## Sustained and targeted delivery of checkpoint inhibitors by metal-organic frameworks for cancer immunotherapy

• January 22, 2021

## Abstract

The major impediments to the implementation of cancer immunotherapies are the sustained immune effect and the targeted delivery of these therapeutics, as they have life-threatening adverse effects. In this work, biomimetic metal-organic frameworks [zeolitic imidazolate frameworks (ZIFs)] are used for the controlled delivery of nivolumab (NV), a monoclonal antibody checkpoint inhibitor that was U.S. Food and Drug Administration–approved back in 2014. The sustained release behavior of NV-ZIF has shown a higher efficacy than the naked NV to activate T cells in hematological malignancies. The system was further modified by coating NV-ZIF with cancer cell membrane to enable tumor-specific targeted delivery while treating solid tumors. We envisage that such a biocompatible and biodegradable immunotherapeutic delivery system may promote the development and the translation of hybrid superstructures into smart and personalized delivery platforms.

## INTRODUCTION

Over the past decade, metal-organic frameworks (MOFs) have been actively used as intricately engineered platforms for biomedical applications (1719). Zeolitic imidazolate frameworks (ZIF-8)—a subclass of MOFs—are crystalline solids based on Zn2+ ion subunits coordinated to organic 2-methylimidazole (mIM) ligands, resulting in the formation of highly porous structures (20). ZIF-8 has recently emerged as a potential candidate for on-demand drug delivery applications due to its biocompatibility, remarkable LC, superior stability under physiological conditions (no premature drug release), pH responsiveness, and tunable drug release properties (21, 22). In comparison to other delivery vehicles, ZIFs can encapsulate different size and charge therapeutic hosts with a high LC reproducibly and deliver them on demand. Therefore, they were used in the delivery of current breakthrough proteins such as small interfering RNA (23), CRISPR-Cas9 ribonucleoprotein (24, 25), and catalytic enzymes (26, 27). Here, we developed an efficient strategy for the sustained release and high loading of the PD-1 inhibitor, nivolumab (NV), using ZIF-8 (NV-ZIF) with the capability of working on both hematological malignancies and solid tumors (Fig. 1). Targeted delivery, in the case of solid tumors, was achieved by using cell-specific membrane coating (CC), as this technique has proved viable for enhancing targeted cancer therapy (25, 28, 29).

## RESULTS

### Design and characterization of NV-ZIF

In a typical experiment for the biomimetically mineralized growth of ZIF-8, an aqueous solution containing mIM (2.5 M, 0.9 ml) and NV (1 mg·ml−1) was mixed with a separate aqueous solution of zinc (Zn) nitrate (0.5 M, 0.1 ml) at room temperature for 20 min. The solution immediately turned opaque, indicating crystal formation. Cryogenic transmission electron microscopy (cryo-TEM) and TEM micrographs clearly illustrated octahedral crystals with an average diameter ranging between 102 and 160 nm (Fig. 2A). The energy-dispersive x-ray spectroscopy elemental mapping revealed a uniform distribution of ZIF-8–associated elements, Zn, nitrogen (N), and carbon (C); and NV-associated elements, oxygen (O) and sulfur (S) (Fig. 2B).

Our powder x-ray diffraction (PXRD) results demonstrate that the embedded NV did not result in any change in ZIF-8 crystallinity (Fig. 3A), which is consistent with other reported MOF-based protein carriers (18, 26, 30). The LC and loading efficiency (LE) were then evaluated using the Bradford assay. The LC and LE were found to be 5.07 ± 1% and 80 ± 3%, respectively. NV content in NV-ZIF was also estimated by thermogravimetric analysis (TGA), and the results were comparable to those obtained by the Bradford assay (fig. S1, A and B). Comparing the thermogram of NV-ZIF with that of ZIF-8 gives information about the formulation’s composition. The TGA spectrum of the NV-ZIF showed a weight loss of 6.1% between 10° and 150°C due to the loss of the adsorbed moisture. The 23% loss observed between 250° and 444.6°C can be attributed to the pyrolysis of the carboxyl or hydroxyl groups, which most probably originated from the NV decomposition. The final range of temperature from 320° to 600°C resulted in an obvious mass loss of 52.9%, which we assigned to the removal of the organic linker molecules and the collapse of ZIF-8. The interaction between ZIF-8 and NV delayed the pyrolysis process of NV that is coved by the in situ growth of ZIF-8. Unlike NV-ZIF, ZIF-8 showed a weight loss of 3.7% between 10° and 150°C, 21.6% between 250° and 444.6°C, and 47.89% from 320° to 600°C. The 23% weight loss between 250° and 444°C supports the presence of the NV. On the basis of TGA analysis, we carried out a calcination process at 320°C for 2 hours to confirm that NV is majorly embedded at the surface of the framework, as previously reported for protein-embedded MOFs (31). The TEM image of NV-ZIF after calcination supports the existence of small cavities (fig. S1C) resulting from the removal of NV molecules and their aggregates. The ultraviolet–visible–near-infrared (IR) (UV-Vis-NIR) spectrum of NV-ZIF clearly showed the absorbance band of NV at 280 nm. The embedded NV resulted in reducing its symmetry; therefore, a red shift was observed (fig. S1D) as previously reported (32). The embedded NV was also confirmed by Fourier transform IR (FT-IR) spectrum through the absence of the vibrational band at 1660 cm−1, characteristic of COO group of NV (fig. S1E). We further investigated the possible coordination of NV and Zn2+. Therefore, different ratios of NV and Zn2+ (0.1:1 and 1:1) were stirred for 20 min at room temperature. Zn nitrate showed an absorbance band at 300 nm according to our UV spectrum (fig. S2A). When NV was mixed with Zn at different ratios, an absorbance peak at 280 nm appeared for NV only, indicating that no coordination occurred between Zn2+ and NV. The increase in the absorbance of NV is attributed to the increase in NV concentration. The FT-IR analysis showed no shift in the vibrational band at 1700 cm−1 for the COOH and 3400 cm−1 for the N─H, supporting no coordination with Zn2+ and validating the importance of mIM in the NV-ZIF formation (fig. S2B).

To monitor the loading and release, we labeled NV with rhodamine B, Rh (RNV) (fig. S3). By measuring the Rh fluorescence intensity, the majority of NV was embedded in ZIF-8 (fig. S3, A and B). Moreover, the sustained release of RNV from NV-ZIF at varying pH values in phosphate-buffered saline (PBS) was monitored by fluorescence spectroscopy (Fig. 3, B and C). NV-ZIF exhibited a slow sustained release at acidic pH (6.5), and approximately 50% of RNV was released within 12 hours. More stable release of small dosages of encapsulated RNV was observed after 24 hours, reaching more than 70% of RNV release from ZIF-8 (Fig. 3B and fig. S3D). Scanning electron microscopy images of the release process show a gradual dissociation of NV-ZIF at acidic conditions over time. NV-ZIF morphology remarkably changed after 12 hours, extrapolating the 50% release of RNV from ZIF-8 (fig. S3E). In contrast, less than 25% of RNV was released over 3 days under physiological conditions at pH 7.3, and the system exhibited an excellent colloidal stability for over 6 months (Fig. 3B and fig. S3C). Such slow and controlled release behavior is intended to improve treatment outcomes. Next, we evaluated the in vitro cytotoxicity of NV, ZIF-8, and NV-ZIF by cell counting kit-8 (CCK-8) against human embryonic kidney (HEK), HeLa, and Michigan Cancer Foundation-7 (MCF-7). As expected, no obvious cytotoxicity was observed, supporting NV-ZIF biocompatibility (fig. S4, A to C). Our system exhibited excellent NV sustained release performances and triggered pH responsiveness, which is consistent with previously reported drug-loaded ZIF-8 delivery systems (30, 32).

