A sequence motif within chromatin entry sites directs MSL establishment on the Drosophila X chromosome.

The Drosophila MSL complex associates with active genes specifically on the male X chromosome to acetylate histone H4 at lysine 16 and increase expression approximately 2-fold. To date, no DNA sequence has been discovered to explain the specificity of MSL binding. We hypothesized that sequence-specific targeting occurs at "chromatin entry sites," but the majority of sites are sequence independent. Here we characterize 150 potential entry sites by ChIP-chip and ChIP-seq and discover a GA-rich MSL recognition element (MRE). The motif is only slightly enriched on the X chromosome ( approximately 2-fold), but this is doubled when considering its preferential location within or 3' to active genes (>4-fold enrichment). When inserted on an autosome, a newly identified site can direct local MSL spreading to flanking active genes. These results provide strong evidence for both sequence-dependent and -independent steps in MSL targeting of dosage compensation to the male X chromosome.

Antigenic Cartography Indicates That the Omicron BA.1 and BA.4/BA.5 Variants Remain Antigenically Distant to Ancestral SARS-CoV-2 after Sputnik V Vaccination Followed by Homologous (Sputnik V) or Heterologous (Comirnaty) Revaccination.

The rapid emergence of evasive SARS-CoV-2 variants is an ongoing challenge for COVID-19 vaccinology. Traditional virus neutralization tests provide detailed datasets of neutralization titers against the viral variants. Such datasets are difficult to interpret and do not immediately inform of the sufficiency of the breadth of the antibody response. Some of these issues could be tackled using the antigenic cartography approach. In this study, we created antigenic maps using neutralization titers of sera from donors who received the Sputnik V booster vaccine after primary Sputnik V vaccination and compared them with the antigenic maps based on serum neutralization titers of Comirnaty-boosted donors. A traditional analysis of neutralization titers against the WT (wild-type), Alpha, Beta, Delta, Omicron BA.1, and BA.4/BA.5 variants showed a significant booster humoral response after both homologous (Sputnik V) and heterologous (Comirnaty) revaccinations against all of the studied viral variants. However, despite this, a more in-depth analysis using antigenic cartography revealed that Omicron variants remain antigenically distant from the WT, which is indicative of the formation of insufficient levels of cross-neutralizing antibodies. The implications of these findings may be significant when developing a new vaccine regimen.

Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs.

Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. Here we identify chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. Our results reveal an extensive list of known and novel HP1a-interacting proteins, of which we selected three for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein-protein interactions, we found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. We propose that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms.

VAV1-overexpressing YT cells display improved cytotoxicity against malignant cells.

Immunotherapy based on adoptive transfer of genetically engineered T- and NK-cells is an area of active ongoing research and has proven highly efficacious for patients with certain B-cell malignancies. Use of NK cells and NK cell lines as carriers of chimeric antigen receptors (CARs) appears particularly promising, as this opens an opportunity for moving the therapy from autologous to the allogeneic (universal) format. This "off-the-shelf" approach is thought to significantly reduce the price of the treatment and make it available to many more patients in need. Yet, the efficacy of CAR-NK cells in vivo presently remains low, and boosting the activity of CAR NK cells via stronger tumor homing, resistance to tumor microenvironment, as well as greater cytotoxicity may translate into improved patient outcomes. Here, we established a derivative of a human NK cell line YT overexpressing a positive regulator of cytotoxicity, VAV1. Activity of YT-VAV1 cells obtained was assayed in vitro against several cancer cell lines and primary patient-derived cancer cells. YT-VAV1 cells outperform parental YT cells in terms of cytotoxicity.

Conservation and de novo acquisition of dosage compensation on newly evolved sex chromosomes in Drosophila.

Dosage compensation has arisen in response to the evolution of distinct male (XY) and female (XX) karyotypes. In Drosophila melanogaster, the MSL complex increases male X transcription approximately twofold. X-specific targeting is thought to occur through sequence-dependent binding to chromatin entry sites (CESs), followed by spreading in cis to active genes. We tested this model by asking how newly evolving sex chromosome arms in Drosophila miranda acquired dosage compensation. We found evidence for the creation of new CESs, with the analogous sequence and spacing as in D. melanogaster, providing strong support for the spreading model in the establishment of dosage compensation.

Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification.

