Killing Activity Research Articles

Utilization of Platelet-Rich Plasma (PRP) in the Performance of Blood Cultures

Sepsis is a leading cause of death and morbidity. A precise microbiologic diagnosis in these patients reduces mortality, morbidity, hospital length of stay and costs. Current blood culture technology is woefully inadequate in the detection of circulating bacteria, with detection rates in the range of only 32 to 71%. Platelets play a key role in innate immunity. Platelets interact directly with innate immune cells, exert important immunomodulatory effects, and promote leucocyte migration to sites of infection. During active bacterial infection, platelets release platelet factor -4 which binds onto a variety of bacterial surfaces, thus rendering bacteria more susceptible to opsonization by leucocytes. Platelet surface Toll-like receptors play a key role in regulating bacterial phagocytosis by leucocytes. FcgammaRllA mediates immune complex mediated platelet activation for killing of opsonized bacteria. Platelets also have been observed to “corral” circulating bacteria by forming hollow spheres around them. For these reasons, we performed a prospective pilot study involving 100 patients with suspected sepsis testing the hypothesis that BC performed with PRP is superior to traditional BC performed with whole blood in detecting circulating bacteria. PRP was prepared via centrifugation of blood contained in 3 cc CBC tubes containing EDTA obtained upon admission. PRP and whole blood were injected into separate and identical culture media bottles and cultures were incubated identically in our state-of-the art microbiology laboratory. PRP culture technique detected two patients with circulating bacteria in whom traditional blood cultures were negative: Enterobacter faecalis in an elderly woman with bilateral pneumonia and Burkolderia capecia in an immunocompromised elderly man with pneumonia. In two patients with urosepsis culture results were concordant with identification of E. coli in both techniques. In eight patients, traditional BC technique detected circulating bacteria, wherein PRP-generated cultures were negative: three with Klebsiella pneumoniae, two with Enterococcus faecalis, two with E. Coli, and one with anaerobic Bacteroides Theta-iota group. Three positive cultures generated by traditional methodology were deemed contaminants by I.D. consultation. No contaminants or false positives were detected by the PRP-generated technique. Thus, in this pilot study, evaluating the utility of blood cultures prepared from platelet-rich-plasma, two patients were detected to harbor pathogenic circulating bacteria which was undetected by standard state-of-the-art blood culture techniques. Two distinct disadvantages of BC prepared from PRP relative to traditional BC technique in this study were the time delay in preparing PRP from the patient's CBC tube: these had been stored in the lab for typically 4 to 48 hours before centrifugation and subsequent inoculation, and the dramatic difference in volume represented by PRP vs. traditional BC: typically .5 to 1.1 cc vs. 32 to 40 cc, respectively. An additional disadvantage was the presence of EDTA in CBC tubes, which is confirmed to exert antibacterial properties. The results of this pilot study are intriguing. Further studies comparing PRP-generated BC and traditional BC in the detection of circulating bacteria with emerging identification techniques such as metrogenomic analysis in detecting bacterial DNA, RNA or peptide signatures would be interesting.

CD38-Directed, Single-Chain T Cell-Engager Targets Leukemia Stem Cells through IFNγ-Induced CD38 Expression

Leukemia stem cells (LSCs), enriched in the CD34+CD38- AML population, are considered the main drivers of relapse in acute myeloid leukemia (AML). While more differentiated progenitors and bulk AML blasts reside in the CD34+CD38+ fraction, targeting CD38 in AML is less pursued, as LSCs are mainly CD38-. We show IFNγ (10 ng/ml) induced an increase in CD38 mRNA (mean fold change IFNγ over vehicle = 7.7, p=0.03, n=3) and surface CD38 (mean CD38 High% population IFNγ 56%±17 vs vehicle 16%±10, p<0.0001) in both CD34+ and CD34- AML primary blasts, and decreased the clonogenic activities of primary AML cells (colony forming unit IFNγ 50±36 vs vehicle 216±117, p=0.002, n=4) regardless of cytogenetics. scRNA and total RNA sequencing data in CD34+ AML primary blasts showed that upon IFNγ treatment, CD38 and Interferon Regulatory Factor 1 (IRF-1) were consistently the most upregulated genes compared to vehicle (>2.5 Log 2 Fold Change; p=0; FDR=0). Furthermore, gene set enrichment analysis (FDR=0) indicated IFNγ induced an upregulation of genes involved in apoptosis and DNA repair, and a suppression of genes in the pro-oncogenic Wnt beta catenin pathways. CD38 luciferase-based reporter assays, chromatin immunoprecipitation, and IRF-1 knockdown experiments in AML cells showed that, upon IFNγ treatment, IRF1 is pivotal in CD38 transcriptional regulation. Because CD4+T helper type 1 and CD8+ cytotoxic T cells release IFNγ once engaged on their target, we hypothesized a CD38-directed T cell engager will convert CD34+CD38- LSC into CD34+CD38+ blasts via the secreted IFNγ to kill LSCs and blasts. We created a single chain construct (BN-CD38), inserting a CD38 nanobody between the light and heavy chains of an anti-CD3 Fab using two short peptide linkers. Based on modeling, BN-CD38 reproduces the distance between T cell receptor and the major histocompatibility complex, mimicking the natural immunological synapse. Surface plasmon resonance and cytometry studies showed BN-CD38 had strong binding affinity to both CD38 (K D 4.77xE -10 M) and CD3 (K D 4.27xE -8 M). The IC 50 for BN-CD38 at 1:1 E:T at 24hrs in different CD38+ AML cell lines (n=6) was within the 0.01 to 0.1 ng/ml range, whereas the IC 50 of a non-binding CD38 construct (BN-CD38Mut) was 100-fold higher. By 24hrs of treatment, we noted >60% CD4+ and CD8+ T cell activation (CD69+, CD25+, IFNγ+) with BN-CD38 (e.g., CD69: 60%±27) in contrast to controls (e.g., CD69: IgG 7%±0.8 and BN-CD38Mut 11%±10, p<0.0001). In primary AML samples (n=10) BN-CD38 activated >70% T cells against autologous leukemia cells (CD69: BN-CD38 73%±16, BN-CD38Mut 10%±9, IgG 11%±10, p<0.0001). CyTOF analysis showed BN-CD38 but not controls significantly reduced CD34+CD38+ AML blasts (%change to IgG range: BN-CD38 -27 to -82% vs BN-CD38Mut 1% to 7%, p = 0.01) and CD34+CD38- LSCs (%change to IgG range: BN-CD38 -78 to -99% vs BN-CD38Mut -7 to 11%, p= 0.0003) and expanded CD8+ effector memory (p<0.01), CD8+ terminally differentiated effector memory CD45RA+ (p<0.05), NKT cells (p<0.01), and central memory Tregs (p<0.0001). Anti-human-IFNγ neutralizing antibody reverted BN-CD38 induced expression of CD38 and BN-CD38 killing activity on CD34+CD38- AML cells (p<0.05). BN-CD38 (2.5 mg/kg, i.v, n=6/group) co-injected weekly with healthy 3x10 6 T cells induced near-complete disease eradication in mice xenografted with luciferase-CD38+ THP1 (p=0.003) and U937 (p=0.004) cells. Long-term survival analysis showed 38% of BN-CD38-treated THP-1 engrafted mice achieved complete cure compared to IgG (OS median: BN-CD38 61.5d, control IgG 29d, p=0.0005). Consistently, BN-CD38 suppressed AML cell engraftment, splenomegaly, and prolonged survival in three patient-derived xenografts (PDX) with complex karyotypes including TP53 mutation (PDX1: OS median BN-CD38 undefined, BN-CD38Mut 28d (p=0.002), and IgG 27d (p=0.002); PDX2: OS median BN-CD38 39d, BN-CD38Mut and control IgG 32d (p<0.01); PDX3: OS median BN-CD38 undefined, BN-CD38Mut 92d (p=0.001), and IgG 81d (p=0.0007)). BN-CD38 did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34+ humanized mice. In sum, BN-CD38 induces an effective T cell synapse and IFNγ release, while concomitantly blocking the clonogenicity and inducing CD38 expression on LSCs. These studies suggest a new mechanism to unmask and target LSCs, providing the rationale for using BN-CD38 to effectively treat AML.

