that such a significant modulation of activity in visual cortex would fail to have consequences on perceptual thresholds. 7610

Most human blood ␥␦ T cells react without major histocompatibility complex restriction to small phosphorylated nonpeptide antigens (phosphoantigens) that are abundantly produced by mycobacteria and several other microbial pathogens. Although isopentenyl pyrophosphate has been identified as a mycobacterial antigen for ␥␦ T cells, the structure of several other stimulating compounds with bioactivities around 1000-fold higher than isopentenyl pyrophosphate remains to be elucidated. This paper describes the structural identification of 3-formyl-1-butyl-pyrophosphate as the core of several non-prenyl mycobacterial phosphoantigens bioactive at the nM range. Recognition of this molecule by ␥␦ T cells is very selective and relies on its aldehyde and pyrophosphate groups. This novel pyrophosphorylated aldehyde most probably corresponds to a metabolic intermediate of the non-mevalonate pathway of prenyl phosphate biosynthesis in eubacteria and algae. The reactivity to 3-formyl-1-butyl-pyrophosphate supports the view that human ␥␦ T cells are physiologically devoted to antimicrobial surveillance.Although the vast majority of T lymphocytes recognize via their ␣␤ TCR 1 antigenic peptides associated to major histocompatibility complex molecules, the so-called unconventional T cells that often express ␥␦ TCR recognize their ligands in a different way. The prominent ␥␦ T cell subset in human blood expresses the V␥9/V␦2 TCR and responds to nonpeptide antigens produced by various microbial pathogens, such as mycobacteria. The mycobacterial stimuli for these T cells have been characterized independently by two groups as nonpeptide phosphoesters, collectively referred to as phosphoantigens. On the one hand, isoprenoid-PP such as isopentenyl-PP, dimethylallyl-PP, farnesyl-PP, and geranyl-PP have been characterized as V␥9/V␦2 T cell-stimulating ligands in bioactive fractions from mycobacteria (1-4). On the other hand, we have purified from several mycobacterial species a set of four phosphoantigens composed of two pyrophosphates of an unidentified monoester (X, in the so-called TUBag1 and TUBag2) and of the corresponding X-phosphodiesters of ␥-UTP (5) and ␥-TTP (6) (respectively, TUBag3 and TUBag4). These TUBag compounds have been shown to be active at the nM range (i.e. with bioactivities about 1000-fold higher than that of IPP), thus suggesting that these molecules could account for most of the ␥␦ T cell-stimulating activity recovered from mycobacteria. Poor yields and intrinsic lability of purified TUBag1-4 have considerably slowed the identification of X. However, several biochemical lines of evidence indicated that this mycobacterial X moiety was distinct from prenyl phosphates (5, 7). Accordingly, a molecular analysis of phosphoantigen recognition has evidenced a pattern of TCR ␥␦ cell reactivity that distinguishes alkyl-PP from mycobacterial phosphoantigens (8). To understand the fine specificity of ␥␦ T cell reactivity to mycobacteria, we have identified the hitherto referred-to X moiety as 3-formyl-1-butyl-PP. The activation...

Whereas αβ T cell receptors (TCR) recognize processed antigenic peptides or glycolipids bound respectively to major histocompatibility complex or CD1 molecules, γδ TCR react differently to a broad set of native antigens. A major human γδ T cell subset is activated through a mechanism involving Vγ 9Vδ 2 TCR and structurally unrelated phosphorylated nonpeptide antigens (referred to as phosphoantigens). Here, the structure‐function relationship of the strongest natural and synthetic phosphoantigens stimulating γδ cells was analyzed to elucidate the molecular basis of this unconventional recognition. Besides conformational determinants, we found that chemical reactivity of antigens is critical to their bioactivity. For Vγ 9Vδ 2 T cell activation, both organic and phosphorylated moieties of strong ligands undergo rapid and degradative structural changes. Conversely, analogs that are resistant to degradation specifically antagonize phosphoantigen‐mediated γδ T cell activation. These data suggest a novel mode of antigen perception involving both topological recognition and ligand consumption, which confers highly specific γδ T cell activation by structurally diverse ligands.

Antibody-drug conjugates (ADCs) have demonstrated clinical benefits that have led to the recent FDA approval of KADCYLA and ADCETRIS. Most ADCs that are currently in clinical use or development, including ADCETRIS, are produced by chemical conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, we describe the in vitro and in vivo characterization of four novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as ADCETRIS, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivatives of monomethyl auristatin E (all comprising a cleavable linker) to the glutamine at positions 295 and 297 of cAC10, thereby yielding homogeneous ADCs with a DAR of 4. In vitro cell toxicity experiments using two different CD30-positive cell lines (Karpas 299 and Raji-CD30(+)) revealed comparable EC50 values for ADCETRIS (1.8 ± 0.4 and 3.6 ± 0.6 ng/mL, respectively) and the four cAC10-based ADCs (2.0 ± 0.4 to 4.9 ± 1.0 ng/mL). Quantitative time-dependent in vivo biodistribution studies (3-96 h p.i.) in normal and xenografted (Karpas 299 cells) SCID mice were performed with a selected (125)I-radioiodinated cAC10 ADC and compared with that of (125)I-ADCETRIS. The chemo-enzymatically conjugated, radioiodinated ADC showed higher tumor uptake (17.84 ± 2.2% ID/g 24 h p.i.) than (125)I-ADCETRIS (10.5 ± 1.8% ID/g 24 h p.i.). Moreover, (125)I-ADCETRIS exhibited higher nontargeted liver and spleen uptake. In line with these results, the maximum tolerated dose of the BTG-coupled ADC (>60 mg/kg) was significantly higher than that of ADCETRIS (18 mg/kg) in rats. These results suggest that homogeneous ADCs display improved pharmacokinetics and better therapeutic indexes compared to those of chemically modified ADCs with variable DARs.

Gammadelta-T-lymphocytes contribute to innate immunity and are selectively activated by nonpeptide phosphorylated molecules (so-called phosphoantigens) produced by organisms responsible for causing a broad range of infectious diseases. gammadelta-T-cells are also activated by synthetic phosphoantigens and are cytotoxic to tumor cells. Here we report the synthesis, NMR characterization, and comparative biological evaluation of new pyrophosphate, phosphonate, and pyrophosphonate monoesters whose structures correspond to isosteric analogues and stereoisomers of the highly potent isoprenoid metabolite ( E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate called HDMAPP (hydroxy-dimethyl-allyl pyrophosphate). Both pyrophosphate and pyrophosphonate series elicit promising gammadelta-T-cell stimulatory responses in vitro, the pyrophosphonate ester (C-HDMAPP) being by far more stable than its parent pyrophosphate ester (HDMAPP) with improved ADMET properties and a similar pharmacodynamic profile based on in vivo studies in nonhuman primate. In both series, we found that E-stereoisomers are the most active derivatives and that Z-stereoisomers show very marginal bioactivity levels. These results indicate that the use of bioisosteric analogues of HDMAPP may represent promising new leads for immunotherapy.

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