Demonstrating the Manufacture of Human CAR‐T Cells in an Automated Stirred‐Tank Bioreactor

Chimeric antigen receptor T‐cell (CAR‐T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR‐T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR‐T cells from multiple donors in an automated stirred‐tank bioreactor. The authors successfully produced functional human CAR‐T cells from multiple donors under dynamic conditions in a stirred‐tank bioreactor, resulting in overall cell yields which were significantly better than in static T‐flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR‐T cells are able to proliferate effectively, reaching viable cell densities of >5 × 10⁶ cells ml‐1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T‐flasks and gas‐permeable culture bags). Importantly, engineered T‐cells post‐expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR‐T cell manufacture in stirred‐tank bioreactors and the findings present significant implications and opportunities for larger‐scale allogeneic CAR‐T production.     

CD19‐CAR engineered NK‐92 cells are sufficient to overcome NK cell resistance in B‐cell malignancies

Many B‐cell acute and chronic leukaemias tend to be resistant to killing by natural killer (NK) cells. The introduction of chimeric antigen receptors (CAR) into T cells or NK cells could potentially overcome this resistance. Here, we extend our previous observations on the resistance of malignant lymphoblasts to NK‐92 cells, a continuously growing NK cell line, showing that anti‐CD19‐CAR (αCD19‐CAR) engineered NK‐92 cells can regain significant cytotoxicity against CD19 positive leukaemic cell lines and primary leukaemia cells that are resistant to cytolytic activity of parental NK‐92 cells. The ‘first generation’ CAR was generated from a scFv (CD19) antibody fragment, coupled to a flexible hinge region, the CD3ζ chain and a Myc‐tag and cloned into a retrovirus backbone. No difference in cytotoxic activity of NK‐92 and transduced αCD19‐CAR NK‐92 cells towards CD19 negative targets was found. However, αCD19‐CAR NK‐92 cells specifically and efficiently lysed CD19 expressing B‐precursor leukaemia cell lines as well as lymphoblasts from leukaemia patients. Since NK‐92 cells can be easily expanded to clinical grade numbers under current Good Manufactoring Practice (cGMP) conditions and its safety has been documented in several phase I clinical studies, treatment with CAR modified NK‐92 should be considered a treatment option for patients with lymphoid malignancies.     

Advanced Cell Mapping Visualizations for Single Cell Functional Proteomics Enabling Patient Stratification

Highly multiplexed single‐cell functional proteomics has emerged as one of the next‐generation toolkits for a deeper understanding of functional heterogeneity in cell. Different from the conventional population‐based bulk and single‐cell RNA‐Seq assays, the microchip‐based proteomics at the single‐cell resolution enables a unique identification of highly polyfunctional cell subsets that co‐secrete many proteins from live single cells and most importantly correlate with patient response to a therapy. The 32‐plex IsoCode chip technology has defined a polyfunctional strength index (PSI) of pre‐infusion anti‐CD19 chimeric antigen receptor (CAR)‐T products, that is significantly associated with patient response to the CAR‐T cell therapy. To complement the clinical relevance of the PSI, a comprehensive visualization toolkit of 3D uniform manifold approximation and projection (UMAP) and t‐distributed stochastic neighbor embedding (t‐SNE) in a proteomic analysis pipeline is developed, providing more advanced analytical algorithms for more intuitive data visualizations. The UMAP and t‐SNE of anti‐CD19 CAR‐T products reveal distinct cytokine profiles between nonresponders and responders and demonstrate a marked upregulation of antitumor‐associated cytokine signatures in CAR‐T cells from responding patients. Using this powerful while user‐friendly analytical tool, the multi‐dimensional single‐cell data can be dissected from complex immune responses and uncover underlying mechanisms, which can promote correlative biomarker discovery, improved bioprocessing, and personalized treatment development.     

Non‐viral nucleic acid delivery to the central nervous system and brain tumors

Gene therapy is a rapidly emerging remedial route for many serious incurable diseases, such as central nervous system (CNS) diseases. Currently, nucleic acid medicines, including DNAs encoding therapeutic or destructive proteins, small interfering RNAs or microRNAs, have been successfully delivered to the CNS with gene delivery vectors using various routes of administration and have subsequently exhibited remarkable therapeutic efficiency. Among these vectors, non‐viral vectors are favorable for delivering genes into the CNS as a result of their many special characteristics, such as low toxicity and pre‐existing immunogenicity, high gene loading efficiency and easy surface modification. In this review, we highlight the main types of therapeutic genes that have been applied in the therapy of CNS diseases and then outline non‐viral gene delivery vectors.     

