CD37 is a safe chimeric antigen receptor target to treat acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells in the bone marrow and the peripheral blood. Nearly half of the AML patients relapse after standard induction therapy, and new forms of therapy are urgently needed. Chimeric antigen receptor (CAR) T therapy has so far not been successful in AML due to lack of efficacy and safety. Indeed, the most attractive antigen targets are stem cell markers such as CD33 or CD123. We demonstrate that CD37, a mature B cell marker, is expressed in AML samples, and its presence correlates with the European LeukemiaNet (ELN) 2017 risk stratification. We repurpose the anti-lymphoma CD37CAR for the treatment of AML and show that CD37CAR T cells specifically kill AML cells, secrete proinflammatory cytokines, and control cancer progression in vivo. Importantly, CD37CAR T cells display no toxicity toward hematopoietic stem cells. Thus, CD37 is a promising and safe CAR T cell AML target.

Multiomic analyses uncover immunological signatures in acute and chronic coronary syndromes.

Acute and chronic coronary syndromes (ACS and CCS) are leading causes of mortality. Inflammation is considered a key pathogenic driver of these diseases, but the underlying immune states and their clinical implications remain poorly understood. Multiomic factor analysis (MOFA) allows unsupervised data exploration across multiple data types, identifying major axes of variation and associating these with underlying molecular processes. We hypothesized that applying MOFA to multiomic data obtained from blood might uncover hidden sources of variance and provide pathophysiological insights linked to clinical needs. Here we compile a longitudinal multiomic dataset of the systemic immune landscape in both ACS and CCS (n = 62 patients in total, n = 15 women and n = 47 men) and validate this in an external cohort (n = 55 patients in total, n = 11 women and n = 44 men). MOFA reveals multicellular immune signatures characterized by distinct monocyte, natural killer and T cell substates and immune-communication pathways that explain a large proportion of inter-patient variance. We also identify specific factors that reflect disease state or associate with treatment outcome in ACS as measured using left ventricular ejection fraction. Hence, this study provides proof-of-concept evidence for the ability of MOFA to uncover multicellular immune programs in cardiovascular disease, opening new directions for mechanistic, biomarker and therapeutic studies.

Beyond CAR T cells: exploring alternative cell sources for CAR-like cellular therapies.

Chimeric antigen receptor (CAR)-T cell therapy has led to remarkable clinical outcomes in the treatment of hematological malignancies. However, challenges remain, such as limited infiltration into solid tumors, inadequate persistence, systemic toxicities, and manufacturing insufficiencies. The use of alternative cell sources for CAR-based therapies, such as natural killer cells (NK), macrophages (MΦ), invariant Natural Killer T (iNKT) cells, γδT cells, neutrophils, and induced pluripotent stem cells (iPSC), has emerged as a promising avenue. By harnessing these cells' inherent cytotoxic mechanisms and incorporating CAR technology, common CAR-T cell-related limitations can be effectively mitigated. We herein present an overview of the tumoricidal mechanisms, CAR designs, and manufacturing processes of CAR-NK cells, CAR-MΦ, CAR-iNKT cells, CAR-γδT cells, CAR-neutrophils, and iPSC-derived CAR-cells, outlining the advantages, limitations, and potential solutions of these therapeutic strategies.

Comparative performance of scFv-based anti-BCMA CAR formats for improved T cell therapy in multiple myeloma.

In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.

PGE2 limits effector expansion of tumour-infiltrating stem-like CD8+ T cells.

Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1-4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5-9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2-EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.

Efficient Chimeric Antigen Receptor T-Cell Generation Starting with Leukoreduction System Chambers of Thrombocyte Apheresis Sets.