### Expression of PD-1 and in vitro pH-responsive NV-ZIF

The expression of PD-1 was first examined on Jurkat T cells that were either activated with anti-CD3/anti-CD28 antibodies or left unactivated. Flow cytometric analysis demonstrated that the expression of PD-1 on activated Jurkat T cells (aTCs; >80%, P < 0.005) was significantly higher than that on unactivated Jurkat T cells (10%, P < 0.005) (fig. S5, A and B). To assess NV activity and integrity after loading, we extracted NV from ZIF-8 by adding EDTA to dissociate the ZIF-8 crystals by breaking the coordination bonds between Zn2+ and 2-mIM. The released NV was then incubated with aTC for 30 min and labeled with a secondary phycoerythrin (PE)–labeled anti-human antibody. The fluorescence intensity of the extracted NV shifted to the right; the same shift was observed with free NV–treated aTC that was used as a control (fig. S5C). The same result was obtained when the residual NV in the supernatant was used, confirming that embedding NV in the framework did not affect the activity and the integrity of this antibody. Subsequently, the release of NV from NV-ZIF was tested in vitro by incubating NV-ZIF with aTC for 6 and 12 hours at slightly acidic (pH 6.5) and physiological (pH 7.3) conditions. The PD-1/anti–PD-1 (NV) interaction was detected by staining aTC against PE-labeled anti-human antibody. No obvious difference was detected when aTC was incubated with NV-ZIF for 6 hours (17.3%). Twelve hours post incubation induced the release of NV (fig. S6). A marked increase was observed at pH 6.5 (66.3%) (fig. S6B). On the other hand, treating aTC with free NV for 30 min showed a rapid binding of anti–PD-1 (NV) with PD-1 on aTC (47.7%) (fig. S6A). Such treatment profile is consistent with the one currently used in the clinics, which is associated with increased levels of toxicity (33). Administering the same dose but at a slower rate would help to avoid adverse reactions. Hence, the slow and sustained release behavior of NV-ZIF will help in reducing immune-related life-threatening events associated with free NV delivery.

### Efficacy of NV sustained release on hematological malignancies

Lymphocytes of patients with acute myeloid leukemia (AML) and chronic lymphoid leukemia (CLL) are known to express high levels of PD-1 (34, 35), while PD-L1 was shown to be up-regulated on cancerous cells and antigen-presenting cells (APCs) from these patients (3638). Peripheral blood mononuclear cells (PBMCs) isolated from patients with AML and CLL were initially treated with ZIF-8 to test its effect on T cell proliferation (CD8+ and CD4+) using Ki-67 as a marker for cell proliferation. Our data demonstrated that CD8+ and CD4+ T cells treated with phytohemagglutinin (PHA) resulted in high Ki-67 expression compared to ZIF-8 and the control (fig. S7, A to C), indicating that ZIF-8 has no effect on lymphocyte proliferation. Next, we treated CD8+ T cells with NV-ZIF for 6 and 12 hours. Compared to free NV, NV-ZIF boosted the activity of PHA-stimulated T cells over time. As shown in Figs. 4 and 5, contrary to ZIF-8, NV-ZIF enhanced the activation of CD8+ T cells compared to nontreated cells as evidenced by the higher levels of CD8+ interferon-γ (IFN-γ) and CD8+ tumor necrosis factor–α (TNF-α) T cells. NV-ZIF increased the level of CD8+ IFN-γ and CD8+ TNF-α T cells 12 hours following treatment (Figs. 4 and 5). This is mostly due to the sustained release of NV from the NV-ZIF over time. The level of activation of those cells at 12 hours was either slightly higher or comparable to cells treated with free NV (Figs. 4 and 5). Although we expected that there is higher cytokine release at 12 hours compared to 6 hours, the comparably high levels of cytokines observed for cells treated with NV-ZIF and NV suggest that there was a sufficient release of NV from the NV-ZIF.

### Cell type–specific delivery of NV

Attending to the tumor microenvironment (TME) is crucial when developing therapies for solid tumors. We modified the NV-ZIF to specifically deliver and release NV into the TME, enabling local activation of tumor-specific immune responses and reducing systemic toxicity associated with NV administration. CC membrane was used as a targeting agent in our study, which provided a personalized tumor-specific PD-1 blockade therapy. We previously validated that coating ZIF-8 with CC resulted in preferential accumulation of coated ZIF-8 within CCs from which the membrane was extracted (25). The same protocol was followed for coating NV-ZIF with MCF-7 membrane. TEM micrographs of MCF membrane–coated NV-ZIF (NV-ZIFMCF) showed an octagonal crystal with an average size of 166 nm, and the negative staining of NV-ZIFMCF exhibited rough surface after coating (fig. S8A). The PXRD patterns and intensity of NV-ZIFMCF are similar to those of the NV-ZIF and ZIF-8, which supports that the ZIF-8 crystallinity was maintained after coating (fig. S8B). Zeta potential measurements validated the complexation with CC, as the charge of the NV-ZIF dropped from +11 to −21 mV (fig. S8C). The successful functionalization was further confirmed by SDS–polyacrylamide gel electrophoresis (PAGE), followed by protein staining (fig. S9). The protein profile of the purified CC matches that of the NV-ZIFMCF (fig. S9A), indicating a good retention of the characteristic proteins inherited from the CC. Surface adhesion molecules such as CD44, E-cadherin, and CD49e were detected on CC and NV-ZIFMCF by Western blot (fig. S9B). Biocompatibility of NV-ZIFMCF was tested at different concentrations by incubation with MCF-7 for 24 hours using CCK-8. Compared to the native MCF-7, concentrations below 100 μg·ml−1 were completely safe, whereas high concentrations (100 μg·ml−1) led to a low level of cytotoxicity (fig. S10). To verify the cancer-targeting ability of NV-ZIFMCF, we incubated NV-ZIFMCF with HeLa, HEK, and MCF-7. The results indicated that NV-ZIFMCF accumulated in MCF-7 tumors and that the accumulation steadily increased with longer incubation times (fig. S11, A to C), which is consistent with our previous study (25). The targeted delivery and preferential accumulation of NV-ZIFMCF were further evaluated using homologous 4T1 cancer-bearing mice in vivo. XenoLight DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide) was loaded with NV-ZIF for in vivo imaging purposes. Mice-bearing 4T1 tumors were imaged at different time points (3 and 24 hours) following injection of ZIF particles using In Vivo Imaging System (IVIS). Our data revealed that NV-ZIFMCF exhibited a high accumulation in tumors within 3 hours with prolonged tumor retention. Unlike NV-ZIFMCF, the accumulation of NV-ZIF (uncoated) was low and detected after 24 hours, supporting the efficiency of this targeting strategy (Fig. 6A). Measuring Zn2+ content of the tumor by inductively coupled plasma mass spectrometry (ICP-MS) showed a significant increase in the accumulation of NV-ZIFMCF at tumor site compared to that of NV-ZIF (Fig. 6B). Unlike most developed delivery systems that targeted superficial TME, such as melanoma (39, 40), our engineered NV-ZIFMCF efficiently targets TME inside the body. This highly specific cancer recognition ability of NV-ZIFMCF can extensively enhance the therapeutic effect of NV, as the platform showed negligible toxicity to the animals as verified by the control samples.

### Efficacy of NV sustained release on solid tumors

We injected mice with either NV-ZIFMCF, NV-ZIF, CC-NV, or NV (3 mg·kg−1 per mouse for each injection) on days 3, 6, 9, and 12. The mice were then sacrificed on day 21, and tumor sizes were measured. Results in Fig. 6 (C to F) indicate that the NV-ZIFMCF treatment significantly inhibited tumor growth compared to NV-ZIF, CC-NV, or NV treatment alone. To further test the treatment effect, we observed tumor development over 21 days after various treatments and found that the antitumor activity and survival rate were significantly extended (tumor volume maintained <200 mm3, P < 0.01) after the NV-ZIFMCF treatment (Fig. 6, C to F). The survival time was slightly extended from 42 days for untreated mice to 45 and 49 days for NV-ZIF– and NVMCF-treated mice, respectively (Fig. 6F). Free NV did not show superior tumor survival compared to NV-ZIF and NVMCF, indicating the necessity of efficient NV delivery for effective tumor inhibition. In contrast, NV-ZIFMCF significantly prolonged animal survival (Fig. 6F). Mice treated with NV-ZIFMCF also showed a significantly higher production of IFN-γ and TNF-α (P < 0.001) compared to other treated groups that showed comparable levels of production (Fig. 6G). We measured the mice body weight, and as expected, experimental and control mice did not show an obvious difference in body weight (fig. S12).

Next, we characterized CC proliferation by histological assays. CC damage was detected by hematoxylin and eosin (H&E) staining upon treatment with NV-ZIFMCF (Fig. 7A). The Ki-67 staining results revealed marked reduction in Ki-67 levels after NV-ZIFMCF treatment, resulting in significant inhibition of the proliferation of tumor cells compared to other treatments (Fig. 7B). Of a particular note, functionalizing NV with the targeting agent (CC-NV) did not result in equivalent antitumor activity to that of NV-ZIFMCF. Likewise, NV-ZIF did not exhibit antitumor activity similar to that of NV-ZIFMCF, which supports insufficient NV delivery to TME in both cases. The cancer inhibition ratio reached about 73% after treatment with NV-ZIFMCF, indicating that this strategy made TME more sensitive to immunotherapy. To gain a better understanding of the effects of each treatment regimen on lymphocytes present in the TME, we analyzed the population of FoxP3+ regulatory T cells. These FoxP3+ regulatory T cells act to suppress immune responses and, in this case, antitumor immune responses. T cells from tumor tissues were harvested and analyzed by flow cytometry to determine the percentage of regulatory T cells within the total T cell population in the tumor. The percentage of FoxP3+ cells significantly decreased in NV-ZIFMCF–treated groups compared to the control and ZIF-8, whereas NV-, CC-NV–, and NV-ZIF–treated groups showed some level of reduction (Fig. 7C), supporting the enhanced production of IFN-γ and TNF-α in groups treated with NV-ZIFMCF. These results strongly confirm the anticancer efficacy of NV-ZIFMCF enabled by the inhibition of the regulatory T cells. Collectively, the antitumor activity of CC-NV was comparable to that of NV-ZIF, indicating that sufficient delivery of NV to TME is the key for enhanced NV-ZIFMCF antitumor activity. An extrapolation of these results suggests that tumor-specific delivery of NV results in (i) enriching NV within TME, leading to local inhibition of PD-1; (ii) enhancing the sensitivity of TME to anti–PD-1 blockade therapy; and (iii) systemically activating specific antitumor immune response enabled by the local inhibition of the regulatory T cells.