Novel Pi3kδ Inhibitor Roginolisib Synergizes with the Bcl-2 Inhibitor Venetoclax in Hematological Malignancies

Roginolisib (IOA-244) is a first in class allosteric modulator and non-ATP competitive, selective PI3Kδ inhibitor currently in a Phase 1 clinical study in lymphoma and solid tumors. Initial structural and biochemical studies identified unique chemical structure, excellent selectivity, and excellent PK properties for roginolisib as compared to earlier PI3Kδ inhibitors, while the clinical trial has demonstrated a favorable safety profile. Consistent with prior PI3Kδ inhibitors, roginolisib inhibits the in vitro growth of lymphoma cells. In contrast to other PI3Kδ inhibitors, roginolisib activity is correlated with the expression levels of PIK3CD, suggesting specific and on target cancer cell-intrinsic effects (PMID: 37066023). To identify rational combination strategies, initial pharmacological screening of 474 compounds in combination with roginolisib was carried out using a MTT assay in the lymphoma cell lines HH (cutaneous T cell lymphoma, CTCL) and SP-53 (mantle cell lymphoma, MCL). Initial screening identified that roginolisib synergized with the bcl-2 inhibitor venetoclax in the HH cell line with a combination index value (CI) of 0.003 by the Chou-Talalay method. To further validate this finding, the combination of roginolisib with venetoclax or with another bcl-2 inhibitor S55746 was tested in a broad range of lymphoma cell lines including GRANTA519, JVM2, SP49 (MCL); FARAGE, TMD8 (diffuse large B cell lymphoma); MEC1 (chronic lymphocytic leukemia); MJ (cutaneous T cell lymphoma); and YT (NK lymphoma). The combination showed synergism in all the cell lines (CI 0.81 - 0.05) except SP49 (CI- 1.3). Similarly, the combination of roginolisib with S55746 showed synergism in most of the cell lines (CI- 0.46 - 0.089), additive effect in SP49 (CI- 1) and antagonistic effect in GRANTA519 (CI- 1.3) and FARAGE (CI- 2.17). To assess the tumor-intrinsic effect of roginolisib in patient derived CLL cells, PBMCs isolated from CLL patients responding to prior BTK inhibitor (i) (n=4) or progressed on BTKi (n=6) were tested to check the effect of roginolisib on BCR signalling. Cells were treated with increasing doses of roginolisib from 0.625 μM to 5 μM in the presence of anti-IgM. Other BCRi such as idelalisib, duvelisib and acalabrutinib were used at 5 μM concentrations as positive controls. Roginolisib showed a dose dependent suppression of the activity of BCR downstream kinases such as AKT and ERK. The suppression of phosphorylation of AKT and ERK were comparable with other BCRi tested. Additionally, the apoptosis analysis by annexin/7AAD showed comparable activity of roginolisib with idelalisib, duvelisib or acalabrutinib at 5 μM concentration. To investigate the possible synergy of roginolisib with venetoclax in CLL patient samples who are BTKi responders (n=6) or BTKi progressors (n=8), apoptosis analysis was carried out in patient PBMCs. Viability of CLL cells following 48h of ex vivo treatment with increasing concentrations of roginolisib (0.625 to 5 μM) and venetoclax (1 to 8 nM) alone or in combination was tested. Both the BTKi responders and progressors showed strong synergy with the combination, with slightly better cell killing activity in BTKi progressors ( p=0.007) than responders ( p=0.019). To understand the mechanism by which roginolisib increases the sensitivity to venetoclax, changes in the expression levels of various Bcl-2 family proteins that have been implicated in venetoclax resistance such as Mcl-1, Bcl-xL, Bcl-2, A1 and BIM were measured by immunoblotting. Changes in expression were evaluated in PBMCs from both the BTKi responders (n=8) and progressors (n=7) after 24h treatment with 0.625 and 5 μM concentrations of roginolisib. A significant dose dependent downregulation of Mcl-1 was observed compared to DMSO treated cells in both BTKi responders ( p=0.0033) and progressors ( p= 0.0025). In addition, a significant increase in proapoptotic BIM EL was also observed at both the 0.625 μM ( p=0.0054) and 5 μM ( p=0.0194) concentrations compared to DMSO. In summary, the novel PI3Kδ selective inhibitor roginolisib shows tumor cell killing on various hematologic malignancies including CLL cells from patients with disease progression on BTK inhibitors. In addition, roginolisib synergized with venetoclax in lymphoma cell lines and CLL patient samples. Our data support extending this combination strategy to clinical trials in hematological malignancies.