Introduction: Primary human blood cells represent an essential model system to study physiology and disease. However, human blood is a limited resource. During healthy donor plateletpheresis, the leukoreduction system chamber (LRSC) reduces the leukocyte amount within the subsequent platelet concentrate through saturated, fluidized, particle bed filtration technology. Normally, the LRSC is discarded after apheresis is completed. Compared to peripheral blood, LRSC yields 10-fold mononuclear cell concentration. Methods: To explore if those retained leukocytes are attractive for research purposes, we isolated CD3+ T cells from the usually discarded LRSCs via density gradient centrifugation in order to manufacture CD19-targeted chimeric antigen receptor (CAR) T cells. Results: Immunophenotypic characterization revealed viable and normal CD4+ and CD8+ T-cell populations within LRSC, with low CD19+ B cell counts. Magnetic-activated cell sorting (MACS) purified CD3+ T cells were transduced with CD19 CAR-encoding lentiviral self-inactivating vectors using concentrated viral supernatants. Robust CD19 CAR cell surface expression on transduced T cells was confirmed by flow cytometry. CD19 CAR T cells were further enriched through anti-CAR MACS, yielding 80% CAR+ T-cell populations. In vitro CAR T cell expansion to clinically relevant numbers was achieved. To prove functionality, CAR T cells were co-incubated with the human CD19+ B cell precursor leukemia cell line Nalm6. Compared to unmodified T cells, CD19 CAR T cells effectively eradicated Nalm6 cells. Conclusion: Taken together, we can show that lymphocytes isolated from LRSCs of plateletpheresis sets can be efficiently used for the generation of functional CAR T cells for experimental purposes.

Mutation-specific CAR T cells as precision therapy for IGLV3-21R110 expressing high-risk chronic lymphocytic leukemia.

The concept of precision cell therapy targeting tumor-specific mutations is appealing but requires surface-exposed neoepitopes, which is a rarity in cancer. B cell receptors (BCR) of mature lymphoid malignancies are exceptional in that they harbor tumor-specific-stereotyped sequences in the form of point mutations that drive self-engagement of the BCR and autologous signaling. Here, we use a BCR light chain neoepitope defined by a characteristic point mutation (IGLV3-21R110) for selective targeting of a poor-risk subset of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR) T cells. We develop murine and humanized CAR constructs expressed in T cells from healthy donors and CLL patients that eradicate IGLV3-21R110 expressing cell lines and primary CLL cells, but neither cells expressing the non-pathogenic IGLV3-21G110 light chain nor polyclonal healthy B cells. In vivo experiments confirm epitope-selective cytolysis in xenograft models in female mice using engrafted IGLV3-21R110 expressing cell lines or primary CLL cells. We further demonstrate in two humanized mouse models lack of cytotoxicity towards human B cells. These data provide the basis for advanced approaches of resistance-preventive and biomarker-guided cellular targeting of functionally relevant lymphoma driver mutations sparing normal B cells.

Major immunophenotypic abnormalities in patients with primary adrenal insufficiency of different etiology.

Introduction: Patients with primary adrenal insufficiency (PAI) suffer from increased risk of infection, adrenal crises and have a higher mortality rate. Such dismal outcomes have been inferred to immune cell dysregulation because of unphysiological cortisol replacement. As the immune landscape of patients with different types of PAI has not been systematically explored, we set out to immunophenotype PAI patients with different causes of glucocorticoid (GC) deficiency. Methods: This cross-sectional single center study includes 28 patients with congenital adrenal hyperplasia (CAH), 27 after bilateral adrenalectomy due to Cushing's syndrome (BADx), 21 with Addison's disease (AD) and 52 healthy controls. All patients with PAI were on a stable GC replacement regimen with a median dose of 25 mg hydrocortisone per day. Peripheral blood mononuclear cells were isolated from heparinized blood samples. Immune cell subsets were analyzed using multicolor flow cytometry after four-hour stimulation with phorbol myristate acetate and ionomycin. Natural killer (NK-) cell cytotoxicity and clock gene expression were investigated. Results: The percentage of T helper cell subsets was downregulated in AD patients (Th1 p = 0.0024, Th2 p = 0.0157, Th17 p < 0.0001) compared to controls. Cytotoxic T cell subsets were reduced in AD (Tc1 p = 0.0075, Tc2 p = 0.0154) and CAH patients (Tc1 p = 0.0055, Tc2 p = 0.0012) compared to controls. NKCC was reduced in all subsets of PAI patients, with smallest changes in CAH. Degranulation marker CD107a expression was upregulated in BADx and AD, not in CAH patients compared to controls (BADx p < 0.0001; AD p = 0.0002). In contrast to NK cell activating receptors, NK cell inhibiting receptor CD94 was upregulated in BADx and AD, but not in CAH patients (p < 0.0001). Although modulation in clock gene expression could be confirmed in our patient subgroups, major interindividual-intergroup dissimilarities were not detected. Discussion: In patients with different etiologies of PAI, distinct differences in T and NK cell-phenotypes became apparent despite the use of same GC preparation and dose. Our results highlight unsuspected differences in immune cell composition and function in PAI patients of different causes and suggest disease-specific alterations that might necessitate disease-specific treatment.

