Trogocytosis of CAR molecule regulates CAR-T cell dysfunction and tumor antigen escape.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical response in treating both hematologic malignancies and solid tumors. Although instances of rapid tumor remissions have been observed in animal models and clinical trials, tumor relapses occur with multiple therapeutic resistance mechanisms. Furthermore, while the mechanisms underlying the long-term therapeutic resistance are well-known, short-term adaptation remains less understood. However, more views shed light on short-term adaptation and hold that it provides an opportunity window for long-term resistance. In this study, we explore a previously unreported mechanism in which tumor cells employ trogocytosis to acquire CAR molecules from CAR-T cells, a reversal of previously documented processes. This mechanism results in the depletion of CAR molecules and subsequent CAR-T cell dysfunction, also leading to short-term antigen loss and antigen masking. Such type of intercellular communication is independent of CAR downstream signaling, CAR-T cell condition, target antigen, and tumor cell type. However, it is mainly dependent on antigen density and CAR sensitivity, and is associated with tumor cell cholesterol metabolism. Partial mitigation of this trogocytosis-induced CAR molecule transfer can be achieved by adaptively administering CAR-T cells with antigen density-individualized CAR sensitivities. Together, our study reveals a dynamic process of CAR molecule transfer and refining the framework of clinical CAR-T therapy for solid tumors.

Mechanisms of antigen escape from BCMA- or GPRC5D-targeted immunotherapies in multiple myeloma.

B cell maturation antigen (BCMA) target loss is considered to be a rare event that mediates multiple myeloma (MM) resistance to anti-BCMA chimeric antigen receptor T cell (CAR T) or bispecific T cell engager (TCE) therapies. Emerging data report that downregulation of G-protein-coupled receptor family C group 5 member D (GPRC5D) protein often occurs at relapse after anti-GPRC5D CAR T therapy. To examine the tumor-intrinsic factors that promote MM antigen escape, we performed combined bulk and single-cell whole-genome sequencing and copy number variation analysis of 30 patients treated with anti-BCMA and/or anti-GPRC5D CAR T/TCE therapy. In two cases, MM relapse post-TCE/CAR T therapy was driven by BCMA-negative clones harboring focal biallelic deletions at the TNFRSF17 locus at relapse or by selective expansion of pre-existing subclones with biallelic TNFRSF17 loss. In another five cases of relapse, newly detected, nontruncating, missense mutations or in-frame deletions in the extracellular domain of BCMA negated the efficacies of anti-BCMA TCE therapies, despite detectable surface BCMA protein expression. In the present study, we also report four cases of MM relapse with biallelic mutations of GPRC5D after anti-GPRC5D TCE therapy, including two cases with convergent evolution where multiple subclones lost GPRC5D through somatic events. Immunoselection of BCMA- or GPRC5D-negative or mutant clones is an important tumor-intrinsic driver of relapse post-targeted therapies. Mutational events on BCMA confer distinct sensitivities toward different anti-BCMA therapies, underscoring the importance of considering the tumor antigen landscape for optimal design and selection of targeted immunotherapies in MM.

T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants.

Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.

Dual antigen-targeted off-the-shelf NK cells show durable response and prevent antigen escape in lymphoma and leukemia.

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition, we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies.

Promoting antigen escape from dendritic cell endosomes potentiates anti-tumoral immunity.

The cross-presenting capacity of dendritic cells (DCs) can be limited by non-specific degradation during endosome maturation. To bypass this limitation, we present in this study a new Accum-based formulation designed to promote endosome-to-cytosol escape. Treatment of primary DCs with Accum linked to the xenoantigen ovalbumin (OVA) triggers endosomal damages and enhances protein processing. Despite multiple challenges using ascending doses of tumor cells, DC prophylactic vaccination results in complete protection due to increased levels of effector CD4 and CD8 T cells as well as high production of pro-inflammatory mediators. When combined with anti-PD-1, therapeutic vaccination using both syngeneic and allogeneic Accum-OVA-pulsed DCs triggers potent anti-tumoral responses. The net outcome culminates in increased CD11c, CD8, and NK infiltration along with a high CD8/Treg ratio. These highly favorable therapeutic effects highlight the promising potential of Accum as a distinct and potent technology platform suitable for the design of next generation cell cancer vaccines.

T cells expressing CD5/CD7 bispecific chimeric antigen receptors with fully human heavy-chain-only domains mitigate tumor antigen escape.