Characterization and genomic analysis of a novel E. coli lytic phage with extended lytic activity against S. Enteridis and S. Typhimurium

In order to effectively use of phages as antimicrobial agents for controlling multidrug-resistant E. coli, it is important to understand phage biology. The isolation and research of novel bacteriophages are urgently needed for food safety and animal health. Phage is considered as a novel alternative antibacterial agents and a new way of prevent, control and treat pathogens. In this paper, we isolated and characterized a novel lytic phage from chicken samples, named vB_EscP_vE20 (in brief vE20). We identified vE20 as belonging to the Podoviridae family through morphological and phylogenetic analysis. It had lytic activities against 24 out of 51 different serotypes of clinical E. coli strains, interestingly it also can lyse Salmonella species, such as Salmonella Enteritidis (S. Enteritidis) and Samonella Typhimurium (S. Typhimurium). One step growth curve showed that the latent period and lysis period was 10 min and 60 min, respectively. The burst size of vE20 was about 60 PFU/cell. The phage vE20 survived in a wide range of temperatures (30–60 °C) and pH (3–11). The whole genome size of phage vE20 is 77,938 bps, which is double stranded DNA. There are 121 Open reading frame (ORFs) in total, and the GC-content is 42.17%, without virulence-associated, antibiotic and lysogeny related genes. Phage vE20 has high bactericidal activity in killing E. coli EXG20-1 in cultures containing 4 × 109 cfu/mL in several MOIs culture. The results revealed the promising potential of phage vE20 as attractive candidates for the control of E. coli infections.Graphical

CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma

BackgroundEven acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40–60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed.MethodsIn this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules.ResultsWe successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice.ConclusionsIn this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

Evaluating vaccine-elicited antibody activities against Neisseria gonorrhoeae: cross-protective responses elicited by the 4CMenB meningococcal vaccine.

The bacterial pathogen Neisseria gonorrhoeae is an urgent global health problem due to increasing numbers of infections, coupled with rampant antibiotic resistance. Vaccines against gonorrhea are being prioritized to combat drug-resistant N. gonorrhoeae. Meningococcal serogroup B vaccines such as four-component meningococcal B vaccine (4CMenB) are predicted by epidemiology studies to cross-protect individuals from natural infection with N. gonorrhoeae and elicit antibodies that cross-react with N. gonorrhoeae. Evaluation of vaccine candidates for gonorrhea requires a suite of assays for predicting efficacy in vitro and in animal models of infection, including the role of antibodies elicited by immunization. Here, we present the development and optimization of assays to evaluate antibody functionality after immunization of mice: antibody binding to intact N. gonorrhoeae, serum bactericidal activity, and opsonophagocytic killing activity using primary human neutrophils [polymorphonuclear leukocytes (PMNs)]. These assays were developed with purified antibodies against N. gonorrhoeae and used to evaluate serum from mice that were vaccinated with 4CMenB or given alum as a negative control. Results from these assays will help prioritize gonorrhea vaccine candidates for advanced preclinical to early clinical studies and will contribute to identifying correlates and mechanisms of immune protection against N. gonorrhoeae.

Reconstitution of Natural Killer cells after allogeneic hematopoietic stem cell transplantation is facilitated by Huiyang-Guben decoction through activating the Smad7/Stat3 signal pathway.

Natural Killer (NK) cell is the first batch of re-constructed cell populations after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and its delayed reconstitution inevitably causes poor outcome. The traditional Chinese medicine Huiyang-Guben decoction (HYGB) has been clinically used in patients undergoing allo-HSCT, but its effect on NK cell reconstruction is still unclear. 40 patients with allo-HSCT therapy were randomly divided into the control group and the HYGB group, and were given oral administration of normal saline or HYGB for 4weeks before allo-HSCT, respectively. NK cells were cultured and treated with transforming growth factor β (TGF-β) and HYGB in vitro, and cell viability, cell apoptosis, and the function of NK cells were evaluated. Functional verification experiments were performed by knocking down signal transduction molecule 7 (Smad7) in NK cells before TGF-β and HYGB treatment. Clinical data suggested that HYGB intervention decreased the incidence of acute graft-versus-host disease after allo-HSCT, and increased the proportion of NK cell population. Meanwhile, HYGB improved cell viability, restrained apoptotic cell death, and enhanced cell killing activity of NK cells in patients with allo-HSCT. Notably, we found that HYGB significantly increased the expression level of Smad7 and the phosphorylation level of signal transducer and activator of transcription 3 (Stat3) in NK cells from patients with allo-HSCT. Moreover, HYGB alleviated TGF-β-induced NK cell impairment and re-activated the Smad7/Stat3 signaling in vitro, while silencing Smad7 reversed the protective effect of HYGB on TGF-β-treated NK cells. HYGB promotes NK cell reconstruction and improves NK cell function after allo-HSCT through activating the Smad7/Stat3 signaling pathway.

Isolation of Streptococcus mutans temperate bacteriophage with broad killing activity to S. mutans clinical isolates

Isolation of Streptococcus mutans temperate bacteriophage with broad killing activity to S. mutans clinical isolates

EXTH-77. EXTENDED HALF-LIFE IL-2 SYNERGIZES WITH Α-PD-1 FOR TREATMENT OF PRECLINICAL MURINE GLIOBLASTOMA MODELS

Abstract α-PD-1 checkpoint blockade is a promising immunotherapeutic approach for many solid tumors; however, this strategy is minimally effective for GBM patients as a monotherapy. One potential reason why α-PD-1 checkpoint blockade is ineffective is the tumor microenvironment and systemic derangements of the immune system which are prevalent in GBM patients. We have successfully modeled peripheral immune derangements in experimental murine GBM models. These include low peripheral blood CD4 T cell counts, reduced MHC class II expression on monocytes, and atrophy of primary immune organs in animals harboring gliomas. We therefore hypothesized that extended half-life IL-2, a potent cytokine which promotes the proliferation, differentiation, and killing activity of T cells, could overcome immune derangements and could potentially synergize with α-PD-1 checkpoint blockade therapy. In cohorts of C57Bl/6 mice, we supplemented α-PD-1 checkpoint blockade with multiple bio-engineered extended half-life IL-2 molecules to treat GL261 and CT2A orthotopic glioma models which are normally resistant to monotherapy. We observe a statistically significant increase in survival in GL261-bearing animals receiving the combination of extended half-life IL-2 reagents and αPD-1 therapy relative to control-treated mice. Some animals receiving this combination therapy had a reduction in tumor burden to levels below detection. These long-term survivors underwent a rechallenge experiment with a second inoculation of GL261-Luc and effectively cleared tumor without further therapeutic intervention. This intervention causes a significant shift in the immune profile, including the formation of mature T cell responses, as well as restoring the diminished peripheral immune cell counts in GL261-bearing animals. Finally, combining extended half-life IL-2 with α-PD-1 checkpoint blockade is effective independently of the CD8 T cell response. Together, these data suggest that combination immunotherapies employing extended half-life IL-2 fusion proteins are translational to the clinic and do not require the identification of tumor specific antigens for efficacy

New broad-spectrum and potent antibacterial agents with dual-targeting mechanism: Promoting FtsZ polymerization and disrupting bacterial membranes

The emergence of multidrug-resistant bacteria and the slow development of new antibacterial agents have led to a growing global health crisis. Here, we identified an antibacterial agent possessing 1-methyl-2,5-diphenylpyridin-1-ium core, MA220607, with a dual-targeting mechanism of action (MOA), which exhibited effective killing activity against both Gram-positive (MIC = 0.062–2 μg/mL) and Gram-negative bacteria (MIC = 0.5–4 μg/mL). Moreover, our study revealed that MA220607 could block the formation of bacterial biofilm, which might be the reason for low frequency of resistance. MOA studies showed that MA220607 not only promoted FtsZ protein polymerization, but also increased the permeability of bacterial membranes and altered their proton gradients. In addition, MA220607 had low hemolytic toxicity and could significantly inhibit the growth of bacteria in mice. Molecular dynamics simulations demonstrated that MA220607 could block the transition from the tense (T) to relaxed (R) state of FtsZ protein, thereby perturbing the stepping mechanism of FtsZ protein. Overall, our findings suggest that integrating the dual mechanisms targeting FtsZ protein and cell membranes of bacteria into a single scaffold represents a promising direction for the development of new antibacterial agents.