Mitochondrial dysfunction promotes the transition of precursor to terminally exhausted T cells through HIF-1α-mediated glycolytic reprogramming.

T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy.

Functional enhancement of mesothelin-targeted TRuC-T cells by a PD1-CD28 chimeric switch receptor.

T cells expressing a mesothelin (MSLN)-specific T cell receptor fusion construct (TRuC®), called TC-210, have demonstrated robust antitumor activity in preclinical models of mesothelioma, ovarian cancer, and lung cancer. However, they are susceptible to suppression by the programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) axis and lack intrinsic costimulatory signaling elements. To enhance the function of anti-MSLN TRuC-T cells, chimeric switch receptors (CSRs) have been designed to co-opt the immunosuppressive PD-1/PD-L1 axis and to deliver a CD28-mediated costimulatory signal. Here, we report that coexpression of the PD1-CD28 CSR in TRuC-T cells enhanced T cell receptor signaling, increased proinflammatory effector cytokines, decreased anti-inflammatory cytokines, and sustained effector function in the presence of PD-L1 when compared with TC-210. Anti-MSLN TRuC-T cells engineered to coexpress PD1-CD28 CSRs comprising the ectodomain of PD-1 and the intracellular domain of CD28 linked by the transmembrane domain of PD-1 were selected for integration into an anti-MSLN TRuC-T cell therapy product called TC-510. In vitro, TC-510 showed significant improvements in persistence and resistance to exhaustion upon chronic stimulation by tumor cells expressing MSLN and PD-L1 when compared with TC-210. In vivo, TC-510 showed a superior ability to provide durable protection following tumor rechallenge, versus TC-210. These data demonstrate that integration of a PD1-CD28 CSR into TRuC-T cells improves effector function, resistance to exhaustion, and prolongs persistence. Based on these findings, TC-510 is currently being evaluated in patients with MSLN-expressing solid tumors.

Programmable multispecific DNA-origami-based T-cell engagers.

Multispecific antibodies have emerged as versatile therapeutic agents, and therefore, approaches to optimize and streamline their design and assembly are needed. Here we report on the modular and programmable assembly of IgG antibodies, F(ab) and scFv fragments on DNA origami nanocarriers. We screened 105 distinct quadruplet antibody variants in vitro for the ability to activate T cells in the presence of target cells. T-cell engagers were identified, which in vitro showed the specific and efficient T-cell-mediated lysis of five distinct target cell lines. We used these T-cell engagers to target and lyse tumour cells in vivo in a xenograft mouse tumour model. Our approach enables the rapid generation, screening and testing of bi- and multispecific antibodies to facilitate preclinical pharmaceutical development from in vitro discovery to in vivo proof of concept.

HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice.

The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets.

Rational design of PD-1-CD28 immunostimulatory fusion proteins for CAR T cell therapy.