## DISCUSSION

ICB therapy has shown encouraging preclinical and clinical results to treat different types of tumors. Current delivery methods, however, are not antigen specific and result in the systemic blocking of regulatory pathways, leading to systemic activation of immune cells and limiting therapeutic benefits in many patients. Consequently, there is a tremendous need to improve the safety and efficacy of such treatments. Here, we demonstrated the therapeutic potential of the sustained release and targeted delivery of NV by NV-ZIF and NV-ZIFMCF on both hematological malignancies and solid tumors, respectively.

Hematological malignancies, such as leukemia, involve continuous and systemic contact between the tumor clone and the immune system. Our NV-ZIF release behavior showed a slow release of small doses of NV over time that resulted in improving the antitumor activity with longer incubation by inducing T cell activation to a level comparable to that of free NV. This kind of release behavior is expected to reduce immune-related toxicity and increase patient compliance.

On the other hand, solid tumors are characterized by confining infiltrating lymphocytes in localized tissue. The suppressive nature of TME induces the irresponsiveness to PD-1 blockade therapy. Thus, we coated our NV-ZIF with CC to enable tumor-specific recognition, reducing off-target delivery and immune-related side effects, improving the sensitivity of TME to NV, extending the retention of NV-ZIF within tumor, and eliciting tumor-specific immunity. We used the challenging 4T1 mouse breast cancer model to demonstrate the therapeutic potential of NV-ZIFMCF. Our results showed the superior antitumor activity of NV-ZIFMCF over NV, CC-NV, and NV-ZIF. Of a particular note, functionalizing NV with the targeting agent (CC-NV) did not result in equivalent antitumor activity to that of NV-ZIFMCF. Likewise, NV-ZIF did not exhibit antitumor activity similar to that of NV-ZIFMCF, which supports insufficient NV delivery to TME in both cases. The cancer inhibition ratio reached about 73% after treatment with NV-ZIFMCF, indicating that this treatment regimen made TME more sensitive to immunotherapy. Such treatment could be followed by chemotherapy or radiotherapy to completely eradicate tumor. CC coating could be used as a promising strategy to develop a personalized tumor-specific immune response as shown in previous studies (28). Our strategy has shown that local delivery of NV-ZIFMCF leads to systemic and durable activation of antitumor immune response that has the potential to reduce the risk of metastasis. Unlike most developed delivery systems that targeted superficial TME, such as melanoma (39, 40), our developed NV-ZIFMCF was efficient in targeting TME inside the body. Our strategy shows a great clinical translation potential in patients with both hematological malignancies and solid tumors because all the materials used in this system are biocompatible, and it would be a step toward developing personalized immune therapeutics treatment plans.

In summary, we have successfully loaded NV in ZIF-8 and demonstrated the potential utility of the sustained NV release in hematological malignancies and solid tumors. The sustained release behavior of NV-ZIF has shown its efficacy in activating T cells in AML and CLL. The system was further modified to enable tumor-specific targeted delivery while treating solid tumors by coating NV-ZIF with specific CC membrane. NV-ZIFMCF displayed an enhanced antitumor activity due to the preferential accumulation and prolonged retention of NV-ZIFMCF within TME that resulted in efficient NV delivery. Collectively, this work demonstrates that tackling the sustained and targeted delivery is the way forward for the broader impact of ICB therapy in the fight against cancer.

## MATERIALS AND METHODS

### Fabrication of ZIF-8, NV-ZIF, and NV-ZIFMCF

NV-ZIF was synthesized by stirring NV (1 mg·ml−1) and 2-mIM (2.5 M, 0.9 ml) for 30 min. Zinc nitrate solution (0.5 M, 0.1 ml) was slowly added under mechanical agitation for 20 min. The resulting product was collected by centrifugation and washed three times with deionized water to remove any residues. ZIF-8 was synthesized by slowly adding zinc nitrate solution (0.5 M, 0.1 ml) to 2-mIM (2.5 M, 0.9 ml). The solution was agitated for 20 min. The supernatant of NV-ZIF was collected to calculate the LC and LE by Bradford assay. NV-ZIFMCF was fabricated by mixing 1:1 weight ratio of NV-ZIF and extracted CC membrane in deionized water. The mixture was then transferred into a syringe and successively extruded through 1.0-μm and 800.0- and 450.0-nm polycarbonate membrane. The obtained NV-ZIFMCF in solution was further purified by centrifugation to remove the free CC membrane. The zeta potential of NV-ZIF was performed using a Malvern Zetasizer Nano ZS at 25°C at pH 7.3 in aqueous solutions. PXRD measurements were performed using a Panalytical X’Pert Pro X-ray powder diffractometer using the Cu Kα radiation (40 V, 40 mA, λ = 1.54056 Å) in a θ – θ mode from 20° to 90° (2θ). TEM images were obtained using FEI Tecnai 12 microscope operating at 120 kV. For visualization by TEM, samples were prepared by dropping the solution on a copper grid 300 mesh (Electron Microscopy Sciences, LC 300-Cu). Fluorescence measurements were performed on a Cary Eclipse fluorescence spectrophotometer (Varian). The slits for excitation and emission were set at 10 nm. NV was labeled with rhodamine B (Rh) by N-hydroxysuccinimide (NHS) chemistry. Briefly, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (5 mg) and NHS (2.5 mg) were mixed with NV solution (10 mg·ml−1, 1 ml), and the mixed solution was stirred for 2 hours. Rh (63 μg) was then dissolved in dimethyl sulfoxide (100 μl), and the whole solution was stirred overnight at 4°C in the dark. The dialysis technique was used to remove unreacted EDC, NHS, and Rh.

### Preparation of CC membrane

Human breast adenocarcinoma cell (MCF-7) cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin). Cells were grown in T-175 culture flasks to full confluency and then detached and washed in PBS three times by centrifuging at 500g. Then, they were suspended in a hypotonic lysing buffer consisting of 20 mM tris-HCl (pH 7.5), 10 mM KCl, 2 mM MgCl2, and 1 EDTA-free mini protease inhibitor tablet per 10 ml of solution and disrupted using a Dounce homogenizer with a tight-fitting pestle. The entire solution was subjected to 20 passes before spinning down at 3200g for 5 min. The supernatant was saved, while the pellet was resuspended in hypotonic lysing buffer and subjected to another 20 passes and spun down again. The supernatants were pooled and centrifuged at 20,000g for 20 min, after which the pellet was discarded, and the supernatant was centrifuged again at 100,000g. The pellet containing the plasma membrane material was then washed again in 10 mM tris-HCl (pH 7.5) and 1 mM EDTA. The final pellet was collected and used as a purified CC membrane.

### CC membrane protein characterization

Protein characterization was carried out using the SDS-PAGE method. The cracked CC membrane samples were suspended in lithium dodecyl sulfate loading buffer (Invitrogen). Samples were heated to 90°C for 10 min, and 20 μl of sample was loaded into each well of a NuPAGE Novex 4 to 12% bis-tris minigel, using Mops SDS as the running buffer (Invitrogen) in an XCell SureLock Electrophoresis System based on the manufacturer’s instructions. Protein staining was accomplished using Coomassie Blue (Invitrogen) and destained in water overnight before imaging. For Western blot analysis, the protein was transferred to Protran nitrocellulose membranes (Whatman) using an XCell II Blot Module (Invitrogen) in NuPAGE transfer buffer (Invitrogen) per the manufacturer’s instructions. Membranes were probed using antibodies against CD44 (clone 515; BD Biosciences), E-cadherin (clone 36; BD Biosciences), and CD49e (BD Biosciences), followed by horseradish peroxidase–conjugated anti-mouse immunoglobulin G (Cell Signaling Technology) as the secondary antibody.

