High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models.

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.

Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness.

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

An Open Library of Human Kinase Domain Constructs for Automated Bacterial Expression.

Kinases play a critical role in cellular signaling and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Therapeutics targeting kinases currently account for roughly 50% of cancer drug discovery efforts. The ability to explore human kinase biochemistry and biophysics in the laboratory is essential to designing selective inhibitors and studying drug resistance. Bacterial expression systems are superior to insect or mammalian cells in terms of simplicity and cost effectiveness but have historically struggled with human kinase expression. Following the discovery that phosphatase coexpression produced high yields of Src and Abl kinase domains in bacteria, we have generated a library of 52 His-tagged human kinase domain constructs that express above 2 µg/mL of culture in an automated bacterial expression system utilizing phosphatase coexpression (YopH for Tyr kinases and lambda for Ser/Thr kinases). Here, we report a structural bioinformatics approach to identifying kinase domain constructs previously expressed in bacteria and likely to express well in our protocol, experiments demonstrating our simple construct selection strategy selects constructs with good expression yields in a test of 84 potential kinase domain boundaries for Abl, and yields from a high-throughput expression screen of 96 human kinase constructs. Using a fluorescence-based thermostability assay and a fluorescent ATP-competitive inhibitor, we show that the highest-expressing kinases are folded and have well-formed ATP binding sites. We also demonstrate that these constructs can enable characterization of clinical mutations by expressing a panel of 48 Src and 46 Abl mutations. The wild-type kinase construct library is available publicly via Addgene.

MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes.

Recombinant expression of large, multiprotein complexes is essential and often rate limiting for determining structural, biophysical, and biochemical properties of DNA repair, replication, transcription, and other key cellular processes. Baculovirus-infected insect cell expression systems are especially well suited for producing large, human proteins recombinantly, and multigene baculovirus systems have facilitated studies of multiprotein complexes. In this chapter, we describe a multigene baculovirus system called MacroBac that uses a Biobricks-type assembly method based on restriction and ligation (Series 11) or ligation-independent cloning (Series 438). MacroBac cloning and assembly is efficient and equally well suited for either single subcloning reactions or high-throughput cloning using 96-well plates and liquid handling robotics. MacroBac vectors are polypromoter with each gene flanked by a strong polyhedrin promoter and an SV40 poly(A) termination signal that minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have successfully assembled as many as 10 genes into a single MacroBac vector. Importantly, we have observed significant increases in expression levels and quality of large, multiprotein complexes using a single, multigene, polypromoter virus rather than coinfection with multiple, single-gene viruses. Given the importance of characterizing functional complexes, we believe that MacroBac provides a critical enabling technology that may change the way that structural, biophysical, and biochemical research is done.

Macro-to-micro structural proteomics: native source proteins for high-throughput crystallization.

Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.

The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins.

The Escherichia coli MukB, MukE, and MukF proteins form a bacterial condensin (MukBEF) that contributes to chromosome management by compacting DNA. MukB is an ATPase and DNA-binding protein of the SMC superfamily; however, the structure and function of non-SMC components, such as MukF, have been less forthcoming. Here, we report the crystal structure of the N-terminal 287 amino acids of MukF at 2.9 A resolution. This region folds into a winged-helix domain and an extended coiled-coil domain that self-associate to form a stable, doubly domain-swapped dimer. Protein dissection and affinity purification data demonstrate that the region of MukF C-terminal to this fragment binds to MukE and MukB. Our findings, together with sequence analyses, indicate that MukF is a kleisin subunit for E. coli condensin and suggest a means by which it may organize the MukBEF assembly.