Interleukin‐6 contributes to chemoresistance in MDA‐MB‐231 cells via targeting HIF‐1α

Chemoresistance is a critical challenge in the clinical treatment of triple‐negative breast cancer (TNBC). It has been well documented that inflammatory mediators from tumor microenvironment are involved in the pathogenesis of TNBC and might be related to chemoresistance of cancer cells. In this study, the contribution of interleukin‐6 (IL‐6), one of the principal oncogenic molecules, in chemoresistance of a TNBC cell line MDA‐MB‐231 was first investigated. The results showed that IL‐6 treatment could induce upregulation of HIF‐1α via the activation of STAT3 in MDA‐MB‐231 cells, which consequently contributed to its effect against chemotherapeutic drug‐induced cytotoxicity and cell apoptosis. However, knockdown of HIF‐1α attenuated such effect via affecting the expressions of apoptosis‐related molecules as Bax and Bcl‐2 and drug transporters as P‐gp and MRP1. This study indicated that targeting at IL‐6/HIF‐1α signaling pathway might be an effective strategy to overcome chemoresistance in TNBC therapy.     

In vivo cleavage of transgene donors promotes nuclease‐mediated targeted integration

Targeted DNA integration is commonly used to eliminate position effects on transgene expression. Integration can be targeted to specific sites in the genome via both homology‐based and homology‐independent processes. Both pathways start the integration process with a site‐specific break in the chromosome, typically from a zinc‐finger nuclease (ZFN). We previously described an efficient homology‐independent targeted integration technique that captures short (<100 bp) pieces of DNA at chromosomal breaks created by ZFNs. We show here that inclusion of a nuclease target site on the donor plasmid followed by in vivo nuclease cleavage of both the donor and the chromosome results in efficient integration of large, transgene‐sized DNA molecules into the chromosomal double‐strand break. Successful targeted integration via in vivo donor linearization is demonstrated at five distinct loci in two mammalian cell types, highlighting the generality of the approach. Finally, we show that CHO cells, a cell type recalcitrant to homology‐based integration, are proficient at capture of in vivo‐linearized transgene donors. Moreover, we demonstrate knockout of the hamster FUT8 gene via the simultaneous ZFN‐ or TALE nuclease‐mediated integration of an antibody cassette. Our results enable efficient targeted transgene addition to cells and organisms that fare poorly with traditional homology‐driven approaches. Biotechnol. Bioeng. 2013; 110: 871–880. © 2012 Wiley Periodicals, Inc.     

快速构建CRISPR/Cas9基因组靶向编辑系统

CRISPR/Cas9核酸酶作为一种新的基因组靶向编辑技术,已成功应用于多种动植物基因组修饰研究. CRISPR/Cas9作用后的阳性细胞筛选和富集是该技术的关键之一. 本研究以鸡EAV-HP（endogenous avian retrovirus-HP）基因和MSTN（myostatin）基因为例,从靶位点的选择、表达载体构建、双基因报告载体构建和核酸酶活性验证4个方面,系统研究了CRISPR/Cas9核酸酶技术平台. 结果表明,利用寡聚核苷酸直接退火方法,构建表达载体和报告载体的阳性率分别高达100%和89.5%. 报告载体的PuroR（puromycin resistant gene）和eGFP（enhanced green fluorescent protein）基因的成功表达表明,构建的CRISPR/Cas9系统能有效切割靶序列,并用于后续阳性克隆的筛选和富集. 本方法摒弃了传统分子克隆的PCR扩增和酶切处理目标基因的方法,而是利用寡聚核苷酸直接退火获得含有黏性末端的目标DNA,简化了载体构建过程,低成本且快速获得CRISPR/Cas9基因组靶向编辑系统.     