### LC and LE NV-ZIF

The LC and LE of NV in ZIF-8 nanoparticles were measured with the Bradford method. First, a standard curve of NV at 595 nm was generated. Then, LE and LC of NV in ZIF-8 were obtained by analyzing residual NV in supernatants, which was collected after washing. The LE was calculated as follows

$LC=[mass loaded drug/mass of loaded drug+NPs]×100$

$LE=[mass of drug loaded/mass of initial drug]×100$

### Release of Rh-labeled NV via pH trigger

To evaluate the release of Rh-labeled NV from ZIF-8, the fluorescence signal of Rh-labeled NV was measured by using the microplate spectrophotometer. Aliquots of hydrochloric acid were added to Rh-labeled NV-ZIF (600 μg·ml−1) in PBS to reach a pH of 6.5 at 37°C. PBS only was added to the sample of pH 7.3. The supernatant of the mixture solution was obtained through centrifugation at different time points. The fluorescence of released Rh-labeled NV was monitored by fluorescent spectroscopy (excitation/emission wavelength: 540 nm/625 nm).

### In vitro release and Jurkat activation

Anti-CD3 (mouse anti-human CD3, clone: OKT3; eBioscience) was added in a 24-well plate at a final concentration of 5 μg·ml−1 prepared in PBS (300 μl per well) and incubated for 3 hours in a 37°C incubator supplied with 5% CO2. After incubation, antibody solution was removed from each well. In the same well, 106 cells·ml−1 of Jurkat cells (acute T cell leukemia human, Jurkat, clone E6-1) were resuspended in 2 ml of RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco) and 1% streptomycin (Hyclone). Anti-CD28 (purified NA/LE as described in BD mouse anti-human CD28, BD) was added at a final concentration of 1 μg·ml−1 to each well. Last, interleukin-2 was added at a final concentration of 100 U·ml−1. The cells were then incubated in a 37°C humidified incubator supplied with 5% CO2 for 3 days.

### Cell viability

CCK-8 assay was performed according to the manufacturer’s protocol. Briefly, MCF-7, HEK, and HeLa cells (5 × 103 cells per well) were seeded onto a 96-well plate. After 12 hours, the culture medium was changed, and the cells were incubated with different concentrations (100, 50, 25, 12, 6, and 3 μg·ml−1) of NV, NV-ZIF, and ZIF-8 in 200 μl of DMEM at 37°C for 24 hours. The media was then discarded, and the prepared culture medium containing 10% CCK-8 solution was added into each well, including a negative control of culture media alone. After 3 hours of incubation, the absorbance was measured at 450 nm using a microplate spectrophotometer (xMark Microplate Absorbance Spectrophotometer).

### Specific targeting studies

Flow cytometric assay was used to investigate the specific targeting ability of NV-ZIFMCF. Rh–NV–ZIFMCF was used to track the uptake of the NV-ZIFMCF. Cells were seeded in six-well plates at a density of 5 × 105 cells per single well and cultured for 12 hours in 2 ml of DMEM containing 10% FBS and 1% antibiotics (penicillin-streptomycin). After NV-ZIFMCF (100 μg·ml−1) was co-incubated with the cells for 1, 3, 6, 12, and 24 hours, the cells were washed three times with PBS, detached by trypsin, and lastly collected by centrifugation at 1000 rpm for 5 min. The bottom cells were washed three times with PBS, and then the suspended cells were analyzed by BD LSR II Flow Cytometer equipped with BD FACSDiva (BD Biosciences) software.

### Treating stimulated inflammatory cells with NV, ZIF-8, and NV-ZIF

PBMCs were isolated from blood samples of two patients, one with AML and another with CLL, using a Ficoll gradient (Axis Shield, Norway). Cells were collected in complete RPMI 1640 medium (pH 6.5). PBMCs (106 cells) were then divided into five sets for each treatment and incubated with either ZIF-8 (5 μg·ml−1), NV (10 μg·ml−1), or NV-ZIF (5 μg·ml−1) for 1 hour. Cells were then stimulated with PHA (100 ng·ml−1) for 6 or 12 hours (the last 2 hours in the presence of brefeldin A). Intracellular cytokine staining was performed to determine the ability of CD8+ cells to express cytokines. The cells were surface stained with CD3+ APC (0.2 μg·μl−1; R&D Systems, Minneapolis, MN, USA). They were then fixed in 4% paraformaldehyde, resuspended in 0.25% saponin (S4521; Sigma-Aldrich, Germany), and stained with anti–IFN-γ PE-Cy7 [PE-Cy7 mouse anti-human IFN-γ (BD Biosciences), 0.2 μg·μl−1] and anti–TNF-α–PE-Cy7 [PE-Cy7 mouse anti-human TNF-α (BD Biosciences), 0.2 μg·μl−1] antibodies. Samples were analyzed using a BD LSR II flow cytometer equipped with BD FACSDiva (BD Biosciences) software.

### Animals and tumor models

All animal experiments were carried out in accordance with the Institute of Laboratory Animal Resources guidelines. Ethical approval was granted by the Institutional Animal Care and Use Committee of Zhejiang Academy of Medical Sciences, China.

Female BALB/c mice (4 weeks old, ~20-g body weight) were purchased from the Zhejiang Academy of Medical Sciences and maintained in a pathogen-free environment under controlled temperature (24°C). A total of 0.1 ml of 4T1 cells (5 × 105) was injected into the breast fat pad of the mice. The tumors were allowed to grow to ~100 mm3 before experimentation.

The tumor volume was calculated as (tumor length) × (tumor width)2/2.

For tumor accumulation studies, the mice were randomly divided into two groups (n = 3) and intravenously injected with XenoLight DiR–NV–ZIF or Rh–NV–ZIFMCF [corresponding to NV (3.0 mg·kg−1)]. The fluorescent images were obtained under an IVIS (CRi USA, IVIS: 710 excitation/760 emission). The tumor samples were then collected at the desired time after injection and were digested using concentrated nitric acid. The amount of Zn2+ in the tumors was measured using ICP-MS.

For antitumor activity study, 4T1 tumor-bearing mice were randomly divided into six groups (n = 5) and intravenously injected with (i) 200 μl of physiological saline, (ii) 200 μl of ZIF-8 solution, (iii) 200 μl of NV-ZIF solution, (iv) 200 μl of NV-ZIFMCF solution, (v) 200 μl of NV solution, and (vi) 200 μl of NVMCF solution, respectively. The dosage of NV is 3.0 mg·kg−1. Mice received treatment four times every 3 days. Physiological saline that is used for in vivo application is 1× PBS (0.01 M). Tumor volume and body weight were measured every 3 days. In the histological assay, the tumor tissues were fixed in 4% paraformaldehyde for 24 hours. The specimens were dehydrated in graded ethanol, embedded in paraffin, and cut into 5-mm-thick sections. The fixed sections were deparaffinized and hydrated according to a standard protocol and stained with H&E for microscopic observation. Tumor sections were also stained with antibody against Ki-67 (Abcam, USA) to visualize viable CCs.

Blood samples (0.1 ml) were taken from retro-orbital sinus to isolate serum for analysis, 48 hours after single injection. TNF-α (MTA00B; R&D systems) and IFN-γ (MIF00; R&D systems) were analyzed with enzyme-linked immunosorbent assay kits according to the vendors’ protocols.

To study the immune cells inside tumors, tumors were harvested from mice in different groups and cut into small pieces. After being ground by the rubber end of a syringe in cell strainers, tumor tissues were treated with 0.25% trypsin-EDTA solution for 5 min at 37°C. Then, cells were filtered through nylon mesh filters with a size of 70 μm and washed with PBS. The single-cell suspension was then incubated with anti-CD16/32 (BD Pharmingen; catalog: 553141) to reduce nonspecific binding to the fragment crystallizable region (Fc receptor). Cells were further stained with anti-mouse FOXP3 (eBioscience; catalog: 12-5773-82) antibodies according to the manufacturer’s protocols. Last, flow cytometry was used for cell sorting.

### Statistical analysis

Data are reported as means ± SD. The differences among groups were determined using one- or two-way analysis of variance (ANOVA) analysis. Statistical significance was calculated by one- or two-way ANOVA and Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Acknowledgments: We thank R. Langer, Institute Professor, MIT, for feedback and comments. We acknowledge H. Alrabiah, Associate Professor at the Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University and H. I. Aljohar, Assistant Professor at the Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University for providing nivolumab. Funding: This work was funded by the King Abdulaziz City for Science and Technology (KACST) through the MERS-CoV research grant program (number 20-0004), which is a part of the Targeted Research Program (TRP). Author contributions: N.M.K. conceived the idea. S.K.A. carried out synthesis and characterization of NV-ZIF and NV-ZIFMCF and in vitro studies. S.S.Q., S.S., A.A., R.H., W.B., M.A., MR.A., and J.M. helped in characterization and in vitro studies. Z.M. designed and performed in vivo studies. S.K.A. and N.M.K. designed the experiments and wrote the paper. All authors discussed the results and commented on the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

## Study predicts good passive immunotherapy donors to combat COVID-19

• January 19, 2021

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to spread worldwide. Since the virus first emerged in late-2019, over 95.55 million cases and more than 2 million deaths have been reported.

