Engineered T cells secreting anti-BCMA T cell engagers control multiple myeloma and promote immune memory in vivo.

Multiple myeloma is the second most common hematological malignancy in adults and remains an incurable disease. B cell maturation antigen (BCMA)-directed immunotherapy, including T cells bearing chimeric antigen receptors (CARs) and systemically injected bispecific T cell engagers (TCEs), has shown remarkable clinical activity, and several products have received market approval. However, despite promising results, most patients eventually become refractory and relapse, highlighting the need for alternative strategies. Engineered T cells secreting TCE antibodies (STAb) represent a promising strategy that combines the advantages of adoptive cell therapies and bispecific antibodies. Here, we undertook a comprehensive preclinical study comparing the therapeutic potential of T cells either expressing second-generation anti-BCMA CARs (CAR-T) or secreting BCMAxCD3 TCEs (STAb-T) in a T cell-limiting experimental setting mimicking the conditions found in patients with relapsed/refractory multiple myeloma. STAb-T cells recruited T cell activity at extremely low effector-to-target ratios and were resistant to inhibition mediated by soluble BCMA released from the cell surface, resulting in enhanced cytotoxic responses and prevention of immune escape of multiple myeloma cells in vitro. These advantages led to robust expansion and persistence of STAb-T cells in vivo, generating long-lived memory BCMA-specific responses that could control multiple myeloma progression in xenograft models, outperforming traditional CAR-T cells. These promising preclinical results encourage clinical testing of the BCMA-STAb-T cell approach in relapsed/refractory multiple myeloma.

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2.

Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT , are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT , the bispecific trimerbody TNT DNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNT DNGR-1, but not TNT , protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.

A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response.

Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers.

Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity.

Costimulation of tumor-infiltrating T lymphocytes by anti-4-1BB monoclonal antibodies (mAbs) has shown anti-tumor activity in human trials, but can be associated with significant off-tumor toxicities involving FcγR interactions. Here, we introduce albumin-fused mouse and human bispecific antibodies with clinically favorable pharmacokinetics designed to confine 4-1BB costimulation to the tumor microenvironment. These Fc-free 4-1BB agonists consist of an EGFR-specific VHH antibody, a 4-1BB-specific scFv, and a human albumin sequence engineered for high FcRn binding connected in tandem (LiTCo-Albu). We demonstrate in vitro cognate target engagement, EGFR-specific costimulatory activity, and FcRn-driven cellular recycling similar to non-fused FcRn high-binding albumin. The mouse LiTCo-Albu exhibited a prolonged circulatory half-life and in vivo tumor inhibition, with no indication of 4-1BB mAb-associated toxicity. Furthermore, we show a greater therapeutic effect when used in combination with PD-1-blocking mAbs. These findings demonstrate the feasibility of tumor-specific LiTCo-Albu antibodies for safe and effective costimulatory strategies in cancer immunotherapy.

Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers.

Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.

Alternative mass spectrometry techniques for the validation of the fragmentation pattern of capsaicin and dihydrocapsaicin.

RATIONALE: Capsaicinoids are prevalent secondary metabolites in many natural and synthetic pharmacological compounds. To date, several soft ionization studies related to capsaicinoids have been reported; they all proposed a common fragmentation pattern based on a rearrangement of the aromatic double bonds and the fragmentation of the various positional acyl chains. However, the mechanism has never been validated by high-resolution analyses. Consequently, in this work, a validated fragmentation mechanism of the main capsaicinoids, capsaicin (1) and dihydrocapsaicin (2), is offered. METHODS: In order to propose and validate a common electron ionization (EI) fragmentation mechanism for the target analytes, the following mass spectrometric methods were employed: collision-induced dissociation (CID) by means of linked scans (LS), reinforcing the methodology by high-resolution mass spectrometry (HRMS), in addition to appropriate deuterium-labeled experiments performed using gas chromatography/mass spectrometry (GC/MS) and direct analysis in real time (DART). RESULTS: In a first stage, a common EI fragmentation pattern comprising two pathways was proposed for compounds 1 and 2; then, the suggested mechanism was validated by CID-LS together with HRMS complemented by DART-deuterium-labeling studies. The obtained results are indicative that the corresponding molecular ions were conveniently observed, m/z 305 and m/z 307; it is worth noting that the common base peak is in correspondence with a tropylium ion derivative (m/z 137), as a consequence of a McLafferty rearrangement. In addition to these highlighted fragments, other common ions, m/z 122 and m/z 94, and their corresponding trajectory, were confirmed using the same approach. Finally, the proposed mechanism was complementarily validated by deuterium-labeling studies, taking into account the two exchangeable hydrogens present in the phenolic and the amidic moieties. CONCLUSIONS: A common validated EI fragmentation pattern for both capsaicin and dihydrocapsaicin was established using appropriated mass spectrometric methods together with convenient hydrogen/deuterium labeling. This study provides a new alternative to validate mechanisms of fragmentation of important natural products.