Understanding the composition of epigenetic regulators remains an important challenge in chromatin biology. Traditional biochemical analysis of chromatin-associated complexes requires their release from DNA under conditions that can also disrupt key interactions. Here we develop a complementary approach (BioTAP-XL), in which cross-linking (XL) enhances the preservation of protein interactions and also allows the analysis of DNA targets under the same tandem affinity purification (BioTAP) regimen. We demonstrate the power of BioTAP-XL through analysis of human EZH2, a core subunit of polycomb repressive complex 2 (PRC2). We identify and validate two strong interactors, C10orf12 and C17orf96, which display enrichment with EZH2-BioTAP at levels similar to canonical PRC2 components (SUZ12, EED, MTF2, JARID2, PHF1, and AEBP2). ChIP-seq analysis of BioTAP-tagged C10orf12 or C17orf96 revealed the similarity of each binding pattern with the location of EZH2 and the H3K27me3-silencing mark, validating their physical interaction with PRC2 components. Interestingly, analysis by mass spectrometry of C10orf12 and C17orf96 interactions revealed that these proteins may be mutually exclusive PRC2 subunits that fail to interact with each other or with JARID2 and AEBP2. C10orf12, in addition, shows a strong and unexpected association with components of the EHMT1/2 complex, thus potentially connecting PRC2 to another histone methyltransferase. Similarly, results from CBX4-BioTAP protein pulldowns are consistent with reports of a diversity of PRC1 complexes. Our results highlight the importance of reciprocal analyses of multiple subunits and suggest that iterative use of BioTAP-XL has strong potential to reveal networks of chromatin-based interactions in higher organisms.

Long-range spreading of dosage compensation in Drosophila captures transcribed autosomal genes inserted on X.

Dosage compensation in Drosophila melanogaster males is achieved via targeting of male-specific lethal (MSL) complex to X-linked genes. This is proposed to involve sequence-specific recognition of the X at approximately 150-300 chromatin entry sites, and subsequent spreading to active genes. Here we ask whether the spreading step requires transcription and is sequence-independent. We find that MSL complex binds, acetylates, and up-regulates autosomal genes inserted on X, but only if transcriptionally active. We conclude that a long-sought specific DNA sequence within X-linked genes is not obligatory for MSL binding. Instead, linkage and transcription play the pivotal roles in MSL targeting irrespective of gene origin and DNA sequence.

The epigenome of evolving Drosophila neo-sex chromosomes: dosage compensation and heterochromatin formation.

Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.

Nature and function of insulator protein binding sites in the Drosophila genome.

Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes.

Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context.

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin.

Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the "silencing" marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin-heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both "activation" marks (e.g., H3K4me3 and H3K36me3) and "silencing" marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin.

Serial Llama Immunization with Various SARS-CoV-2 RBD Variants Induces Broad Spectrum Virus-Neutralizing Nanobodies.

The emergence of SARS-CoV-2 mutant variants has posed a significant challenge to both the prevention and treatment of COVID-19 with anti-coronaviral neutralizing antibodies. The latest viral variants demonstrate pronounced resistance to the vast majority of human monoclonal antibodies raised against the ancestral Wuhan variant. Less is known about the susceptibility of the evolved virus to camelid nanobodies developed at the start of the pandemic. In this study, we compared nanobody repertoires raised in the same llama after immunization with Wuhan's RBD variant and after subsequent serial immunization with a variety of RBD variants, including that of SARS-CoV-1. We show that initial immunization induced highly potent nanobodies, which efficiently protected Syrian hamsters from infection with the ancestral Wuhan virus. These nanobodies, however, mostly lacked the activity against SARS-CoV-2 omicron-pseudotyped viruses. In contrast, serial immunization with different RBD variants resulted in the generation of nanobodies demonstrating a higher degree of somatic mutagenesis and a broad range of neutralization. Four nanobodies recognizing distinct epitopes were shown to potently neutralize a spectrum of omicron variants, including those of the XBB sublineage. Our data show that nanobodies broadly neutralizing SARS-CoV-2 variants may be readily induced by a serial variant RBD immunization.

Hybrid Immunity from Gam-COVID-Vac Vaccination and Natural SARS-CoV-2 Infection Confers Broader Neutralizing Activity against Omicron Lineage VOCs Than Revaccination or Reinfection.

