Evidence for an Enzyme-Catalyzed Rauhut-Currier Reaction during the Biosynthesis of Spinosyn A.

The catalog of enzymes known to catalyze the nucleophile-assisted formation of C-C bonds is extremely small, and there is presently no definitive example of a biological Rauhut-Currier reaction. Biosynthesis of the polyketide insecticide spinosyn A in Saccharopolyspora spinosa involves a [4 + 2]-cycloaddition and a subsequent intramolecular C-C bond formation catalyzed by SpnF and SpnL, respectively. Isotope tracer experiments and kinetic isotope effects, however, imply that the SpnL-catalyzed reaction proceeds without initial deprotonation of the substrate. The crystal structure of SpnL exhibits high similarity to SAM-dependent methyltransferases as well as SpnF. The residue Cys60 is also shown to reside in the SpnL active site, and the Cys60Ala SpnL mutant is found to be devoid of activity. Moreover, SpnL is covalently modified at Cys60 and irreversibly inactivated when it is coincubated with a fluorinated substrate analogue designed as a suicide inactivator of nucleophile-assisted C-C bond formation. These results suggest that SpnL catalyzes a biological Rauhut-Currier reaction.

A PSMA-targeted bispecific antibody for prostate cancer driven by a small-molecule targeting ligand.

Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell-recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021).

Switchable CAR-T Cells Outperformed Traditional Antibody-Redirected Therapeutics Targeting Breast Cancers.

Various antibody-redirected immunotherapeutic approaches, including antibody-drug conjugates (ADCs), bispecific antibodies (bsAbs), and chimeric antigen receptor-T (CAR-T) cells, have been devised to produce specific activity against various cancer types. Using genetically encoded unnatural amino acids, we generated a homogeneous Her2-targeted ADC, a T cell-redirected bsAb, and a FITC-modified antibody capable of redirecting anti-FITC CAR-T (switchable CAR-T; sCAR-T) cells to target different Her2-expressing breast cancers. sCAR-T cells showed activity against Her2-expressing tumor cells comparable to that of conventional anti-Her2 CAR-T cells and superior to that of ADC- and bsAb-based approaches. To prevent antigen escape, we designed bispecific sCAR-T cells targeting both the Her2 receptor and IGF1R, which showed an overall improved activity against cancer cells with low Her2 expression. This study increases our understanding of various explored cancer therapeutics and underscores the efficient application of sCAR-T cells as a promising therapeutic option for breast cancer patients with low or heterogeneous antigen expression.

Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer.

Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5). We demonstrate that both switch formats can be readily optimized to redirect CAR-T cells (specific for the corresponding FITC or PNE) to Her2-expressing tumor cells, and afford dose-titratable activation of CAR-T cells ex vivo and complete clearance of the tumor in rodent xenograft models. This strategy may facilitate the application of immunotherapy to solid tumors by affording comparable efficacy with improved safety owing to switch-based control of the CAR-T response.

Versatile strategy for controlling the specificity and activity of engineered T cells.

The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR-T--cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as "switch" molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a "universal" anti-FITC-directed CAR-T cell. Optimization of this CAR-switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR-T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy.

Mechanistic Investigation of the Radical S-Adenosyl-L-methionine Enzyme DesII Using Fluorinated Analogues.

DesII is a radical S-adenosyl-l-methionine (SAM) enzyme that can act as a deaminase or a dehydrogenase depending on the nature of its TDP-sugar substrate. Previous work has implicated a substrate-derived, C3-centered α-hydroxyalkyl radical as a key intermediate during catalysis. Although deprotonation of the α-hydroxyalkyl radical has been shown to be important for dehydrogenation, much less is known regarding the course of the deamination reaction. To investigate the role played by the C3 hydroxyl during deamination, 3-deutero-3-fluoro analogues of both substrates were prepared and characterized with DesII. In neither case was deamination or oxidation observed; however, in both cases deuterium was efficiently exchanged between the substrate analogues and SAM. These results imply that the C3 hydroxyl plays a key role in both reactions­thereby arguing against a 1,2-migration mechanism of deamination­and that homolysis of SAM concomitant with H atom abstraction from the substrate is readily reversible when forward partitioning is inhibited.

An anti-B cell maturation antigen bispecific antibody for multiple myeloma.

The development of immunotherapies for multiple myeloma is critical to provide new treatment strategies to combat drug resistance. We report a bispecific antibody against B cell maturation antigen (BiFab-BCMA), which potently and specifically redirects T cells to lyse malignant multiple myeloma cells. BiFab-BCMA lysed target BCMA-positive cell lines up to 20-fold more potently than a CS1-targeting bispecific antibody (BiFab-CS1) developed in an analogous fashion. Further, BiFab-BCMA robustly activated T cells in vitro and mediated rapid tumor regression in an orthotopic xenograft model of multiple myeloma. The in vitro and in vivo activities of BiFab-BCMA are comparable to those of anti-BCMA chimeric antigen receptor T cell therapy (CAR-T-BCMA), for which two clinical trials have recently been initiated. A BCMA-targeted bispecific antibody presents a promising treatment option for multiple myeloma.