A well-defined NHC-Ir(iii) catalyst for the silylation of aromatic C-H bonds: substrate survey and mechanistic insights.

A well-defined NHC-Ir(iii) catalyst, [Ir(H)2(IPr)(py)3][BF4] (IPr = 1,3-bis-(2,6-diisopropylphenyl)imidazol-2-ylidene), that provides access to a wide range of aryl- and heteroaryl-silanes by intermolecular dehydrogenative C-H bond silylation has been prepared and fully characterized. The directed and non-directed functionalisation of C-H bonds has been accomplished successfully using an arene as the limiting reagent and a variety of hydrosilanes in excess, including Et3SiH, Ph2MeSiH, PhMe2SiH, Ph3SiH and (EtO)3SiH. Examples that show unexpected selectivity patterns that stem from the presence of aromatic substituents in hydrosilanes are also presented. The selective bisarylation of bis(hydrosilane)s by directed or non-directed silylation of C-H bonds is also reported herein. Theoretical calculations at the DFT level shed light on the intermediate species in the catalytic cycle and the role played by the ligand system on the Ir(iii)/Ir(i) mechanism.

Efficient Rhodium-Catalyzed Multicomponent Reaction for the Synthesis of Novel Propargylamines.

[{Rh(µ-Cl)(H)2 (IPr)}2 ] (IPr = 1,3-bis-(2,6-diisopropylphenyl)imidazole-2-ylidene) was found to be an efficient catalyst for the synthesis of novel propargylamines by a one-pot three-component reaction between primary arylamines, aliphatic aldehydes, and triisopropylsilylacetylene. This methodology offers an efficient synthetic pathway for the preparation of secondary propargylamines derived from aliphatic aldehydes. The reactivity of [{Rh(µ-Cl)(H)2 (IPr)}2 ] with amines and aldehydes was studied, leading to the identification of complexes [RhCl(CO)IPr(MesNH2 )] (MesNH2 = 2,4,6-trimethylaniline) and [RhCl(CO)2 IPr]. The latter shows a very low catalytic activity while the former brought about reaction rates similar to those obtained with [{Rh(µ-Cl)(H)2 (IPr)}2 ]. Besides, complex [RhCl(CO)IPr(MesNH2 )] reacts with an excess of amine and aldehyde to give [RhCl(CO)IPr{MesNCHCH2 CH(CH3 )2 }], which was postulated as the active species. A mechanism that clarifies the scarcely studied catalytic cycle of A3 -coupling reactions is proposed based on reactivity studies and DFT calculations.

A bimetallic iridium(ii) catalyst: [{Ir(IDipp)(H)}2][BF4]2 (IDipp = 1,3-bis(2,6-diisopropylphenylimidazol-2-ylidene)).

A new Ir(ii) complex [{Ir(µ-κCNHC,η(6)Dipp-IDipp)(H)}2][BF4]2 has been prepared and fully characterised. This complex acts as a catalyst for the hydroalkynylation of imines according to an unprecedented diiridium-mediated mechanism.

Pyridine-enhanced head-to-tail dimerization of terminal alkynes by a rhodium-N-heterocyclic-carbene catalyst.

A general regioselective rhodium-catalyzed head-to-tail dimerization of terminal alkynes is presented. The presence of a pyridine ligand (py) in a Rh-N-heterocyclic-carbene (NHC) catalytic system not only dramatically switches the chemoselectivity from alkyne cyclotrimerization to dimerization but also enhances the catalytic activity. Several intermediates have been detected in the catalytic process, including the π-alkyne-coordinated Rh(I) species [RhCl(NHC)(η(2)-HC≡CCH2Ph)(py)] (3) and [RhCl(NHC){η(2)-C(tBu)≡C(E)CH=CHtBu}(py)] (4) and the Rh(III)-hydride-alkynyl species [RhClH{-C≡CSi(Me)3}(IPr)(py)2] (5). Computational DFT studies reveal an operational mechanism consisting of sequential alkyne C-H oxidative addition, alkyne insertion, and reductive elimination. A 2,1-hydrometalation of the alkyne is the more favorable pathway in accordance with a head-to-tail selectivity.

Stable preformed chiral palladium catalysts for the one-pot asymmetric reductive amination of ketones.

The application of air stable preformed [(R)-BINAP]PdBr2, [(S)-BINAP]PdBr2, [(R)-Tol-BINAP]PdBr2, and [(S,S)-CHIRAPHOS]PdBr2 complexes in the one-pot asymmetric reductive amination of various carbonyl compounds, leading to chiral amines in very good yields with high enantioselectivities (<99% ee), is reported.