Bispecific CAR-iNKT Immunotherapy for High Risk MLL-Rearranged Acute Lymphoblastic Leukemia

MLL-rearranged CD19+ acute lymphoblastic leukemia ( MLLr-ALL), the commonest infant leukemia, has a high mortality rate (<40% leukemia-free survival). While stem cell transplantation has not improved outcomes, CD19-directed immunotherapeutic approaches, including CAR-T cells, have shown promise. However, as patients can still relapse with CD19-, or lineage-switched disease, alternative strategies are needed. To enhance efficacy and mitigate against immune escape, we aimed to develop iNKT cell-based bispecific CAR immunotherapy. iNKT are rare, CD1d-restricted, glycolipid-reactive T cells that bridge adaptive and innate immunity. Furthermore, allogeneic iNKT protect from aGVHD making them ideal candidates for ‘off-the-shelf’ immunotherapy without need for TCR deletion. To target both CD19 and CD133, which is often co-expressed with CD19 on MLLr blasts and possibly also on CD19-CD133+ leukemia-initiating cells, we developed a CD19-28z/CD133-41BBz bispecific as well as CD19-28z and CD133-4-1BBz monospecific CARs and compared their anti-leukemic activity. In short- and long-term cytotoxicity assays against MLLr leukemic cell lines (SEM, RS4;11, KOPN8) we show that bispecific CAR-iNKT are more potent than their monospecific counterparts. In assays involving single or dual knock-out of CD19 and CD133 in SEM cells, both the CD19 and CD133 moieties of the bispecific CAR were reactive and specific against their respective targets. While bispecific CAR-iNKT retained killing activity against either single target-expressing leukemia cells, monospecific CAR-iNKT only killed leukemia cells expressing their cognate target. In line with the in vitro findings, in xenograft assays, a low dose of 10 6 bispecific CAR-iNKT cells significantly prolonged survival of luc-SEM leukemia-bearing mice as compared to monospecific CAR-iNKT-treated and untreated control animals (Fig 1A). Of note, in vivo as well as in vitro, bispecific CAR-iNKT very effectively eliminated high and low CD19/CD133 co-expressing, but not, as expected, CD19-CD133- disease. Next, we compared bispecific CAR-iNKT vs their CAR-T counterparts. In vitro, CAR-iNKT from three donors demonstrated higher killing activity than CAR-T, while in vivo, compared to 10 6 CAR-T, CAR-iNKT cells induced significantly improved survival of leukemia-bearing mice (Fig 1B). Of note, while CAR-T cells were not detectable in the BM and spleen at sacrifice, CAR-iNKT were readily detectable in the organs of CAR-iNKT cell-treated animals, suggesting they persist longer. Remarkably, mice treated with 5x10 6 bispecific CAR-iNKT cells survived >60 days without any evidence of leukemia by serial bioluminescence assay, or in the bone marrow, spleen (flow-cytometry) and leptomeninges (immunohistochemistry) upon culling. Since SEM cells do not express CD1d, we explored alternative mechanism(s) that would account for the more potent anti-leukemic activity of CAR-iNKT cells. We found that CAR-iNKT express significantly higher baseline levels of the activating receptor NKG2D than CAR-T cells. Upon co-culture with SEM cells, nearly all CAR-iNKT but only a fraction of CAR-T express NKG2D, and a blocking NKG2D Ab significantly abrogated the superior cytotoxic effect of CAR-iNKT cells. Consistent with an NKG2D-dependent mechanism, mRNA of one or more NKG2D ligands is expressed by primary MLLr blasts as well as leukemia cell lines. Since CD133 is expressed on hematopoietic stem and progenitor cells (HSPC), the potential hematopoietic toxicity of bispecific CAR-iNKT cells was tested in a series of experiments involving cord blood humanisation of NSGS mouse hematopoiesis. We found that transfer of 10 7 bispecific CAR-iNKT cells significantly but transiently depleted engrafted CB CD19+ B cells. However, phenotypic analysis showed that long term engraftment levels were similar for all mature cell lineages, CD34+ HSPC and specifically for Lin-CD34+CD38- HSC. We conclude that bispecific CAR-iNKT cell immunotherapy is a very effective treatment for pre-clinical aggressive MLLr-ALL, it outperforms CAR-T cell immunotherapy in an NKG2D-dependent manner and has the potential to protect from immune escape, leptomeningeal disease and lineage switch without discernible hematological toxicity. These findings provide the basis for clinical development of bispecific CD19/CD133 CAR-iNKT cells as an ‘off-the-shelf’ treatment for MLLr-ALL.

Bivalent BCMA Binding and Low Affinity CD3 T-Cell Engagement By Abbv-383 Drives Sustained Activation with Reduced T-Cell Exhaustion in Preclinical Models of Multiple Myeloma