Bispecific chimeric antigen receptor T-cell (CAR-T) therapies have shown promising results in clinical trials for advanced B-cell malignancies. However, it is challenging to broaden the success of bispecific CAR-T therapies to treat refractory/relapse (r/r) T-cell leukemia/lymphoma because targeting multiple T-cell-expressing antigens leads to exacerbated CAR-T cell fratricide and potential safety concerns. Fully human heavy chain variable (FHVH) antibodies that specifically target CD5 or CD7 were screened and constructed to CD5/CD7 bispecific CARs. A truncated Epidermal growth factor receptor were integrated into CAR constructs to address safety concerns. To tackle the fratricidal issue of CAR-T cells targeting T-cell-pan marker(s), CRISPR/Cas9-based CD5 and CD7 genes knockout were performed before lentiviral transduction of bispecific CARs. Functional comparison between different bispecific CAR structures: tandem CARs and dual CAR were performed in vitro and in vivo to determine the optimal construct suitable for addressing T-cell malignancy antigen escape in clinical setting. Knockout of CD5 and CD7 prevents fratricide of CD5/CD7 bispecific CAR-T cells, and FHVH-derived CD5/CD7 bispecific CAR-T cells demonstrate potent antitumor activity in vitro and in vivo. The fratricide-resistant FHVH-derived CD5/CD7 bispecific CAR-T cells have potent antitumor activity against T-cell malignancies, and tandem CARs are more effective than dual CAR in preventing tumor escape in heterogeneous leukemic cells. The meaningful clinical efficacy and safety of tandem CD5/CD7 CAR-T cells deserve to be explored urgently.

Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies.

Downregulation of surface epitopes causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Here we demonstrate that mRNA encoding CD22 undergoes aberrant splicing in B-ALL. We describe the plasma membrane-bound CD22 Δex5-6 splice isoform, which is resistant to chimeric antigen receptor (CAR) T cells targeting the third immunoglobulin-like domain of CD22. We also describe splice variants skipping the AUG-containing exon 2 and failing to produce any identifiable protein, thereby defining an event that is rate limiting for epitope presentation. Indeed, forcing exon 2 skipping with morpholino oligonucleotides reduced CD22 protein expression and conferred resistance to the CD22-directed antibody-drug conjugate inotuzumab ozogamicin in vitro. Furthermore, among inotuzumab-treated pediatric patients with B-ALL, we identified one nonresponder in whose leukemic blasts Δex2 isoforms comprised the majority of CD22 transcripts. In a second patient, a sharp reduction in CD22 protein levels during relapse was driven entirely by increased CD22 exon 2 skipping. Thus, dysregulated CD22 splicing is a major mechanism of epitope downregulation and ensuing resistance to immunotherapy. SIGNIFICANCE: The mechanism(s) underlying downregulation of surface CD22 following CD22-directed immunotherapy remains underexplored. Our biochemical and correlative studies demonstrate that in B-ALL, CD22 expression levels are controlled by inclusion/skipping of CD22 exon 2. Thus, aberrant splicing of CD22 is an important driver/biomarker of de novo and acquired resistance to CD22-directed immunotherapies. See related commentary by Bourcier and Abdel-Wahab, p. 87. This article is highlighted in the In This Issue feature, p. 85.

Splicing-Mediated Antigen Escape from Immunotherapy for B-cell Malignancies.

SUMMARY: In this issue of Blood Cancer Discovery, Zheng and colleagues identify that alternative RNA splicing of CD22 within B-cell acute lymphoblastic leukemia can result in antigen escape from CD22-targeted immunotherapies. Drug-resistant isoforms of CD22 exist within leukemic cells pretreatment and can influence response to the CD22-directed antibody-drug conjugate inotuzumab ozogamicin, the immunotoxin moxetumomab pasudotox, as well as anti-CD22 chimeric antigen receptor T cells. See related article by Zheng et al., p. 103 (7).

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern.

BACKGROUND: The emergence of new SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) that harbor mutations in the viral S protein raised concern about activity of current vaccines and therapeutic antibodies. Independent studies have shown that mutant variants are partially or completely resistant against some of the therapeutic antibodies authorized for emergency use. METHODS: We employed hybridoma technology, ELISA-based and cell-based S-ACE2 interaction assays combined with authentic virus neutralization assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize the new variants of SARS-CoV-2. FINDINGS: AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). In addition, AX677 is able to bind Omicron Spike protein just like the wild type Spike. The combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. Prophylactic administration of AX290 and AX677, either individually or in combination, effectively reduced viral burden and inflammation in the lungs, and prevented disease in a mouse model of SARS-CoV-2 infection. INTERPRETATION: The virus-neutralizing properties were fully reproduced in chimeric mouse-human versions of the antibodies, which may represent a promising tool for COVID-19 therapy. FUNDING: The study was funded by AXON Neuroscience SE and AXON COVIDAX a.s.