Molecular cloning and expression analysis of bovine programmed death‐1

Recent work has shown that PD‐1, an immune inhibitory receptor, is involved in mechanisms for down‐regulating immune responses during tumor progression or chronic viral infection. However, in the case of bovine diseases, there have been no reports on this molecule due to lack of information about bovine PD‐1. In this study, we performed identification and preliminary characterization of the bovine PD‐1 gene in two breeds of cattle. We cloned full cDNA sequences encoding for PD‐1 from both Holstein‐Friesian and Japanese Black breeds, and found that both of the genes encoded a 282‐amino acid protein, which had a signal sequence, transmembrane domain and an immunoreceptor tyrosine‐based inhibitory motif. This bovine PD‐1 showed 72.9% and 65.6% homology to human and mouse PD‐1, respectively, both of which have been well characterized and documented. Quantitative real‐time PCR analysis showed that bovine PD‐1 is expressed predominantly in T‐cells (such as CD4⁺ and CD8⁺ cells) and among PBMCs, and is strongly upregulated on T‐cell stimulation via ConA. A limited number of cattle were tested yet, as expected, the degree of PD‐1 mRNA expression in CD4⁺ and CD8⁺ T‐cells was greater in cattle with bovine leukemia virus‐induced lymphoma than in uninfected cattle. Further studies to characterize the functions of bovine PD‐1 are therefore warranted, in order to elucidate the mechanism of the immunosuppression associated with progression of several diseases and therapy in cattle.     

An EBV‐based plasmid can replicate and maintain in stem cells

Viral vectors have a wide range of applications in biology, particularly in gene therapy. Based on their integration capacity, viral vectors are classified as either integrating or non‐integrating vectors. Although integrating vectors, such as lentivectors, have the ability to direct prolonged expression of exogenous genes, manipulation of the host genome is an inappropriate feature of these gene delivery tools. Non‐integrating vectors, such as episomal replicating plasmids, can replicate and persist in host cells for long periods without any chromosomal interruption. These advantages made them good tools for gene induction purposes in gene therapy and basic studies. Due to the necessity of gene induction in stem cells for study of mammalian development and targeted differentiation, the use of integrating vectors for prolonged expression of genes of interest has been developed. Application of replicating plasmids can overcome some drawbacks associated with integrating vectors, although replication and maintenance of these plasmids can differ between cell types. Previously, it has been shown that such plasmids can be maintained in human embryonic stem cells for more than one month, but the rate of the plasmid replication during the host cell cycle has not been elucidated. In the present study, we showed that an EBV‐based plasmid can replicate simultaneously with host in pluripotent and multipotent human and mouse stem cells and can be sustained for long time periods in dividing cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1579–1585, 2015     

Protective effect of Agrimonia pilosa polysaccharides on dexamethasone‐treated MC3T3‐E1 cells via Wnt/β‐Catenin pathway

A water‐soluble polysaccharide (APP‐AW) was isolated from Agrimonia pilosa and prepared to three sulphated derivatives (S1, S2 and S3). The results showed that pre‐treatment with APP‐AW, S1, S2 and S3 each at the concentration of 50 μg/mL for 48 hours was able to prevent cytotoxicity induced by 1 μmol/L dexamethasone (Dex) in MC3T3‐E1 cells via inhibition of apoptosis, which is in line with the findings in flow cytometry analysis. Meanwhile, the decreased ALP activity, collagen content, mineralization, BMP2, Runx2, OSX and OCN protein expression in DEX‐treated MC3T3‐E1 cells were reversed by the addition of APP‐AW, S1, S2 and S3. Moreover, APP‐AW, S1, S2 and S3 rescued DEX‐induced increase of Bax, cytochrome c and caspase‐3 and decrease of Bcl‐2, Wnt3, β‐catenin and c‐Myc protein expression in MC3T3‐E1 cells. Our findings suggest that pre‐treatment with APP‐AW, S1, S2 and S3 could significantly protect MC3T3‐E1 cells against Dex‐induced cell injury via inhibiting apoptosis and activating Wnt/β‐Catenin signalling pathway, thus application of these polysaccharides may be a promising alternative strategy for steroid‐induced avascular necrosis of the femoral head (SANFH) therapy.     