Introduction: ABBV-383 is an IgG4-based CD3-bispecific T-cell engager (TCE) that has shown promising efficacy and a favorable safety profile in heavily pre-treated patients with relapsed/refractory multiple myeloma (MM). ABBV-383 possesses a unique combination of a low-affinity CD3δε antibody coupled to a bivalent, high-affinity BCMA engager arm in a 2+1 format. Prior pre-clinical studies demonstrated that ABBV-383 induces potent redirected T-cell killing of MM tumors with reduced cytokine release. Here, we provide additional evidence that bivalent BCMA binding by ABBV-383 reduces the negative impact of soluble BCMA and low affinity CD3 T-cell engagement drives sustained T-cell activation with reduced immune exhaustion. Methods: Structural variants of ABBV-383 were created, including 1) the bivalent BCMA targeting antibody of ABBV-383 replaced by proximal and distal monovalent VH targeting molecules, and 2) BCMA antibody structures coupled to high-and low affinity CD3 variants ( Figure). For functional testing, ABBV-383 and the structural variants were added to PBMCs or T-cells from healthy donors to serve as effector cells in co-culture with BCMA-positive MM tumor cell lines. Tumor cell killing and T-cell activation were determined by flow cytometry, and cytokine production was assessed using Luminex instrumentation. Recombinant soluble BCMA (sBCMA) was utilized in the experimental models to evaluate the extent of BCMA TCE suppression. To define the relationship between CD3-affinity and T-cell exhaustion, multi-dimensional flow cytometry was performed in long-term, 28-day co-cultures with irradiated tumor cells replated every 7 days. Results: T-cell binding was shifted by >100x with substitution of the high affinity CD3 antibody compared to ABBV-383. While all antibody variants tested induced T-cell specific MM cell killing in a BCMA-dependent manner, the negative impact of soluble BCMA on tumor killing was reduced with ABBV-383 due to its bivalent interaction with tumor cells compared to the monovalent TCE formats. In addition, the low CD3 binding affinity of ABBV-383 lowered T-cell activation antigen expression and reduced inflammatory cytokine production but resulted in similar maximal cytotoxicity of tumor cells in co-cultures of healthy and MM patient samples compared to high-affinity CD3 targeting. While maximal tumor cytotoxicity was similar, ABBV-383 was found to significantly delay the induction of a critical exhaustion-inducing transcription factor, TOX, in long-term assays. T-cell population changes were observed as early as 3 days with decreased effector memory cells (CD45RA-CD62L-) and increased naïve T-cell populations (CD62L+CD45RA+) compared to high-affinity CD3 targeting. On day 21, the high-affinity CD3 x bivalent BCMA molecule resulted in 85% of the CD8+ T-cell population expressing the transcription factor TOX compared to only 15% for ABBV-383 co-cultures. TOX induces several gene families with chromatin-modulating capacity that drive the distinct transcriptional trajectory leading to T-cell dysfunction during chronic stimulation. Simultaneous expression of multiple checkpoint inhibitory proteins is indicative of T-cell exhaustion. Triple positive CD8+ T-cells expressing LAG3, PD-1 and CTLA4 were significantly reduced by ABBV-383 versus the high-affinity CD3 variant ( Figure). Conclusions: High avidity BCMA binding coupled to low affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383 in MM. Here, we show that the presence of bivalent-BCMA reduced the negative impact of soluble BCMA, reduced inflammatory cytokine production, and dampened the emergence of TOX+ CD8+ T-cells indicating that ABBV-383 structure maximizes its clinical potential as a safe and effective MM therapy.

CYT-338 NK Cell Engager Mutispecific Antibody Engagement of NKp46 Activation Receptor Overcomes Anti-CD38 Mab Mediated Fratricide of NK Cells and Accords Enhanced Cancer Immunotherapy

Background and Significance: Despite the clinical success of the first anti-CD38 targeted monoclonal antibody, daratumumab, approved for the treatment of multiple myeloma (MM), significant challenges remain such as CD38 antigen loss/internalization and/or natural killer (NK) cell fratricide resulting in resistance to treatment over time. Study Design and Methods: To overcome the above-mentioned challenges, Cytovia has utilized its proprietary multifunctional tetravalent Flex Engager TM bispecific antibody IgG scaffold platform that contains a flexible linker for simultaneous engagement of effector immune cells and tumors targets. Through this, we generated CYT-338, an NK cell engager mutispecific antibody that targets CD38 on MM tumors and NKp46 and CD16 activation receptors on NK cells to mediate potent NK cell redirected killing of MM tumor cells more efficaciously than daratumumab. In serial killing assays against MM tumors, CYT-338 reversed dysfunctional NK cells to its original activation state and prevented exhaustion of killing activity of NK cells over time. In addition, we have also previously reported that CYT-338 has a higher potency over daratumumab for antibody dependent cellular phagocytosis (ADCP) of MM tumors. Preclinically,CYT-338 shows significantly reduced NK cell fratricide compared to daratumumab but the reason for this was unknown. We hypothesized that CYT-338 could overcome anti-CD38 mAb mediated fratricide of NK cells by engaging NKp46 activation receptor. To this end we incubated peripheral blood NK cells (PBNK) with either anti-CD38 mAb, daratumumab, CYT-338 or human IgG1 isotype control either in the presence or absence of blocking anti-NKp46 mAb (IgG1) for 24 hrs at 37 oC and stained with Annexin V apoptosis and Zombie NIR viability dye and analyzed NK fratricide by flow cytometry. We also evaluated the ability of anti-NKp46 mAb to inhibit daratumumab-mediated fratricide of NK cells under the same conditions. Current Status: Through functional preclinical investigations, we have been able to deconvolute the novel molecular mechanisms whereby NKp46 and CD16 co-engagement signals by CYT-338 on NK cells are able to overcome the CD38 apoptotic fratricide signals induced by the single anti-CD38 mAb. Specifically, we have found that blockade of CYT-338 binding to NKp46 by anti-NKp46 IgG1 antibody reversed the fratricide protection provided by CYT-338, indicating that the fratricide protection was mediated by NKp46 activation signals. In addition, the single anti-NKp46 mAb was able to inhibit daratumumab-mediated NK cell fratricide providing direct evidence that daratumumabmediated fratricide can also be reversed by engagement of NKp46 activation receptor. Conclusions: The unique NKp46 activation mechanism provided by CYT-338 for reducing NK cell fratricide and maintaining NK cell levels together with the enhanced potency including reversal of NK cell dysfunction makes it an attractive best-in-class anti-CD38 therapeutic against MM compared to daratumumab. These results support the development of CYT-338 in both monotherapy and combination with other complementary immunotherapy agents being developed or approved for MM. A first-in-human Phase 1 study targeting patients with relapsed/refractory MM is in development in the U.S.

A Novel Fc-Optimized Antibody Drug Conjugate Targeting CD7 As a Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia

Despite progress in improving treatment regimens for patients with T-cell acute lymphoblastic leukemia (T-ALL), the therapeutic options are still limited, and especially antibody-based immunotherapy is not established. The CD7 antigen represents a promising target structure in T-ALL since it is strongly expressed in different T-ALL subtypes including early T-cell precursor (ETP)-ALL. Therefore, different approaches are currently pursued for targeting CD7, including CAR-T cell therapy. Due to its high internalization capacity CD7 also represents an ideal target structure for antibody drug conjugates (ADC). Here, a novel antibody engineering approach for CD7-targeting was evaluated in vitro and in xenograft mouse models of T-ALL. A CD7 antibody was optimized for its ability to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc-domain. In addition, the Fc-engineered antibody was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via an enzymatic-cleavable linker (mc-vc-PABC). The Fc-optimized ADC, CD7-DE-vcMMAE, as well as the unconjugated antibody, CD7-DE, showed improved binding to activating Fcγ receptors (FcγRIIa, FcγRIIIa) compared to a CD7 antibody lacking the DE-modification. This resulted in potent ADCC- and ADCP-activity against different T-ALL cell lines mediated by NK-cells or macrophages as effector cells (EC 50 ADCC, 1-7 pM). In contrast to the CD7-DE antibody, CD7-DE-vcMMAE showed strong cytotoxic effects independently of immune effector cell engagement by releasing its cytotoxic compound into T-ALL cells in an antigen-restricted manner, thereby inducing G2/M cell cycle arrest and apoptosis. CD7-DE-vcMMAE was active at subnanomolar concentrations demonstrating dose-dependent cytotoxic effects in six T-ALL cell lines (IC 50 0.2 - 1 nM). The extent of maximum growth inhibition ranged between 54-98 % and correlated with CD7 antigen density. Yet, although CD7-DE-vcMMAE did not kill CD7-negative cells directly, the linker design facilitated bystander killing activity. Thus, in co-culture experiments using CEM cells and CEM-CD7-knockout cells mimicking CD7-antigen escape, the ADC demonstrated significant killing of neighbouring CD7-negative cells, thereby extending its mode of action. The antitumor activity of the CD7-ADC was further investigated in xenograft mouse models of T-ALL. In a first model, CEM cells were injected subcutaneously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and CD7-DE-vcMMAE treatment was evaluated in comparison to the unconjugated antibody CD7-DE. Treatment with CD7-DE-vcMMAE led to a significantly reduced tumor growth in comparison to CD7-DE or untreated animals (p<0.05; see panel A). A preclinical phase II-like patient-derived xenograft (PDX) study employing eight randomly selected T-ALL-PDX samples from pediatric and adult patients was conducted. PDX-cells were injected intravenously into NSG mice and treatment was started when the leukemia load reached 1 % human blasts in the peripheral blood, reflecting an overt leukemia situation. Animals receiving therapy with CD7-DE-vcMMAE showed significant prolongation of median survival in comparison to animals treated with a similarly designed control ADC targeting an irrelevant antigen (control-DE-vcMMAE) or which were left untreated (median survival time; CD7-DE-vc-MMAE: 82 days; untreated/control-DE-vcMMAE: 60 days; p<0.05; see panel B). Importantly, no leukemic blasts were found in the peripheral blood, spleen or bone marrow in animals treated with CD7-DE-vcMMAE and surviving the experimental period of 150 days. Together, the novel ADC CD7-DE-vcMMAE showed a unique set of Fc effector functions, potent direct growth inhibitory effects, bystander killing activity and efficacy in xenograft models of T-ALL. These results exhibit CD7-DE-vcMMAE as a promising therapeutic strategy and form the basis for new approaches in the treatment of T-ALL.

Identification and Functional Validation of Neoantigen-Specific T Cells in Pediatric Patients with Fusion-Derived Acute Leukemias

Leukemia emerges from the accumulation of genetic mutations in hematopoietic cells, resulting in formation of altered proteins. Some of these mutant proteins (neoantigens) are consequently processed and presented as neoepitopes through major histocompatibility complex (MHC) molecules expressed on the surface of leukemia cells, permitting tumor-specific immune targeting. While adoptive T cell therapies against high mutation burden tumors have shown promising results, less is known about leveraging tumor-specific T cells as an effective approach in pediatric patients, including high-risk leukemia. In this study, peripheral blood or bone marrow samples were obtained from 34 leukemia patients aged 0 -14 years old. The patient tumors had founder fusion genes, including 15 patients with acute lymphoblastic leukemia (ALL), 15 patients with acute myeloid leukemia (AML), and 4 patients with mixed phenotype acute leukemia (MPAL). Samples were collected at the time of diagnosis or relapse. While leukemia blasts were the most abundant cellular component in these samples, the frequency of lymphocytes ranged between 1%- 25%, with a median of 3.5%. KMT2A rearrangements were expressed with either AFF1, MLLT1, MLLT3, MLLT4, MLLT6, or MLLT10 fusion partner in 73.5% of the patients' samples in this study. This included (14/15) ALL, (10/15) AML, and (1/4) MPAL patients . T cells were enriched and expanded for a total of 14 days using a rapid expansion protocol culture system. This approach was successful on all tested samples irrespective of their mutation backgrounds or initial lymphocyte count, with a range of 133-4957fold increases in total cell number and average frequencies of 59.7% and 26.1% for CD4+ and CD8+ T cell, respectively. We further screened the T cell response by examining the expression of 4-1BB, OX40, and IFN-g upon coculture with their autologous leukemia cells, and additionally validated the T cell killing activity against the blasts by a cell killing assay using standard protocol. We found that the expanded T cells exhibited a robust immune activation when cocultured with the autologous leukemia cells from ALL patients with KMT2A rearrangements, indicating an endogenous cell response in all patients in the ALL cohort (100%). However, we observed a considerably reduced reactivity in the samples from the AML and MPAL patients. Interestingly, we observed that such diminished reactivity was associated with a marked downregulation in the expression of MHC class I and II molecules, and that pretreating the leukemia blasts with IFN-γ prior to coculturing with the expanded T cells was sufficient to induce the expression of MHC molecules and subsequent activation of the expanded T cells. Single-cell TCR sequencing of the sorted, tumor-reactive T cells identified the major clonotypes among these reactive T cell clones and the top 4 - 6 clonotypes were selected from 5 patients to evaluate reactivity against the leukemia blasts and examine their specificity against the corresponding driving fusions. We further generated TCR-expressing clones and subsequently cocultured each clone with leukemia blasts to validate their antigen recognition and reactivity by cytotoxicity assay. Interestingly, and in line with the findings from investigations on solid tumors, the leukemia- reactive T cells were consistently characterized by higher expression of both PD1 and CD39 (PD1 hi CD39 +), indicating the capability of utilizing both markers for the enrichment of T cell prior to expansion and subsequent implementation. Despite the high-risk prognosis and low mutation burden in our patient cohort, our findings demonstrate that patient T cells contain endogenous neoantigen-specific clones which can be therapeutically leveraged for targeting leukemia blasts. These results support the utilization of adoptive T cell therapy in the context of hematological malignancies especially given the less restrictive spatial segregation between tumor-specific T cells and leukemia cells compared to solid tumors, where T cell infiltration is a prerequisite for antitumor immunity. Overall, these findings suggest the possibility of leveraging leukemia-associated lymphocytes, fusion oncogene vaccines, and TCR-based therapy as novel immunotherapeutic approaches for mitigating pediatric leukemia, particularly in high-risk patients.