BACKGROUND: In many situations, the therapeutic efficacy of CAR T cells is limited due to immune suppression and poor persistence. Immunostimulatory fusion protein (IFP) constructs have been advanced as a tool to convert suppressive signals into stimulation and thus promote the persistence of T cells, but no universal IFP design has been established so far. We now took advantage of a PD-1-CD28 IFP as a clinically relevant structure to define key determinants of IFP activity. METHODS: We compared different PD-1-CD28 IFP variants in a human leukemia model to assess the impact of distinctive design choices on CAR T cell performance in vitro and a xenograft mouse model. RESULTS: We observed that IFP constructs that putatively exceed the extracellular length of PD-1 induce T-cell response without CAR target recognition, rendering them unsuitable for tumour-specific therapy. IFP variants with physiological PD-1 length ameliorated CAR T cell effector function and proliferation in response to PD-L1+ tumour cells in vitro and prolonged survival in vivo. Transmembrane or extracellular CD28 domains were found to be replaceable by corresponding PD-1 domains for in vivo efficacy. CONCLUSION: PD-1-CD28 IFP constructs must mimic the physiological interaction of PD-1 with PD-L1 to retain selectivity and mediate CAR-conditional therapeutic activity.

A microbiota-modulated checkpoint directs immunosuppressive intestinal T cells into cancers.

Antibiotics (ABX) compromise the efficacy of programmed cell death protein 1 (PD-1) blockade in cancer patients, but the mechanisms underlying their immunosuppressive effects remain unknown. By inducing the down-regulation of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the ileum, post-ABX gut recolonization by Enterocloster species drove the emigration of enterotropic α4ß7+CD4+ regulatory T 17 cells into the tumor. These deleterious ABX effects were mimicked by oral gavage of Enterocloster species, by genetic deficiency, or by antibody-mediated neutralization of MAdCAM-1 and its receptor, α4ß7 integrin. By contrast, fecal microbiota transplantation or interleukin-17A neutralization prevented ABX-induced immunosuppression. In independent lung, kidney, and bladder cancer patient cohorts, low serum levels of soluble MAdCAM-1 had a negative prognostic impact. Thus, the MAdCAM-1-α4ß7 axis constitutes an actionable gut immune checkpoint in cancer immunosurveillance.

Mechanisms and strategies for safe chimeric antigen receptor T-cell activity control.

The clinical application of chimeric antigen receptor (CAR) T-cell therapy has rapidly changed the treatment options for terminally ill patients with defined blood-borne cancer types. However, CAR T-cell therapy can lead to severe therapy-associated toxicities including CAR-related hematotoxicity, ON-target OFF-tumor toxicity, cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Just as CAR T-cell therapy has evolved regarding receptor design, gene transfer systems and production protocols, the management of side effects has also improved. However, because of measures taken to abrogate adverse events, CAR T-cell viability and persistence might be impaired before complete remission can be achieved. This has fueled efforts for the development of extrinsic and intrinsic strategies for better control of CAR T-cell activity. These approaches can mediate a reversible resting state or irreversible T-cell elimination, depending on the route chosen. Control can be passive or active. By combination of CAR T-cells with T-cell inhibiting compounds, pharmacologic control, mostly independent of the CAR construct design used, can be achieved. Other strategies involve the genetic modification of T-cells or further development of the CAR construct by integration of molecular ON/OFF switches such as suicide genes. Alternatively, CAR T-cell activity can be regulated intracellularly through a self-regulation function or extracellularly through titration of a CAR adaptor or of a priming small molecule. In this work, we review the current strategies and mechanisms to control activity of CAR T-cells reversibly or irreversibly for preventing and for managing therapy-associated toxicities.

Dual TLR9 and PD-L1 targeting unleashes dendritic cells to induce durable antitumor immunity.