Clinical determinants of relapse following CAR-T therapy for hematologic malignancies: Coupling active strategies to overcome therapeutic limitations.

The advent of chimeric antigen receptor (CAR)-T cell therapy has been hailed as a major breakthrough in the treatment of B cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL). While multiple promising CAR-T cell clinical trials continue to receive approval from the FDA and the Chinese Clinical Trial Register (ChiCTR), many hematologic malignancies patients nonetheless experience disease relapse following treatment as a consequence of genetic mutations, antigen escape, lineage switching, poor CAR-T cell persistence, CAR T cell exhaustion, and immunogenicity against CAR T cells. In this article, we summarize the structural characteristics of CAR constructs and discuss clinical factors known to be related to relapse following CAR-T cell treatment. By better understanding the mechanistic basis for such disease recurrence, it will be possible to fully realize the potential of this potent therapeutic modality in the future. This review will focus on current activate strategies aimed at overcoming known limitations to CAR-T cell therapy in an effort to improve hematologic malignancies patient outcomes.

A new self-attenuated therapeutic influenza vaccine that uses host cell-restricted attenuation by artificial microRNAs.

New strategies are urgently needed for developing vaccines and/or anti-viral drugs against influenza viruses, because antigenic shift and drift inevitably occurs in circulating strains each year, and new strains resistant to anti-viral drugs have recently emerged. In our study, we designed and incorporated artificial microRNAs (amiRNAs) into the NA segment of rescued influenza viruses to separately target two host genes, Cdc2-like kinase 1 (CLK1) and SON DNA binding protein (SON), which were found to play an essential role in virus replication. Mouse epithelial fibroblast (MEF) or human lung carcinoma A549 cells infected with engineered influenza PR8 viruses expressing amiR-30CLK1 (PR8-amiR-30CLK1) or amiR-93SON (PR8-amiR-93SON) had reduced expression of host proteins CLK1 and SON, respectively. All engineered influenza viruses functioned as attenuated vaccines, induced significantly higher antibody responses, and provided greater protective efficacy. In addition, they were found to be safe, based on the mouse weight changes and clinical signs observed. In contrast to the engineered viruses targeting SON, mice treated with engineered viruses targeting CLK1 recovered from weight loss and survived lethal infection by 6 h after lethal-dose PR8 infection, suggesting that our PR8-amiR-30CLK1 self-attenuated influenza virus (SAIV) could be used as a new therapeutic influenza vaccine.

Therapeutic potential of CAR T cell in malignancies: A scoping review.

Although tremendous advancements in cancer therapy over the last several years, cancer still is a complex illness to cure. Traditional cancer treatments, including chemotherapy, radiotherapy, and surgery, have a poor therapeutic effect, emphasizing the significance of employing innovative treatments like activated cell therapy. Chimeric antigen receptor T cell is one of the most prevalent types of activated cell therapy have been developed to direct T lymphocytes toward cancers (CAR-T cells). CAR-T cells therapy has illustrated poor impact versus solid tumors despite the remarkable success in patients suffering from hematological malignancies. CAR-T cells must overcome various hurdles to obtain full responses to solid tumors, including growth, stability, trafficking, and destiny inside tumors. As a result, novel treatment methods will entail overcoming the challenges that CAR-T cells face in solid tumors. The use of CAR-T cells in combination with other therapeutic approaches such as chemotherapy, radiotherapy, immuno-checkpoint inhibitors, and oncolytic viruses can promote the effectiveness of CAR-T cell therapy for the treatment of solid tumors. However, more research is needed to determine the safety and effectiveness of these therapies. CAR-T cell treatment success rates vary by type of disease, but are predicted to reach up to 90% in patients with leukemia. However, since this kind of immunotherapy is still in its infancy, there is much to learn about its efficacy. This review provided an in-depth examination of CAR-T cell therapy and its success and failure as a cancer treatment approach. We also discuss combination therapies with CAR-T Cell.

Bispecific CAR T Cells against EpCAM and Inducible ICAM-1 Overcome Antigen Heterogeneity and Generate Superior Antitumor Responses.

Adoptive transfer of chimeric antigen receptor (CAR) T cells has demonstrated unparalleled responses in hematologic cancers, yet antigen escape and tumor relapse occur frequently. CAR T-cell therapy for patients with solid tumors faces even greater challenges due to the immunosuppressive tumor environment and antigen heterogeneity. Here, we developed a bispecific CAR to simultaneously target epithelial cell adhesion molecule (EpCAM) and intercellular adhesion molecule 1 (ICAM-1) to overcome antigen escape and to improve the durability of tumor responses. ICAM-1 is an adhesion molecule inducible by inflammatory cytokines and elevated in many types of tumors. Our study demonstrates superior efficacy of bispecific CAR T cells compared with CAR T cells targeting a single primary antigen. Bispecific CAR T achieved more durable antitumor responses in tumor models with either homogenous or heterogenous expression of EpCAM. We also showed that the activation of CAR T cells against EpCAM in tumors led to upregulation of ICAM-1, which rendered tumors more susceptible to ICAM-1 targeting by bispecific CAR T cells. Our strategy of additional targeting of ICAM-1 may have broad applications in augmenting the activity of CAR T cells against primary tumor antigens that are prone to antigen loss or downregulation.

Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control.

Treating solid malignancies with chimeric antigen receptor (CAR) T cells typically results in poor responses. Immunomodulatory biologics delivered systemically can augment the cells' activity, but off-target toxicity narrows the therapeutic window. Here we show that the activity of intratumoural CAR T cells can be controlled photothermally via synthetic gene switches that trigger the expression of transgenes in response to mild temperature elevations (to 40-42 °C). In vitro, heating engineered primary human T cells for 15-30 min led to over 60-fold-higher expression of a reporter transgene without affecting the cells' proliferation, migration and cytotoxicity. In mice, CAR T cells photothermally heated via gold nanorods produced a transgene only within the tumours. In mouse models of adoptive transfer, the systemic delivery of CAR T cells followed by intratumoural production, under photothermal control, of an interleukin-15 superagonist or a bispecific T cell engager bearing an NKG2D receptor redirecting T cells against NKG2D ligands enhanced antitumour activity and mitigated antigen escape. Localized photothermal control of the activity of engineered T cells may enhance their safety and efficacy.

CD19/CD22 Dual-Targeted CAR T-cell Therapy for Relapsed/Refractory Aggressive B-cell Lymphoma: A Safety and Efficacy Study.

Chimeric antigen receptor (CAR) T-cell therapies that target either CD19 or CD22 alone have potent antilymphoma effects. However, antigen escape-mediated relapse often occurs. CAR T cells targeting both CD19 and CD22 may overcome this limitation. In this study, we developed bispecific CAR T cells simultaneously recognizing CD19- and CD22-expressing targets and assessed their safety and efficacy profiles in patients with relapsed/refractory aggressive B-cell lymphoma. Twenty-four patients were screened, and 16 were found eligible for the study. CAR T-cell-associated toxicities were recorded. Responses, overall survival (OS), and progression-free survival (PFS) were assessed. Of the 16 eligible patients, 14 (87.5%) achieved objective response and 10 (62.5%) achieved complete response (CR). The 2-year OS and PFS rates were 77.3% and 40.2%, respectively. Achieving CR (P = 0.046) and the number of prior chemotherapy lines (n = 2; P = 0.047) were independent prognostic factors associated with favorable PFS. The 2-year OS and PFS among patients who achieved CR were higher than among those who did not (P = 0.015 and P < 0.001, respectively). The 2-year PFS among patients who received two prior lines of chemotherapy was higher than that among patients who received more than two lines of chemotherapy (P = 0.049); OS did not differ between the groups. Severe grade 4 cytokine-release syndrome (CRS) was observed in 1 patient; 4 and 11 patients had grades 1 and 2 CRS, respectively. No patients developed neurotoxicity. CD19/CD22 dual-targeted CAR T cells may be a safe, potent antilymphoma cell-based targeted immunotherapy.

Anti-CD19 CAR T Cells That Secrete a Biparatopic Anti-CLEC12A Bridging Protein Have Potent Activity Against Highly Aggressive Acute Myeloid Leukemia In Vitro and In Vivo.

Refractory acute myeloid leukemia (AML) remains an incurable malignancy despite the clinical use of novel targeted therapies, new antibody-based therapies, and cellular therapeutics. Here, we describe the preclinical development of a novel cell therapy that targets the antigen CLEC12A with a biparatopic bridging protein. Bridging proteins are designed as "CAR-T cell engagers," with a CAR-targeted protein fused to antigen binding domains derived from antibodies. Here, we created a CD19-anti-CLEC12A bridging protein that binds to CAR19 T cells and to the antigen CLEC12A. Biparatopic targeting increases the potency of bridging protein-mediated cytotoxicity by CAR19 T cells. Using CAR19 T cells that secrete the bridging protein we demonstrate potent activity against aggressive leukemic cell lines in vivo This CAR-engager platform is facile and modular, as illustrated by activity of a dual-antigen bridging protein targeting CLEC12A and CD33, designed to counter tumor heterogeneity and antigen escape, and created without the need for extensive CAR T-cell genetic engineering. CAR19 T cells provide an optimal cell therapy platform with well-understood inherent persistence and fitness characteristics.