SARS-CoV-2 has a relatively high mutation rate, with the frequent emergence of new variants of concern (VOCs). Each subsequent variant is more difficult to neutralize by the sera of vaccinated individuals and convalescents. Some decrease in neutralizing activity against new SARS-CoV-2 variants has also been observed in patients vaccinated with Gam-COVID-Vac. In the present study, we analyzed the interplay between the history of a patient's repeated exposure to SARS-CoV-2 antigens and the breadth of neutralization activity. Our study includes four cohorts of patients: Gam-COVID-Vac booster vaccinated individuals (revaccinated, RV), twice-infected unvaccinated individuals (reinfected, RI), breakthrough infected (BI), and vaccinated convalescents (VC). We assessed S-protein-specific antibody levels and the ability of sera to neutralize lentiviral particles pseudotyped with Spike protein from the original Wuhan variant, as well as the Omicron variants BA.1 and BA.4/5. Individuals with hybrid immunity (BI and VC cohorts) exhibited significantly higher levels of virus-binding IgG and enhanced breadth of virus-neutralizing activity compared to individuals from either the revaccination or reinfection (RV and RI) cohorts. These findings suggest that a combination of infection and vaccination, regardless of the sequence, results in significantly higher levels of S-protein-specific IgG antibodies and the enhanced neutralization of SARS-CoV-2 variants, thereby underscoring the importance of hybrid immunity in the context of emerging viral variants.

VH3-53/66-Class RBD-Specific Human Monoclonal Antibody iB20 Displays Cross-Neutralizing Activity against Emerging SARS-CoV-2 Lineages.

Immune evasion of SARS-CoV-2 undermines current strategies tocounteract the pandemic, with the efficacy of therapeutic virus-neutralizing monoclonal antibodies (nAbs) being affected the most. In this work, we asked whether two previously identified human cross-neutralizing nAbs, iB14 (class VH1-58) and iB20 (class VH3-53/66), are capable of neutralizing the recently emerged Omicron (BA.1) variant. Both nAbs were found to bind the Omicron RBD with a nanomolar affinity, yet they displayed contrasting functional features. When tested against Omicron, the neutralizing activity of iB14 was reduced 50-fold, whereas iB20 displayed a surprising increase in activity. Thus, iB20 is a unique representative of the VH3-53/66-class of nAbs in terms of breadth of neutralization, which establishes it as a candidate for COVID-19 therapy and prophylactics.

Functional Profiling of In Vitro Reactivated Memory B Cells Following Natural SARS-CoV-2 Infection and Gam-COVID-Vac Vaccination.

Both SARS-CoV-2 infection and vaccination have previously been demonstrated to elicit robust, yet somewhat limited immunity against the evolving variants of SARS-CoV-2. Nevertheless, reports performing side-by-side comparison of immune responses following infection vs. vaccination have been relatively scarce. The aim of this study was to compare B-cell response to adenovirus-vectored vaccination in SARS-CoV-2-naive individuals with that observed in the COVID-19 convalescent patients six months after the first encounter with the viral antigens. We set out to use a single analytical platform and performed comprehensive analysis of serum levels of receptor binding domain (RBD)-specific and virus-neutralizing antibodies, frequencies of RBD-binding circulating memory B cells (MBCs), MBC-derived antibody-secreting cells, as well as RBD-specific and virus-neutralizing activity of MBC-derived antibodies after Gam-COVID-Vac (Sputnik V) vaccination and/or natural SARS-CoV-2 infection. Overall, natural immunity was superior to Gam-COVID-Vac vaccination. The levels of neutralizing MBC-derived antibodies in the convalescent patients turned out to be significantly higher than those found following vaccination. Our results suggest that after six months, SARS-CoV-2-specific MBC immunity is more robust in COVID-19 convalescent patients than in Gam-COVID-Vac recipients. Collectively, our data unambiguously indicate that natural immunity outperforms Gam-COVID-Vac-induced immunity six months following recovery/vaccination, which should inform healthcare and vaccination decisions.

Memory B Cells Induced by Sputnik V Vaccination Produce SARS-CoV-2 Neutralizing Antibodies Upon Ex Vivo Restimulation.

The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, the effects of Sputnik V on B cell immunity need to be explored in detail. We performed comprehensive profiling of humoral and B cell responses in a cohort of vaccinated subjects (n = 22), and demonstrate that Sputnik vaccination results in robust B cell immunity. We show that B memory cell (MBC) and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not. 85 days after the first dose of the vaccine, ex vivo stimulated MBCs from the vast majority of Sputnik V vaccinees produced antibodies that robustly neutralized the Wuhan Spike-pseudotyped lentivirus. MBC-derived antibodies from all previously infected and some of the naïve vaccine recipients could also cross-neutralize Beta (B.1.351) variant of SARS-CoV-2. Virus-neutralizing activity of MBC-derived antibodies correlated well with that of the serum antibodies, suggesting the interplay between the MBC and long-lived plasma cell responses. Thus, our in-depth analysis of MBC responses in Sputnik V vaccinees complements traditional serological approaches and may provide important outlook into future B cell responses upon re-encounter with the emerging variants of SARS-CoV-2.