Many countries have commenced targeted vaccination efforts to control the spread of the virus and immunize vulnerable groups. However, vaccine rollout may still lag behind ongoing infections, as fast-spreading new variants threaten many countries. Finding an effective therapy to help patients fight the infection remains crucial.

Passive immunotherapy treatment, wherein SARS-CoV-2-neutralizing antibodies (nAbs) from the plasma of recovered patients are administered to acutely sick patients, is a promising method for COVID-19 treatment in severe cases.

A team of researchers at the University of Washington, Fred Hutchinson Cancer Research Center, and the National Institute of Health, USA, recently studied the neutralizing antibodies from patients recovering from COVID-19 to investigate which clinical factors predict good passive immunotherapy donors.

In the study, published in the Journal of Clinical Investigation, the research team measured SARS-CoV-2-nAb titers in the plasma of 250 people with SARS-CoV-2 infection.

## Convalescent plasma

In the USA, convalescent plasma therapy for COVID-19 patients was approved under emergency use authorization by the Food and Drug Administration (FDA) on August 23, 2020. This mode of therapy uses antibody-containing blood from recovered individuals to help promote passive immunity in severely ill patients still battling the infection.

Blood donated by people who have recovered from COVID-19 is processed to remove blood cells, leaving behind the plasma and neutralizing antibodies against SARS-CoV-2.

The plasma given to severely ill patients will help boost the body’s immune system. In a recent phase II clinical trial in Argentina, scientists found that convalescent plasma with high levels of neutralizing antibodies, particularly when given early in the infection, had a marked beneficial health impact.

Yet, not all SARS-CoV-2-infected people produce a strong neutralizing antibody response. Hence, convalescent plasma from donors should be screened for SARS-CoV-2-neutralizing antibody activity to make sure the recovered patients are suitable donors.

## The study

In the current study, the researchers tested the serum of 250 potential convalescent plasma donors with confirmed SARS-CoV-2 infection for the presence of SARS-CoV-2 spike protein S1 domain, nucleoprotein (NP), and for neutralizing antibodies.

The team found that among the participants, 97 percent were seropositive on one or more assays. About 60 percent of the donors had nAb titers. The correlates of higher nAb titer included old age, male, and severity of the illness. Also, patients with more severe COVID-19 symptoms, like the difficulty of breathing and fever, had higher levels of neutralizing antibodies against SARS-CoV-2.

Moreover, a longer period between the infection and antibody screening had decreased nAb titers. The study results showed that severe COVID-19 illness produces higher levels of antibodies than less severe illness. This also means that the neutralizing antibodies in the blood of recovered patients wane over time.

COVID-19 seems to be one of a group of infections where the sicker one is, and presumably the more virus and therefore the more antigen that is around, the higher the levels of antibody,” Dr. David Koelle of the Koelle Laboratory, University of Washington, said in a statement.

He explained that the potential cause of this discrepancy is that the immune system in people who had a severe illness, was not effective in stopping it. There is a probable temporal race between the proliferation of the virus and host adaptive immunity.

The researchers concluded that nAb titers correlated with disease severity, sex, and age. Also, they suggested that commercially available SARS-CoV-2 immunoglobulin G (IgG) results can become an alternative for nAb testing.

Functional nAb levels were found to decline and a small proportion of persons recovered from COVID-19 lack adaptive immune responses,” they added.

Source:

Journal reference:

## Researchers Discover Way to Boost Immunotherapy Against Breast Cancer

• January 10, 2021

A diagram showing the various strategies that could enhance the activity of CAR T cells against breast cancer. Credit: © 2020 Xu et al. Originally published in Journal of Experimental Medicine. DOI: 10.1084/jem.20200844

Boosting immune system T cells to effectively attack solid tumors, such as breast cancers, can be done by adding a small molecule to a treatment procedure called chimeric antigen receptor-T (CAR-T) cell therapy, according to a study by researchers at the UNC Lineberger Comprehensive Cancer Center. The boost helps recruit more immune cells into battle at the tumor site. The findings are published today (December 31, 2020) in the Journal of Experimental Medicine.

CAR-T immunotherapy, in which T cells are modified in the laboratory to express chimeric antigen receptors, CARs, that in turn target surface proteins on cancer cells, has been most effective in the treatment of patients with B-cell leukemia or lymphoma. But this new research, conducted in mouse models, points to the potential for using CAR-T therapy effectively against solid tumors as well.

“We know that CAR T cells are safe for patients with solid tumors but so far they have not been able to cause significant tumor regression in the overwhelming majority of people treated,” said Jonathan S. Serody, MD, the Elizabeth Thomas Professor of Medicine, Microbiology and Immunology and director of the Immunotherapy Program at UNC Lineberger. “Now we may have a new approach to make CAR T cells work in solid tumors, which we think could be a game-changer for therapies aimed at an appreciable number of cancers.”

UNC Lineberger Comprehensive Cancer Center’s Jonathan S. Serody, MD, and colleagues report that adding a small molecule to a chimeric antigen receptor-T (CAR-T) cell therapy can help immune system T cells to effectively attack solid tumors, such as breast cancers. The boost helps recruit more immune cells into battle at the tumor site, according to the study published in the Journal of Experimental Medicine. Credit: UNC Lineberger Comprehensive Cancer Center

Serody is the paper’s corresponding author and Nuo Xu, PhD, formerly a graduate student at UNC Lineberger and UNC School of Medicine, is the first author.

For CAR-T cell therapy to be effective, T cells infused back into patients have to be able to migrate to the site of a tumor. In treating patients with non-solid tumors, such as lymphomas, CAR T cells home in on bone marrow and other organs that make up the lymphatic system. But for solid tumors, such as breast cancer, that is usually not the case. Even if they do migrate to the tumor, they don’t persist and expand well there due to the nature of the microenvironment surrounding such tumors, noted Serody.

So Serody and colleagues looked for ways to direct the lab-expanded cells toward the site of solid tumors. They focused on Th17 and Tc17 cells, which are known to have longer persistence in the micro-environment that surrounds a tumor, in part due to their better survival capabilities. To boost accumulation of Th17 and Tc17 cells near solid tumors, they turned to two small molecules that can activate an immune response: the stimulator of interferon genes (STING) agonists DMXAA and cGAMP.

DMXAA, which worked well in the investigator’s mouse studies, has not provided benefit in human clinical trials as it does not activate human STING. The other STING agonist however, cGAMP, does activate human STING and is known to boost the human immune system. It also works well in mice.

In Serody’s experiments, mice injected with cGAMP exhibited enhanced proliferation of T cells and those cells migrated to the tumor site. The end result was a significant decrease in tumor growth and enhanced survival.

“We hope to be able to study cGAMP in humans fairly soon,” concluded Serody. “We will look to see if we can produce improvements in the treatment of head and neck cancers first, and if that proves promising, move into other forms of cancer by using CAR T cells generated by one of our colleagues here at UNC.”

UNC Lineberger is one of a select few academic centers in the United States with the scientific, technical and clinical capabilities to develop and deliver CAR-T immunotherapy to patients. The cancer center currently has nine CAR-T clinical trials open and is developing new trials to treat a number of solid tumors, including ovarian and head and neck cancer. It also offers patients commercially available CAR-T therapies.

Reference: “STING agonist promotes CAR T cell trafficking and persistence in breast cancer” by Nuo Xu, Douglas C. Palmer, Alexander C. Robeson, Peishun Shou, Hemamalini Bommiasamy, Sonia J. Laurie, Caryn Willis, Gianpietro Dotti, Benjamin G. Vincent, Nicholas P. Restifo and Jonathan S. Serody, 31 December 2020, Journal of Experimental Medicine.
DOI: 10.1084/jem.20200844

In addition to Serody and Xu, the paper’s other authors are Alexander C. Robeson, PhD, Peishun Shou, PhD, Hemamalini Bommiasamy, PhD, Sonia J. Laurie, PhD, Caryn Willis, MS, Gianpietro Dotti, MD, and Benjamin Vincent, MD, UNC Lineberger and UNC School of Medicine; Douglas C. Palmer, PhD, National Cancer Institute; and Nicholas P. Restifo, MD, Lyell Immunophara, Inc., formerly of the National Cancer Institute.