Novel CD123xCD3 Bispecific Igm Antibody, Igm-2537, Potently Induces T-Cell Mediated Cytotoxicity of Acute Myeloid Leukemia Cells In Vivo and in Vitro with Minimal Cytokine Release

Introduction: New therapeutics are needed for the effective treatment of acute myeloid leukemia (AML), as standard chemotherapeutics are poorly tolerated and ~50% of patients relapse primarily due to the incomplete elimination of leukemia stem cells (LSCs). CD123, the IL-3Rα chain, is highly expressed on leukemic blasts and LSCs, and is further increased in patients with poor prognostic factors. Bispecific T-cell engager (TCE) antibodies and CAR-T cells targeting CD123 have shown promising clinical efficacy in AML patients, but cytokine release syndrome limits their therapeutic window and remains a major safety concern. IGM-2537 is a novel pentameric IgM bispecific TCE antibody engineered with ten anti-CD123 binding sites, and an anti-CD3ε single chain Fv domain fused to the joining chain to engage T-cells. Here, we report that IGM-2537 not only potently induces T-cell mediated cytolytic killing of AML cells in vitro and in vivo but also demonstrates minimal cytokine release, and little to no effect on normal hematopoietic progenitor cells and human vascular endothelial cells (HUVECs) with in vitro and ex vivo studies. Additionally, an improved tolerability, excellent pharmacodynamic (PD) response and lower cytokine release was observed with a cynomolgus cross-reactive IgM TCE in monkeys. Materials and Methods:Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity and epitope mapping, respectively. T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 target cytotoxicity, T cell activation and cytokine release. Ex vivo AML colony formation assays were used to assess the activity on AML blasts. Human MV-4-11 and MOLM-13 xenograft tumor models in humanized NSG dKO mice were utilized to determine in vivo anti-tumor activity of IGM-2537. Healthy human PBMCs and bone marrow (BM) samples as well as HUVECs were used in ex vivo assays to address potential safety concerns of IGM-2537 mediated effects on hematopoietic progenitor cells and HUVECs, respectively. Lastly, to confirm that a CD123 directed IgM-based TCE molecule can potentially provide better safety profile in vivo, a cynomolgus-cross reactive IgM TCE comparator was assessed in monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound with high affinity and avidity to CD123. In vitro, IGM-2537 engaged both CD123 and CD3 to induce potent T-cell activation and T-cell mediated cytotoxicity of CD123 + AML cells, and autologous CD123 + basophils and plasmacytoid dendritic cells (pDCs). Though IGM-2537 demonstrated comparable maximal killing activity to a comparator IgG TCE, IGM-2537 demonstrated minimal cytokine release. In ex vivo patient-derived AML or normal BM colony formation assays, IGM-2537 eliminated AML colony forming cells at physiologically relevant effector/target (E/T) ratios but had little or no effect on normal hematopoietic progenitors. In addition, IGM-2537 demonstrated no cytotoxicity against HUVECs regardless of their CD123 expression at basal levels or at maximal levels upregulated by TNF-a and IFN-g, in contrast to a comparator IgG TCE. In vivo, IGM-2537 completely inhibited tumor growth in two AML xenograft tumor models, a subcutaneous MV-4-11 model, and a systemic MOLM-13 model in humanized NSG dKO mice. To further evaluate the potency and safety of the CD123xCD3 IgM bispecific TCE format in vivo, a cynomolgus cross-reactive CD123xCD3 IgM bispecific TCE was evaluated for tolerability and PD responses in cynomolgus monkeys using single doses. All animals tolerated the cross-reactive bispecific IgM TCE well at doses up to 10 mg/kg (the maximal dose level evaluated), a dose 100-fold greater than published doses of a comparator IgG TCE. Complete depletion of CD123 + basophils and substantial reductions of pDCs were seen in blood and BM with minimal to no cytokine induction. In summary, IGM-2537 demonstrated potent in vitro and in vivo T-cell mediated cytotoxicity of AML cell lines with minimal cytokine induction. The encouraging preclinical safety profiles were further supported by little/no effect on normal hematopoietic progenitor cells and HUVECs. These preclinical data support the clinical development of IGM-2537 for the treatment of AML and other hematological malignancies.

Targeting Distinct Epitopes of FCRL5 Using CAR-T Generated with Sequences Derived from Newly Identified FCRL5 Humanized Antibodies Resulted in Potent Anti-Myeloma Activity in Vitro and In Vivo

Despite improvement in treatment options for multiple myeloma (MM) patients, including targeted immunotherapies, MM remains incurable. Given the potential for a single CAR-T treatment to generate long-term remission and the optimal expression profile of FCLR5 (expressed on most MM with limited expression on normal cells), we generated new FCLR5 antibodies and obtained single chain variable fragment (scFv) sequences to develop FCRL5 CAR-Ts. The extracellular domain of FCRL5 has 9 Ig domain subunits (D1-9). We immunized ATX mice (Alloy Therapeutics), genetically engineered to produce humanized antibodies, with human membrane proximal D9 of FCRL5 since cell surface membrane proximal epitopes enhance membrane synapse interactions. Engaging D1-8 could also be effective for targeting FCRL5 by CAR-T, so we immunized mice with full-length FCRL5 and screened for clones binding to D1-8. Because we found FCRL5 expression on primary MM cells was universal but modest and variable, we prioritized screening the 40 antibodies we identified (23 bound to D9 and 17 bound to D1-8) for binding to MOLP-2 cells, known to endogenously express low levels of FCRL5 using flow cytometry. The thirteen best binders (highest mean fluorescence intensity; MFI) were next tested for binding to the MM.1S cell line modified to overexpress FCRL5. All 13 antibodies bound with high efficiency. We generated CAR-T (4-1BB CD3z) for the top seven clones (four D9; three D1-8 binders) and assessed cytolytic activity on MOLP-2 target cells modified to express luciferase (MOLP2-CG) and OPM2 cells modified to express luciferase and high levels of full length human FCRL5 (OPM2-CG-FCRL5). Effector (E) CAR-T cells were incubated with target (T) cells at a range of E:T ratios for 48 hours and bioluminescence (BLI) was used to determine cytolytic activity. Most clones showed high killing activity. We tested clones 977 (D1-8), 942, 947 and 980 (D9 binders) for in vivo studies due to highest performance in vitro. NSG mice were engrafted with 1x10 6 OPM2-CG-FCRL5 cells i.v. into tail veins and treated 14 days later i.v. with 2x10 6 FCRL5 CAR-T or BCMA CAR-T, the current lead CAR-T target in MM as an independent positive control. We found significant extension of survival compared to controls (Kaplan Meier, 942: P<.001, 947 P<.003, 977 P<.0002, 980 P<.008 BCMA P<.001) and reduced tumor burden (longitudinal BLI) in all CAR-T treated mice compared to untreated and mice treated with non-transduced T cell controls in this aggressive tumor model ( Figure 1). Co-targeting BCMA and FCRL5 may prevent antigen escape since it targets cells expressing either or both FCRL5 and BCMA. We generated a bi-targeted CAR targeting both BCMA and FCRL5 (clone D1-8 977). To confirm each scFv was active, we ran in vitro killing assays using BCMA knockout (KO)-OPM2-CG FCRL5 (BCMA-FCRL5+), OPM2-CG (BCMA+ FCLR5-) targets and found killing of each cell type by our bi-targeted CAR-T, confirming both scFvs were active. BCMA-FCRL5 CAR-T killed OPM2-CG FCRL5 and MOLP-2-CG cells (both BCMA+FCRL5+) but not BCMA KO OPM2 (BCMA-FCLR5-) cells. The best performing BCMA CAR-T in the clinic targets two BCMA epitopes- our scFv's targeting different domains of FCLR5 opens the opportunity for bi-targeting the FCLR5 protein itself. We generated an FCRL5-FCRL5 bi-targeted CAR-T using clone 977 (D1-8) and 980 (D9) and demonstrate activity of this CAR-T on OPM2-CG-FCRL5 cells; ongoing studies will determine if each scFv is active and whether in vivo activity is superior to single epitope targeting in vivo. Finally, blocking studies of the D9 antibodies showed potential for multiple epitopes within that domain which may facilitate co-targeting D9. We tested four FCRL5 CAR-T (4-1BB CD3z) predicted to target distinct D9 epitopes and observed high cytolytic activity in killing assays using OPM2-CG-FCRL5 target cells. We tested each in vivo by injecting NSG mice in with OPM2-CG and 18 days later treated with 2x10 6 FCRL5 CAR-T. As above, we observed significant extension of survival and reduced tumor burden compared to controls. Future studies will assess feasibility of D9 co-targeting. Together, our data demonstrate efficacy of FCRL5 CAR-T using novel sequences targeting FCLR5 both within and outside of D9 and support development of an FCRL5 CAR-T clinical candidate for use in relapsed/refractory MM patients.