Anti-Ad26 humoral immunity does not compromise SARS-COV-2 neutralizing antibody responses following Gam-COVID-Vac booster vaccination.

Replication-incompetent adenoviral vectors have been extensively used as a platform for vaccine design, with at least four anti-COVID-19 vaccines authorized to date. These vaccines elicit neutralizing antibody responses directed against SARS-CoV-2 Spike protein and confer significant level of protection against SARS-CoV-2 infection. Immunization with adenovirus-vectored vaccines is known to be accompanied by the production of anti-vector antibodies, which may translate into reduced efficacy of booster or repeated rounds of revaccination. Here, we used blood samples from patients who received an adenovirus-based Gam-COVID-Vac vaccine to address the question of whether anti-vector antibodies may influence the magnitude of SARS-CoV-2-specific humoral response after booster vaccination. We observed that rAd26-based prime vaccination with Gam-COVID-Vac induced the development of Ad26-neutralizing antibodies, which persisted in circulation for at least 9 months. Our analysis further indicates that high pre-boost Ad26 neutralizing antibody titers do not appear to affect the humoral immunogenicity of the Gam-COVID-Vac boost. The titers of anti-SARS-CoV-2 RBD IgGs and antibodies, which neutralized both the wild type and the circulating variants of concern of SARS-CoV-2 such as Delta and Omicron, were independent of the pre-boost levels of Ad26-neutralizing antibodies. Thus, our results support the development of repeated immunization schedule with adenovirus-based COVID-19 vaccines.

Isolation of a panel of ultra-potent human antibodies neutralizing SARS-CoV-2 and viral variants of concern.

In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC100 below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.

Challenges and Prospects of Chimeric Antigen Receptor T-cell Therapy for Metastatic Prostate Cancer.

CONTEXT: Progress achieved in the treatment of prostate cancer (PCa) with surgical, radiation, and hormonal therapies has drastically reduced mortality from this disease. Yet, patients with advanced PCa have few, if any, curative options. Recent success in treating patients with hematological malignancies of B-cell origin using T cells engineered to express chimeric antigen receptors (CARs) has inspired multiple groups worldwide to adapt this approach to the problem of late-stage PCa. OBJECTIVE: To summarize the available clinical results for CAR T-cell therapy of PCa and discuss future technological advancements in the CAR T-cell field that may help patients with metastatic PCa. EVIDENCE ACQUISITION: A literature review was conducted of clinical trial data, abstracts presented at recent oncology conferences, as well as reports highlighting critical bottlenecks of CAR T-cell therapy that became apparent from preclinical and clinical studies. EVIDENCE SYNTHESIS: Current understanding of why CAR T-cell therapy may fail, particularly in the context of solid cancers, is as follows. First, a CAR design that provides potent activity and persistence of engineered T cells in the hostile tumor microenvironment is a must. The choice of the targetable epitope(s) is critical to counteract tumor antigen escape. Preclinical and clinical evidence indicates that the efficacy of CAR T-cell therapy can be enhanced significantly in combination with other therapeutic approaches. We propose that several improvements to CAR design and patient conditioning, such as unbiased identification of novel PCa-specific CAR targets, use of next-generation (multispecific, resistant to the tumor microenvironment, and with prolonged persistence) CAR T-cell products, and combination therapies may translate into improved patient outcomes and more durable responses. CONCLUSIONS: Although significant preclinical experience of testing CAR T cells in solid cancer models has identified important technological and biological bottlenecks, information from clinical trials, particularly those focusing on the PCa, will be instrumental to the rational design of advanced CAR T therapies that will be both safe and effective in patients with advanced PCa. PATIENT SUMMARY: So far, chimeric antigen receptor (CAR) T-cell therapy has not shown significant activity in patients with metastatic prostate cancer (PCa). CAR T-cell products used for such trials represent one of the pioneering efforts to adapt this technology to the problem of metastatic PCa. In retrospect, both CAR design and cell composition appear to have been suboptimal to expect strong patient responses. Given the impressive results of CAR-based approaches observed in preclinical models of solid cancers, emerging CAR T-cell products are expected to be more successful in the clinic. Here, we discuss the challenges that need to be overcome to boost the efficacy of PCa-targeted CAR T-cell therapy and call for dialogue between clinicians and cell biologists to address these challenges.