This work was supported by grants from the National Cancer Institute (P50-CA058223) and the University Cancer Research Fund.

## UCSF researchers discover a new way to control immune system’s ‘natural killer’ cells

• January 10, 2021

UC San Francisco scientists have discovered a new way to control the immune system’s “natural killer” (NK) cells, a finding with implications for novel cell therapies and tissue implants that can evade immune rejection. The findings could also be used to enhance the ability of cancer immunotherapies to detect and destroy lurking tumors.

The study, published January 8, 2021 in the Journal of Experimental Medicine, addresses a major challenge for the field of regenerative medicine, said lead author Tobias Deuse, MD, the Julien I.E. Hoffman, MD, Endowed Chair in Cardiac Surgery in the UCSF Department of Surgery.

“As a cardiac surgeon, I would love to put myself out of business by being able to implant healthy cardiac cells to repair heart disease,” said Deuse, who is interim chair and director of minimally invasive cardiac surgery in the Division of Adult Cardiothoracic Surgery. “And there are tremendous hopes to one day have the ability to implant insulin-producing cells in patients with diabetes or to inject cancer patients with immune cells engineered to seek and destroy tumors. The major obstacle is how to do this in a way that avoids immediate rejection by the immune system.”

Deuse and Sonja Schrepfer, MD, PhD, also a professor in the Department of Surgery’s Transplant and Stem Cell Immunobiology Laboratory, study the immunobiology of stem cells. They are world leaders in a growing scientific subfield working to produce “hypoimmune” lab-grown cells and tissues — capable of evading detection and rejection by the immune system. One of the key methods for doing this is to engineer cells with molecular passcodes that activate immune cell “off switches” called immune checkpoints, which normally help prevent the immune system from attacking the body’s own cells and modulate the intensity of immune responses to avoid excess collateral damage.

Schrepfer and Deuse recently used gene modification tools to engineer hypoimmune stem cells in the lab that are effectively invisible to the immune system. Notably, as well as avoiding the body’s learned or “adaptive” immune responses, these cells could also evade the body’s automatic “innate” immune response against potential pathogens.

To achieve this, the researchers adapted a strategy used by cancer cells to keep innate immune cells at bay: They engineered their cells to express significant levels of a protein called CD47, which shuts down certain innate immune cells by avtivating a molecular switch found on these cells, called SIRPα. Their success became part of the founding technology of Sana Biotechnology, Inc, a company co-founded by Schrepfer, who now directs a team developing a platform based on these hypoimmune cells for clinical use.

But the researchers were left with a mystery on their hands — the technique was more successful than predicted. In particular, the field was puzzled that such engineered hypoimmune cells were able to deftly evade detection by NK cells, a type of innate immune cell that isn’t supposed to express a SIRPα checkpoint at all.

NK cells are a type of white blood cell that acts as an immunological first responder, quickly detecting and destroying any cells without proper molecular ID proving they are “self” — native body cells or at least permanent residents — which takes the form of highly individualized molecules called MHC class I (MHC-I).

When MHC-I is artificially knocked out to prevent transplant rejection, the cells become susceptible to accelerated NK cell killing, an immunological rejection that no one in the field had yet succeeded in inhibiting fully. Deuse and Schrepfer’s 2019 data, published in Nature Biotechnology, suggested they might have stumbled upon an off switch that could be used for that purpose.

All the literature said that NK cells don’t have this checkpoint, but when we looked at cells from human patients in the lab we found SIRPα there, clear as day. We can clearly demonstrate that stem cells we engineer to overexpress CD47 are able to shut down NK cells through this pathway.”

Sonja Schrepfer, MD, PhD, Professor, Department of Surgery’s Transplant and Stem Cell Immunobiology Laboratory

To explore their data, Deuse and Schrepfer approached Lewis Lanier, PhD, a world expert in NK cell biology. At first Lanier was sure there must be some mistake, because several groups had looked for SIRPα in NK cells already and it wasn’t there. But Schrepfer was confident in her team’s data.

“Finally it hit me,” Schrepfer said. “Most studies looking for checkpoints in NK cells were done in immortalized lab-grown cell lines, but we were studying primary cells directly from human patients. I knew that must be the difference.”

Further examination revealed that NK cells only begin to express SIRPα after they are activated by certain immune signaling molecules called cytokines. As a result, the researchers realized, this inducible immune checkpoint comes into effect only in already inflammatory environments and likely functions to modulate the intensity of NK cells’ attack on cells without proper MHC class I identification.

“NK cells have been a major barrier to the field’s growing interest in developing universal cell therapy products that can be transplanted “off the shelf” without rejection, so these results are extremely promising,” said Lanier, chair and J. Michael Bishop Distinguished Professor in the Department of Microbiology and Immunology.

In collaboration with Lanier, Deuse and Schrepfer comprehensively documented how CD47-expressing cells can silence NK cells via SIRPα. While other approaches can silence some NK cells, this was the first time anyone has been able to inhibit them completely. Notably, the team found that NK cells’ sensitivity to inhibition by CD47 is highly species-specific, in line with its function in distinguishing “self” from potentially dangerous “other”.

As a demonstration of this principle, the team engineered adult human stem cells with the rhesus macaque version of CD47, then implanted them into rhesus monkeys, where they successfully activated SIRPα in the monkeys’ NK cells, and avoided killing the transplanted human cells. In the future the same procedure could be performed in reverse, expressing human CD47 in pig cardiac cells, for instance, to prevent them from activating NK cells when transplanted into human patients.

“Currently engineered CAR T cell therapies for cancer and fledgling forms of regenerative medicine all rely on being able to extract cells from the patient, modify them in the lab, and then put them back in the patient. This avoids rejection of foreign cells, but is extremely laborious and expensive,” Schrepfer said. “Our goal in establishing a hypoimmune cell platform is to create off-the shelf products that can be used to treat disease in all patients everywhere.”

The findings could also have implications for cancer immunotherapy, as a way of boosting existing therapies that attempt to overcoming the immune checkpoints cancers use to evade immune detection. “Many tumors have low levels of self-identifying MHC-I protein and some compensate by overexpressing CD47 to keep immune cells at bay,” said Lanier, who is director of the Parker Institute for Cancer Immunotherapy at the UCSF Helen Diller Family Comprehensive Cancer Center. “This might be the sweet spot for antibody therapies that target CD47.”

## Scientists gain new insight into how the immune system can be better used to destroy cancer cells

• January 10, 2021

Scientists at the University of Southampton’s Centre for Cancer Immunology have gained new insight into how the immune system can be better used to find and kill cancer cells.

Working with BioInvent International, a team led by Professor Mark Cragg and Dr Jane Willoughby from the Antibody and Vaccine Group, based at the Centre, have shown that antibodies, designed to target the molecule OX40, give a more active immune response when they bind closer to the cell membrane and can be modified to attack cancer in different ways.

OX40 is a ‘co-receptor’ that helps to stimulate the production of helper and killer T-cells during an immune response. One of the ways cancer avoids detection is by suppressing immune responses to stop functional tumour specific T-cells from being produced.

In the study, which has been published in Journal for ImmunoTherapy of Cancer, the team also discovered that switching the antibody’s isotype (the part of the antibody that engages with cells of the immune system) could change the way the antibody worked.

When the mIgG2a isotype was used, the antibody could delete cells called Treg cells which are suppressive in the immune system. When the mIgG1 isotype was present, the antibody could stimulate killer T-cells to increase and therefore kill more cancer cells.

Clinical trials with anti-OX40 antibodies have shown that the body can tolerate these drugs but unfortunately have also shown disappointing clinical responses. We need to understand why this is.

This new data shows us that when there is a cancer with lots of Tregs we could use the equivalent of the m2IgGa isotype and in patients where we feel we need better cytotoxic T cells we could use the equivalent of a mIgG1 isotype to boost the immune response. This information is important for developing the next generation of OX40 antibodies that we hope will be more effective in treating patients with cancer.”

Mark Cragg, Professor, University of Southampton

Source:

Journal reference:

Griffiths, J., et al. (2020) Domain binding and isotype dictate the activity of anti-human OX40 antibodies. Journal for ImmunoTherapy of Cancer. doi.org/10.1136/jitc-2020-001557.

## Engineered stem cells that evade immune detection could boost cell therapy and I-O

• January 8, 2021

Sana Biotechnology was founded in 2018 with a mission of solving some of the most difficult challenges in gene and cell therapy. Toward that end, the company is engineering “hypoimmune stem cells” that can evade detection and destruction by the immune system.

Now, some of Sana’s founders, who are scientists at the University of California, San Francisco (UCSF), are describing how these engineered stem cells are able to shut down the immune system’s natural killer (NK) cells. They believe their findings could enhance the development of implantable cell therapies, as well as cancer immunotherapies, they reported in the Journal of Experimental Medicine.