The next‐generation BET inhibitor,PLX51107, delays melanoma growth in a CD8‐mediated manner

Epigenetic agents such as bromodomain and extra‐terminal region inhibitors (BETi) slow tumor growth via tumor intrinsic alterations; however, their effects on antitumor immunity remain unclear. A recent advance is the development of next‐generation BETi that are potent and display a favorable half‐life. Here, we tested the BETi, PLX51107, for immune‐based effects on tumor growth in BRAF V600E melanoma syngeneic models. PLX51107 delayed melanoma tumor growth and increased activated, proliferating, and functional CD8+ T cells in tumors leading to CD8+ T‐cell‐mediated tumor growth delay. PLX51107 decreased Cox2 expression, increased dendritic cells, and lowered PD‐L1, FasL, and IDO‐1 expression in the tumor microenvironment. Importantly, PLX51107 delayed the growth of tumors that progressed on anti‐PD‐1 therapy; a response associated with decreased Cox2 levels, decreased PD‐L1 expression on non‐immune cells, and increased intratumoral CD8+ T cells. Thus, next‐generation BETi represent a potential first‐line and secondary treatment strategy for metastatic melanoma by eliciting effects, at least in part, on antitumor CD8+ T cells.     

Ectodomain of coxsackievirus and adenovirus receptor genetically fused to epidermal growth factor mediates adenovirus targeting to epidermal growth factor receptor-positive cells

Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.     

Targeted gene delivery to CD117‐expressing cells in vivo with lentiviral vectors co‐displaying stem cell factor and a fusogenic molecule

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc.     

In vitro reconstitution of Cascade‐mediated CRISPR immunity in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR)‐encoded immunity in Type I systems relies on the Cascade (CRISPR‐associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4‐Cas (CRISPR‐associated) system (St‐CRISPR4‐Cas), we isolated an effector complex (St‐Cascade) containing 61‐nucleotide CRISPR RNA (crRNA). We show that St‐Cascade, guided by crRNA, binds in vitro to a matching proto‐spacer if a proto‐spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (?1 position) of the proto‐spacer. In the presence of a correct PAM, St‐Cascade binding to the target DNA generates an R‐loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R‐loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St‐CRISPR4‐Cas and other Type I systems.     

Targeting miR‐193a‐AML1‐ETO‐β‐catenin axis by melatonin suppresses the self‐renewal of leukaemia stem cells in leukaemia with t (8;21) translocation

AML1‐ETO, the most common fusion oncoprotein by t (8;21) in acute myeloid leukaemia (AML), enhances hematopoietic self‐renewal and leukemogenesis. However, currently no specific therapies have been reported for t (8;21) AML patients as AML1‐ETO is still intractable as a pharmacological target. For this purpose, leukaemia cells and AML1‐ETO‐induced murine leukaemia model were used to investigate the degradation of AML1‐ETO by melatonin (MLT), synthesized and secreted by the pineal gland. MLT remarkedly decreased AML1‐ETO protein in leukemic cells. Meanwhile, MLT induced apoptosis, decreased proliferation and reduced colony formation. Furthermore, MLT reduced the expansion of human leukemic cells and extended the overall survival in U937T‐AML1‐ETO‐xenografted NSG mice. Most importantly, MLT reduced the infiltration of leukaemia blasts, decreased the frequency of leukaemia stem cells (LSCs) and prolonged the overall survival in AML1‐ETO‐induced murine leukaemia. Mechanistically, MLT increased the expression of miR‐193a, which inhibited AML1‐ETO expression via targeting its putative binding sites. Furthermore, MLT decreased the expression of β‐catenin, which is required for the self‐renewal of LSC and is the downstream of AML1‐ETO. Thus, MLT presents anti‐self‐renewal of LSC through miR‐193a‐AML1‐ETO‐β‐catenin axis. In conclusion, MLT might be a potential treatment for t (8;21) leukaemia by targeting AML1‐ETO oncoprotein.     

Characterization of age‐associated exhausted CD8+ T cells defined by increased expression of Tim‐3 and PD‐1

Aging is accompanied by altered T‐cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD‐1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T‐cell exhaustion and aging‐associated immune dysfunction. In this study, we demonstrate that T‐cell immunoglobulin mucin domain‐3 (Tim‐3), another exhaustion marker, is up‐regulated on aged T cells, especially CD8⁺ T cells. Tim‐3‐expressing cells also produced PD‐1, but Tim‐3⁺PD‐1⁺ CD8⁺ T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim‐3^?PD‐1⁺ cells. Tim‐3⁺PD‐1⁺ CD8⁺ T cells showed more evident properties associated with exhaustion than Tim‐3^?PD‐1⁺ CD8⁺ T cells: an exhaustion‐related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL‐10 and induced normal CD8⁺ T cells to produce IL‐10, which might contribute to immune dysregulation in aged mice. The generation of Tim‐3‐expressing CD8⁺ T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8⁺ T‐cell population with age‐associated exhaustion was distinguishable by its expression of Tim‐3. These results provide clues for understanding the alterations that occur in T‐cell populations with age and for improving dysfunctions related to the aging of the immune system.     