Mechanistic studies of the radical S-adenosylmethionine enzyme DesII with TDP-D-fucose.

DesII is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the C4-deamination of TDP-4-amino-4,6-dideoxyglucose through a C3 radical intermediate. However, if the C4 amino group is replaced with a hydroxy group (to give TDP-quinovose), the hydroxy group at C3 is oxidized to a ketone with no C4-dehydration. It is hypothesized that hyperconjugation between the C4 C-N/O bond and the partially filled p orbital at C3 of the radical intermediate modulates the degree to which elimination competes with dehydrogenation. To investigate this hypothesis, the reaction of DesII with the C4-epimer of TDP-quinovose (TDP-fucose) was examined. The reaction primarily results in the formation of TDP-6-deoxygulose and likely regeneration of TDP-fucose. The remainder of the substrate radical partitions roughly equally between C3-dehydrogenation and C4-dehydration. Thus, changing the stereochemistry at C4 permits a more balanced competition between elimination and dehydrogenation.

Bispecific small molecule-antibody conjugate targeting prostate cancer.

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII.

The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, 0.22 ± 0.03, when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of 1.8 ± 0.2, yielded a KIE of 8 ± 2 on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.

Mechanistic studies of the radical S-adenosyl-L-methionine enzyme DesII: EPR characterization of a radical intermediate generated during its catalyzed dehydrogenation of TDP-D-quinovose.

DesII, a radical S-adenosyl-l-methionine (SAM) enzyme from Streptomyces venezuelae, catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D-glucose to TDP-3-keto-4,6-dideoxy-D-glucose in the desosamine biosynthetic pathway. DesII can also catalyze the dehydrogenation of TDP-D-quinovose to the corresponding 3-keto sugar. Similar to other radical SAM enzymes, DesII catalysis has been proposed to proceed via a radical mechanism. This hypothesis is now confirmed by EPR spectroscopy with the detection of a TDP-D-quinovose radical intermediate having a g-value of 2.0025 with hyperfine coupling to two spin 1/2 nuclei, each with a splitting constant of 33.6 G. A significant decrease in the EPR line width is observed when the radical is generated in reactions conducted in D(2)O versus H(2)O. These results are consistent with a C3 α-hydroxyalkyl radical in which the p-orbital harboring the unpaired electron spin at C3 is periplanar with the C-H bonds at both C2 and C4.

Stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical SAM enzyme DesII.

DesII from Streptomyces venezuelae is a radical SAM (S-adenosyl-l-methionine) enzyme that catalyzes the deamination of TDP-4-amino-4,6-dideoxy-d-glucose to form TDP-3-keto-4,6-dideoxy-d-glucose in the biosynthesis of TDP-d-desosamine. DesII also catalyzes the dehydrogenation of the nonphysiological substrate TDP-D-quinovose to TDP-3-keto-6-deoxy-d-glucose. These properties prompted an investigation of how DesII handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions. This work was facilitated by the development of an enzymatic synthesis of TDP-4-amino-4,6-dideoxy-d-glucose that couples a transamination equilibrium to the thermodynamically favorable oxidation of formate. In this study, DesII is found to consume SAM versus TDP-sugar with stoichiometries of 0.96 +/- 0.05 and 1.01 +/- 0.05 in the deamination and dehydrogenation reactions, respectively, using Na(2)S(2)O(4) as the reductant. Importantly, no significant change in stoichiometry is observed when the flavodoxin/flavodoxin NADP(+) oxidoreductase/NADPH reducing system is used in place of Na(2)S(2)O(4). Moreover, there is no evidence of an uncoupled or abortive process in the deamination reaction, as indicated by the observation that dehydrogenation can take place in the absence of an external source of reductant whereas deamination cannot. Mechanistic and biochemical implications of these results are discussed.

Characterization and mechanistic studies of DesII: a radical S-adenosyl-L-methionine enzyme involved in the biosynthesis of TDP-D-desosamine.

D-desosamine (1) is a 3-(N,N-dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin (2), neomethymycin (3), pikromycin (4), and narbomycin (5) produced by Streptomyces venezuelae . It plays an essential role in conferring biological activities to its parent aglycones. Previous genetic and biochemical studies of the biosynthesis of desosamine in S. venezuelae showed that the conversion of TDP-4-amino-4,6-dideoxy-D-glucose (8) to TDP-3-keto-4,6-dideoxy-D-glucose (9) is catalyzed by DesII, which is a member of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily. Here, we report the purification and reconstitution of His(6)-tagged DesII, characterization of its [4Fe-4S] cluster using UV-vis and EPR spectroscopies, and the capability of flavodoxin, flavodoxin reductase, and NADPH to reduce the [4Fe-4S](2+) cluster. Also included are a steady-state kinetic analysis of DesII-catalyzed reaction and an investigation of the substrate flexibility of DesII. Studies of deuterium incorporation into SAM using TDP-[3-(2)H]-4-amino-4,6-dideoxy-D-glucose as the substrate provides strong evidence for direct hydrogen atom transfer to a 5'-deoxyadenosyl radical in the catalytic cycle. The fact that hydrogen atom abstraction occurs at C-3 also sheds light on the mechanism of this intriguing deamination reaction.