The ability to evade NK cells could enhance a range of experimental treatments, including implants of insulin-producing cells for patients with diabetes and cardiac cells to repair heart damage. These cells are typically rejected by the immune system—a problem hypoimmune stem cells were designed to circumvent.

The UCSF team used gene modification technology to design the cells so they avoid the immune responses that are either built into the body’s defense system or learned. The researchers achieved that feat by engineering the cells to express the protein CD47, which shuts down innate immune cells by activating signal regulatory protein alpha, or SIRP-alpha.

The researchers were surprised to discover that the hypoimmune stem cells were able to escape NK cells, even though NK cells were not previously known to express SIRP-alpha. Rather than studying lab-grown cell lines, they took cells directly from patients. That’s where they found SIRP-alpha.

What’s more, the UCSF team discovered that NK cells begin to express SIRP-alpha after they are activated by cytokines that are typically abundant in inflammatory states.

To further prove out the utility of engineered stem cells, the UCSF researchers implanted cells with rhesus macaque CD47 into monkeys. They documented the activation of SIRP-alpha in NK cells. Those NK cells did not kill the transplanted cells.

A similar technique could be used, but in reverse, to implant pig cardiac cells into people, the UCSF team argued. If human CD47 were engineered into pig heart cells, they could be implanted into people without risking rejection by NK cells, they suggested.

Sana made waves in 2018 when it raised a whopping \$700 million in a single venture round from the likes of Arch Venture Partners, Flagship Pioneering and Bezos Expeditions. “We believe that one of, if not the most, important thing happening in medicine over the next several decades is the ability to modulate genes, use cells as medicines, and engineer cells,” said Steve Harr, president and CEO of Sana, at the time.

Sana did not provide materials or funding for the new study, but it is now developing the hypoimmune stem cell technology for clinical testing.

The UCSF team believes their findings could also boost cancer immunotherapy. The engineered cells could help combat checkpoints that allow tumors to evade immune detection, they said.

“Many tumors have low levels of self-identifying MHC-I protein and some compensate by overexpressing CD47 to keep immune cells at bay,” said Lewis Lanier, Ph.D., director of the Parker Institute for Cancer Immunotherapy at the UCSF Helen Diller Family Comprehensive Cancer Center, in a statement. “This might be the sweet spot for antibody therapies that target CD47.”

## New cell therapy can boost immunotherapy against breast cancer

• January 4, 2021

Boosting immune system T cells to effectively attack solid tumors, such as breast cancers, can be done by adding a small molecule to a treatment procedure called chimeric antigen receptor-T (CAR-T) cell therapy, according to a study by researchers at the UNC Lineberger Comprehensive Cancer Center. The boost helps recruit more immune cells into battle at the tumor site. The findings are published in the Journal of Experimental Medicine.

CAR-T immunotherapy, in which T cells are modified in the laboratory to express chimeric antigen receptors, CARs, that in turn target surface proteins on cancer cells, has been most effective in the treatment of patients with B-cell leukemia or lymphoma. But this new research, conducted in mouse models, points to the potential for using CAR-T therapy effectively against solid tumors as well.

“We know that CAR T cells are safe for patients with solid tumors but so far they have not been able to cause significant tumor regression in the overwhelming majority of people treated,” said Jonathan S. Serody, MD, the Elizabeth Thomas Professor of Medicine, Microbiology and Immunology and director of the Immunotherapy Program at UNC Lineberger. “Now we may have a new approach to make CAR T cells work in solid tumors, which we think could be a game-changer for therapies aimed at an appreciable number of cancers.”

Serody is the paper’s corresponding author and Nuo Xu, PhD, formerly a graduate student at UNC Lineberger and UNC School of Medicine, is the first author.

For CAR-T cell therapy to be effective, T cells infused back into patients have to be able to migrate to the site of a tumor. In treating patients with non-solid tumors, such as lymphomas, CAR T cells home in on bone marrow and other organs that make up the lymphatic system. But for solid tumors, such as breast cancer, that is usually not the case. Even if they do migrate to the tumor, they don’t persist and expand well there due to the nature of the microenvironment surrounding such tumors, noted Serody.

So Serody and colleagues looked for ways to direct the lab-expanded cells toward the site of solid tumors. They focused on Th17 and Tc17 cells, which are known to have longer persistence in the micro-environment that surrounds a tumor, in part due to their better survival capabilities. To boost accumulation of Th17 and Tc17 cells near solid tumors, they turned to two small molecules that can activate an immune response: the stimulator of interferon genes (STING) agonists DMXAA and cGAMP.

DMXAA, which worked well in the investigator’s mouse studies, has not provided benefit in human clinical trials as it does not activate human STING. The other STING agonist however, cGAMP, does activate human STING and is known to boost the human immune system. It also works well in mice.

In Serody’s experiments, mice injected with cGAMP exhibited enhanced proliferation of T cells and those cells migrated to the tumor site. The end result was a significant decrease in tumor growth and enhanced survival.

We hope to be able to study cGAMP in humans fairly soon. We will look to see if we can produce improvements in the treatment of head and neck cancers first, and if that proves promising, move into other forms of cancer by using CAR T cells generated by one of our colleagues here at UNC.”

Jonathan S. Serody, MD, Corresponding Author, UNC Lineberger Comprehensive Cancer Center

UNC Lineberger is one of a select few academic centers in the United States with the scientific, technical and clinical capabilities to develop and deliver CAR-T immunotherapy to patients. The cancer center currently has nine CAR-T clinical trials open and is developing new trials to treat a number of solid tumors, including ovarian and head and neck cancer. It also offers patients commercially available CAR-T therapies.

## Study points the way to boost immunotherapy against breast cancer, other solid tumors — ScienceDaily

• December 31, 2020

Boosting immune system T cells to effectively attack solid tumors, such as breast cancers, can be done by adding a small molecule to a treatment procedure called chimeric antigen receptor-T (CAR-T) cell therapy, according to a study by researchers at the UNC Lineberger Comprehensive Cancer Center. The boost helps recruit more immune cells into battle at the tumor site. The findings are published in the Journal of Experimental Medicine.

CAR-T immunotherapy, in which T cells are modified in the laboratory to express chimeric antigen receptors, CARs, that in turn target surface proteins on cancer cells, has been most effective in the treatment of patients with B-cell leukemia or lymphoma. But this new research, conducted in mouse models, points to the potential for using CAR-T therapy effectively against solid tumors as well.

“We know that CAR T cells are safe for patients with solid tumors but so far they have not been able to cause significant tumor regression in the overwhelming majority of people treated,” said Jonathan S. Serody, MD, the Elizabeth Thomas Professor of Medicine, Microbiology and Immunology and director of the Immunotherapy Program at UNC Lineberger. “Now we may have a new approach to make CAR T cells work in solid tumors, which we think could be a game-changer for therapies aimed at an appreciable number of cancers.”

Serody is the paper’s corresponding author and Nuo Xu, PhD, formerly a graduate student at UNC Lineberger and UNC School of Medicine, is the first author.

For CAR-T cell therapy to be effective, T cells infused back into patients have to be able to migrate to the site of a tumor. In treating patients with non-solid tumors, such as lymphomas, CAR T cells home in on bone marrow and other organs that make up the lymphatic system. But for solid tumors, such as breast cancer, that is usually not the case. Even if they do migrate to the tumor, they don’t persist and expand well there due to the nature of the microenvironment surrounding such tumors, noted Serody.

So Serody and colleagues looked for ways to direct the lab-expanded cells toward the site of solid tumors. They focused on Th17 and Tc17 cells, which are known to have longer persistence in the micro-environment that surrounds a tumor, in part due to their better survival capabilities. To boost accumulation of Th17 and Tc17 cells near solid tumors, they turned to two small molecules that can activate an immune response: the stimulator of interferon genes (STING) agonists DMXAA and cGAMP.

DMXAA, which worked well in the investigator’s mouse studies, has not provided benefit in human clinical trials as it does not activate human STING. The other STING agonist however, cGAMP, does activate human STING and is known to boost the human immune system. It also works well in mice.

In Serody’s experiments, mice injected with cGAMP exhibited enhanced proliferation of T cells and those cells migrated to the tumor site. The end result was a significant decrease in tumor growth and enhanced survival.

“We hope to be able to study cGAMP in humans fairly soon,” concluded Serody. “We will look to see if we can produce improvements in the treatment of head and neck cancers first, and if that proves promising, move into other forms of cancer by using CAR T cells generated by one of our colleagues here at UNC.”

UNC Lineberger is one of a select few academic centers in the United States with the scientific, technical and clinical capabilities to develop and deliver CAR-T immunotherapy to patients. The cancer center currently has nine CAR-T clinical trials open and is developing new trials to treat a number of solid tumors, including ovarian and head and neck cancer. It also offers patients commercially available CAR-T therapies.