HMQ‐T‐F2 exert antitumour effects by upregulation of Axin in human cervical HeLa cells

Looking for novel, effective and less toxic therapies for cervical cancer is of significant importance. In this study, we reported that HMQ‐T‐F2(F2) significantly inhibited cell proliferation and transplantable tumour growth. Mechanistically, HMQ‐T‐F2 inhibited HeLa cell growth through repressing the expression and nuclear translocation of β‐catenin, enhancing Axin expression, as well as downregulating the Wnt downstream targeted proteins. Knock‐down of a checkpoint β‐catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of β‐catenin, was used to treat HeLa cells and the results demonstrated that HMQ‐T‐F2 inhibited proliferation and migration via the inhibition of the Wnt/β‐catenin pathway.     

Building Lithiophilic Ion‐Conduction Highways on Garnet‐Type Solid‐State Li+ Conductors

The integration of highly conductive solid‐state electrolytes (SSEs) into solid‐state cells is still a challenge mainly due to the high impedance existing at the electrolyte/electrode interface. Although solid‐state garnet‐based batteries have been successfully assembled with the assistance of an intermediate layer between the garnet and the Li metal anode, the slow discharging/charging rates of the batteries inhibits practical applications, which require much higher power densities. Here, a crystalline sulfonated‐covalent organic framework (COF) thin layer is grown on the garnet surface via a simple solution process. It not only significantly improves the lithiophilicity of garnet electrolytes via the lithiation of the COF layer with molten Li, but also creates effective Li⁺ diffusion “highways” between the garnet and the Li metal anode. As a result, the interfacial impedance of symmetric solid‐state Li cells is significantly decreased and the cells can be operated at high current densities up to 3 mA cm^?2, which is difficult to achieve with current interfacial modification technologies for SSEs. The solid‐state Li‐ion batteries using LiFePO₄ cathodes, Li anodes, and COF‐modified garnet electrolytes thus exhibit a significantly improved rate capability.     

Targeted adenoviral vectors

Replication-defective vectors based on human adenovirus serotypes 2 and 5 (Ad2 and Ad5) possess a number of attributes which favor their use as gene delivery vehicles in gene therapy applications. However, the widespread distribution of the primary cellular receptor for Ad, the coxsackievirus and adenovirus receptor (CAR), allows Ad vectors to infect a broad range of cells in the host. Conversely, a number of tissues which represent important targets for gene therapy, such as the airway epithelium and cancer cells, are refractory to Ad infection due a paucity of CAR. Thus, there is a strong rationale for the development of CAR-independent Ad vectors capable of enhanced specificity and efficiency of gene transfer to target cells. In this article we review the approaches which have been employed to generate tropism-modified Ad vectors. These targeting strategies have led to improvements in the safety and efficacy of Ad vectors and have the potential to yield an increased therapeutic benefit in the human clinical context.     

A stable meta‐carborane enables the generation of boron‐rich peptide agonists targeting the ghrelin receptor

Boron neutron capture therapy (BNCT) is a binary cancer therapy, which combines the biochemical targeting of a boron‐containing drug with the regional localization of radiation treatment. Although the concept of BNCT has been known for decades, the selective delivery of boron into tumor cells remains challenging. G protein‐coupled receptors that are overexpressed on cancer cells in combination with peptidic ligands can be potentially used as shuttle system for a tumor‐directed boron uptake. In this study, we present the generation of short, boron‐rich peptide conjugates that target the ghrelin receptor. Expression of the ghrelin receptor on various cancer cells makes it a viable target for BNCT. We designed a novel hexapeptide super‐agonist that was modified with different specifically synthesized carborane monoclusters and tested for ghrelin receptor activation. A meta‐carborane building block with a mercaptoacetic acid linker was found to be optimal for peptide modification, owing to its chemical stability and a suitable activation efficacy of the conjugate. The versatility of this carborane for the development of peptidic boron delivery agents was further demonstrated by the generation of highly potent, boron‐loaded conjugates using the backbone of the known ghrelin receptor ligands growth hormone releasing peptide 6 and Ipamorelin.