BACKGROUND: Although immune checkpoint inhibitors have been a breakthrough in clinical oncology, these therapies fail to produce durable responses in a significant fraction of patients. This lack of long-term efficacy may be due to a poor pre-existing network linking innate and adaptive immunity. Here, we present an antisense oligonucleotide (ASO)-based strategy that dually targets toll-like receptor 9 (TLR9) and programmed cell death ligand 1 (PD-L1), aiming to overcome resistance to anti-PD-L1 monoclonal therapy. METHODS: We designed a high-affinity immunomodulatory IM-TLR9:PD-L1-ASO antisense oligonucleotide (hereafter, IM-T9P1-ASO) targeting mouse PD-L1 messenger RNA and activating TLR9. Then, we performed in vitro and in vivo studies to validate the IM-T9P1-ASO activity, efficacy, and biological effects in tumors and draining lymph nodes. We also performed intravital imaging to study IM-T9P1-ASO pharmacokinetics in the tumor. RESULTS: IM-T9P1-ASO therapy, unlike PD-L1 antibody therapy, results in durable antitumor responses in multiple mouse cancer models. Mechanistically, IM-T9P1-ASO activates a state of tumor-associated dendritic cells (DCs), referred to here as DC3s, which have potent antitumor potential but express the PD-L1 checkpoint. IM-T9P1-ASO has two roles: it triggers the expansion of DC3s by engaging with TLR9 and downregulates PD-L1, thereby unleashing the antitumor functions of DC3s. This dual action leads to tumor rejection by T cells. The antitumor efficacy of IM-T9P1-ASO depends on the antitumor cytokine interleukin-12 (IL-12), produced by DC3s, and Batf3, a transcription factor required for DC development. CONCLUSIONS: By simultaneously targeting TLR9 and PD-L1, IM-T9P1-ASO amplifies antitumor responses via DC activation, leading to sustained therapeutic efficacy in mice. By highlighting differences and similarities between mouse and human DCs, this study could serve to develop similar therapeutic strategies for patients with cancer.

Bispecific antibodies redirect synthetic agonistic receptor modified T cells against melanoma.

BACKGROUND: Melanoma is an immune sensitive disease, as demonstrated by the activity of immune check point blockade (ICB), but many patients will either not respond or relapse. More recently, tumor infiltrating lymphocyte (TIL) therapy has shown promising efficacy in melanoma treatment after ICB failure, indicating the potential of cellular therapies. However, TIL treatment comes with manufacturing limitations, product heterogeneity, as well as toxicity problems, due to the transfer of a large number of phenotypically diverse T cells. To overcome said limitations, we propose a controlled adoptive cell therapy approach, where T cells are armed with synthetic agonistic receptors (SAR) that are selectively activated by bispecific antibodies (BiAb) targeting SAR and melanoma-associated antigens. METHODS: Human as well as murine SAR constructs were generated and transduced into primary T cells. The approach was validated in murine, human and patient-derived cancer models expressing the melanoma-associated target antigens tyrosinase-related protein 1 (TYRP1) and melanoma-associated chondroitin sulfate proteoglycan (MCSP) (CSPG4). SAR T cells were functionally characterized by assessing their specific stimulation and proliferation, as well as their tumor-directed cytotoxicity, in vitro and in vivo. RESULTS: MCSP and TYRP1 expression was conserved in samples of patients with treated as well as untreated melanoma, supporting their use as melanoma-target antigens. The presence of target cells and anti-TYRP1 × anti-SAR or anti-MCSP × anti-SAR BiAb induced conditional antigen-dependent activation, proliferation of SAR T cells and targeted tumor cell lysis in all tested models. In vivo, antitumoral activity and long-term survival was mediated by the co-administration of SAR T cells and BiAb in a syngeneic tumor model and was further validated in several xenograft models, including a patient-derived xenograft model. CONCLUSION: The SAR T cell-BiAb approach delivers specific and conditional T cell activation as well as targeted tumor cell lysis in melanoma models. Modularity is a key feature for targeting melanoma and is fundamental towards personalized immunotherapies encompassing cancer heterogeneity. Because antigen expression may vary in primary melanoma tissues, we propose that a dual approach targeting two tumor-associated antigens, either simultaneously or sequentially, could avoid issues of antigen heterogeneity and deliver therapeutic benefit to patients.