Authors and Disclosures

In addition to Serody and Xu, the paper’s other authors are Alexander C. Robeson, PhD, Peishun Shou, PhD, Hemamalini Bommiasamy, PhD, Sonia J. Laurie, PhD, Caryn Willis, MS, Gianpietro Dotti, MD, and Benjamin Vincent, MD, UNC Lineberger and UNC School of Medicine; Douglas C. Palmer, PhD, National Cancer Institute; and Nicholas P. Restifo, MD, Lyell Immunophara, Inc., formerly of the National Cancer Institute.

This work was supported by grants from the National Cancer Institute (P50-CA058223) and the University Cancer Research Fund.

## Study points the way to boost immunotherapy against breast cancer, other solid tumors

• December 31, 2020

CHAPEL HILL, NC–Boosting immune system T cells to effectively attack solid tumors, such as breast cancers, can be done by adding a small molecule to a treatment procedure called chimeric antigen receptor-T (CAR-T) cell therapy, according to a study by researchers at the UNC Lineberger Comprehensive Cancer Center. The boost helps recruit more immune cells into battle at the tumor site. The findings are published in the Journal of Experimental Medicine.

CAR-T immunotherapy, in which T cells are modified in the laboratory to express chimeric antigen receptors, CARs, that in turn target surface proteins on cancer cells, has been most effective in the treatment of patients with B-cell leukemia or lymphoma. But this new research, conducted in mouse models, points to the potential for using CAR-T therapy effectively against solid tumors as well.

“We know that CAR T cells are safe for patients with solid tumors but so far they have not been able to cause significant tumor regression in the overwhelming majority of people treated,” said Jonathan S. Serody, MD, the Elizabeth Thomas Professor of Medicine, Microbiology and Immunology and director of the Immunotherapy Program at UNC Lineberger. “Now we may have a new approach to make CAR T cells work in solid tumors, which we think could be a game-changer for therapies aimed at an appreciable number of cancers.”

Serody is the paper’s corresponding author and Nuo Xu, PhD, formerly a graduate student at UNC Lineberger and UNC School of Medicine, is the first author.

For CAR-T cell therapy to be effective, T cells infused back into patients have to be able to migrate to the site of a tumor. In treating patients with non-solid tumors, such as lymphomas, CAR T cells home in on bone marrow and other organs that make up the lymphatic system. But for solid tumors, such as breast cancer, that is usually not the case. Even if they do migrate to the tumor, they don’t persist and expand well there due to the nature of the microenvironment surrounding such tumors, noted Serody.

So Serody and colleagues looked for ways to direct the lab-expanded cells toward the site of solid tumors. They focused on Th17 and Tc17 cells, which are known to have longer persistence in the micro-environment that surrounds a tumor, in part due to their better survival capabilities. To boost accumulation of Th17 and Tc17 cells near solid tumors, they turned to two small molecules that can activate an immune response: the stimulator of interferon genes (STING) agonists DMXAA and cGAMP.

DMXAA, which worked well in the investigator’s mouse studies, has not provided benefit in human clinical trials as it does not activate human STING. The other STING agonist however, cGAMP, does activate human STING and is known to boost the human immune system. It also works well in mice.

In Serody’s experiments, mice injected with cGAMP exhibited enhanced proliferation of T cells and those cells migrated to the tumor site. The end result was a significant decrease in tumor growth and enhanced survival.

“We hope to be able to study cGAMP in humans fairly soon,” concluded Serody. “We will look to see if we can produce improvements in the treatment of head and neck cancers first, and if that proves promising, move into other forms of cancer by using CAR T cells generated by one of our colleagues here at UNC.”

UNC Lineberger is one of a select few academic centers in the United States with the scientific, technical and clinical capabilities to develop and deliver CAR-T immunotherapy to patients. The cancer center currently has nine CAR-T clinical trials open and is developing new trials to treat a number of solid tumors, including ovarian and head and neck cancer. It also offers patients commercially available CAR-T therapies.

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Authors and Disclosures

In addition to Serody and Xu, the paper’s other authors are Alexander C. Robeson, PhD, Peishun Shou, PhD, Hemamalini Bommiasamy, PhD, Sonia J. Laurie, PhD, Caryn Willis, MS, Gianpietro Dotti, MD, and Benjamin Vincent, MD, UNC Lineberger and UNC School of Medicine; Douglas C. Palmer, PhD, National Cancer Institute; and Nicholas P. Restifo, MD, Lyell Immunophara, Inc., formerly of the National Cancer Institute.

This work was supported by grants from the National Cancer Institute (P50-CA058223) and the University Cancer Research Fund.

Serody has grant support from NCI, National Heart, Lung, and Blood Institute, Merck Inc., Glaxo Smith Kline, and Carisma Therapeutics. He receives consulting fees from PIQUE Therapeutics. Vincent discloses consulting fees and equity in GeneCentric Therapeutics. Dotti holds patents in the field of T cell engineering and has sponsored research agreements with Bluebird Bio, Cell Medica and Bellicum Pharmaceutical. Dotti also serves on the scientific advisory board of MolMed S.p.A and Bellicum Pharmaceutical. Serody, Restifo and Xu have filed for intellectual property protection for the use of STING agonists to enhance CAR-T cell therapy in solid tumors. No other disclosures were reported.

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## Regeneron scientists target cell recycling to boost cancer immunotherapy

• December 18, 2020

Autophagy is the process by which cellular debris is reused to make new components. This waste-disposal system is important for normal cell survival, but scientists at Regeneron have found a drawback: Autophagy can also protect tumor cells from being targeted by the immune system.

Using a CRISPR screening technology, the Regeneron researchers showed deleting an autophagy-related gene called Rb1cc1 could sensitize tumor cells to killing by the immune system’s T cells, thereby boosting the effect PD-1 and CTLA-4 checkpoint inhibitors in mice. The results were published in Science Immunology.

The data pointed to a new role for autophagy in cancer, opening the possibility of using autophagy inhibitors to enhance the efficacy of immuno-oncology drugs in more patients, the researchers said in the study.

To identify genes that modulate the susceptibility of tumors to T-cell-mediated killing, the Regeneron scientists performed a genomewide CRISPR/Cas9 screen in mouse colon adenocarcinoma cells. They found that the signaling of the inflammatory molecule TNF-alpha played a key role in tumor cell death—a finding that’s consistent with previous studies.

In contrast, autophagy in tumor cells appeared to protect them from T cell-mediated death, the team found. The researchers went on to show that removing three key autophagy genes—Rb1cc1, Atg9a and Atg12—sensitized the cancer cells to T-cell killing.

The Regeneron team dug deeper and found that deleting Rb1cc1 while blocking TNF-alpha with an antibody had a very limited effect on cancer cell killing, suggesting that the protective effect of autophagy is mediated mainly through the TNF-alpha pathway.

RELATED: Agios helps identify genes that allow cancer to escape the immune system

The researchers further tested the findings in a mouse model of breast cancer. Again, knocking out Rb1cc1 in the animals led to increased killing of tumor cells. The team treated the mice with a combination of PD-1 and CTLA-4 antibodies and showed that the cocktail completely cleared Rb1cc1-lacking tumors but only had a modest effect on the growth of control tumors. Similar results were observed in colon cancer models.

Even in tumors without the Rb1cc1 gene, simultaneously knocking out the TNF-alpha receptor limited the anti-cancer effect of immunotherapy in mice, the team showed, indicating that TNF-alpha and autophagy are indeed essential for the killing of cancer cells by T cells.

Other research teams have investigated the role of autophagy in cancer immunotherapy. Collaborators from the University of Toronto and Agios Pharmaceuticals identified the Fitm2 gene as a candidate for inhibition to boost the effect of immuno-oncology treatments. They also discovered that simultaneously removing both autophagy-related Atg12 and Atg5 genes helped cells resist T-cell killing, while targeting either one of them alone did not.

RELATED: AACR: Attacking pancreatic cancer by thwarting its survival strategies

Tyme Therapeutics recently showed its investigational drug racemetyrosine (SM-88) could disrupt autophagy in pancreatic cancer cells and reduce levels of regulatory T cells and M2 macrophages, which are suppressors of the immune response against cancer.

In their study, the Regeneron scientists also found that inhibiting mTOR signaling—a strategy underlying such drugs as Novartis’ Afinitor—increased autophagic activity and protected tumor cells from T-cell killing.

Overall, the findings suggest targeting the autophagy pathway may be a promising strategy to make tumors more vulnerable to T-cell-based immunotherapies, potentially allowing more patients to benefit from those powerful drugs, the researchers wrote in the study.