Phase I Open-Label Single-Arm Study of Sscart-19 in Adult Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia in China

Background: Although CAR-T therapy has shown amazing efficacy in the treatment of hematologic malignancies in recent years, it is also accompanied by high-frequency of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), especially in acute lymphoblastic leukemia patients. Therefore, it is not recommended to enroll patients with active CNS involvement in CAR-T therapy. ssCART-19 is an autologous CD19-specific chimeric antigen receptor T-cell with short hairpin RNA-IL-6 gene silencing element. ssCART-19 can simultaneously express CAR structure and IL-6 silencing element. CAR structure can recognize and bind CD19 positive tumor cells, enhance T cell amplification, and finally exert T cell killing activity, such as releasing cytokines and perforin. Post-transcriptional expression of short hairpin RNA-IL-6 gene silencing element can recognize mRNA of IL-6, inhibit the expression of endogenous IL-6 in CAR-T cells through RNA interference mechanism, and then reduce the stimulation or activation intensity of downstream immune cells (mainly monocytes), with the control of the overall release intensity of cytokines, it reduces the incidence of severe CRS and ICANS, at the end reduces the clinical risk of CAR-T therapy. Methods: The trial of ssCART-19 in adult patients with R/R B-cell ALL (NCT04825496) is a single-arm, open-label Phase 1 study conducted in China. Patients underwent leukapheresis to obtain T cells for ssCART-19 manufacturing. Patients received fractionated infusions split over 3 days (10%; 30%; 60%) after lymphodepleting chemotherapy (3 days fludarabine and cyclophosphamide). Adverse events (AEs) were graded according to CTCAE, v5.0; CRS and ICANS were graded according to ASTCT Consensus Grading. The overall response rate(ORR) of complete response (CR) and CR with incomplete hematological recovery (CRi) within 3 months, duration of remission, relapse-free survival, and OS were assessed as secondary endpoints. Results : ssCART-19 was administered at 3 doses levels (DL) of 1×10 6/kg, 5×10 6/kg, or 10×10 6/kg. As of June 30th 2023 data cutoff (the median follow-up 15.9 months), 13 patients enrolled and underwent leukapheresis, 12 patients were received ssCART-19 treatment, 5 patients in DL1, 6 patients in DL2, and 1 patients in DL3. ssCART-19 was manufactured using serum-free culture system and no manufacture failure was reported. All 12 patients diagnosed with B-cell ALL were relapsed (75.0%) and refractory (25.0%) to multiple lines of prior therapy (100.0% patients had received 2 or more therapies). Five patients (41.7%) had more than 50% blasts in the bone marrow. All 12 patients did not have DLT events within 28 days. The most common ssCART-19-related AEs were transient neutropenia (100.0%), lymphopenia (100.0%), leukopenia (100.0%), anaemia (100.0%) and thrombocytopenia (83.3%). Adverse events of special interest (AESI) were CRS and ICANS. Among the 12 patients, 9 patients (75.0%) developed CRS, and 3 patients (25.0%) were grade 3 CRS. Notably, none of the 12 patients were occurred ICANS. No death cases were reported due to CRS or ICANS. A 83.3% overall response rate was achieved in efficacy-evaluable patients (n=12), with 7 complete response (CR, 58.3%), and 3 CR with incomplete hematological recovery (CRi,25.0%). The median duration of remission, relapse-free survival and OS have not been reached. Among them, 6 patients were in ongoing remission for over 12 months. Notably, one of the 12 patients (S012) had central nervous system infiltration with grade 2 before ssCART-19 infusion(Figure 1). After ssCART-19 therapy, the patient had no ICANS and got continuous remission. Robust CAR T-cell expansion occurred in all patients with a median time to T max of 12.5 days (range 9-14d), and peak copy number (C max) of 52495 (194-348000) copies/μg DNA. Conclusions ： ssCART-19 CAR-T cell therapy can effectively reduce the incidence of severe CRS and ICANS, especially control the occurrence of ICANS. And ssCART-19 achieved a high rate of remission in adult patients with R/R B-cell ALL. The ORR within 3 months were 83.3%. And, ssCART-19 shows good clinical efficacy in the treatment of leukemia patients with central nervous system infiltration. Thus, with the combination of short hairpin RNA-IL-6 gene silencing element, ssCART-19 can greatly improve the safety of CAR-T therapy, especially in central nervous system infiltration patients.

Alginate microbeads and hydrogels delivering meropenem and bacteriophages to treat Pseudomonas aeruginosa fracture-related infections

Bacteriophage (phage) therapy has shown promise in treating fracture-related infection (FRI); however, questions remain regarding phage efficacy against biofilms, phage-antibiotic interaction, administration routes and dosing, and the development of phage resistance. The goal of this study was to develop a dual antibiotic-phage delivery system containing hydrogel and alginate microbeads loaded with a phage cocktail plus meropenem and evaluate efficacy against muti-drug resistant Pseudomonas aeruginosa. Two phages (FJK.R9–30 and MK.R3–15) displayed enhanced antibiotic activity against P. aeruginosa biofilms when tested in combination with meropenem. The antimicrobial activity of both antibiotic and phage was retained for eight days at 37 °C in dual phage and antibiotic loaded hydrogel with microbeads (PA-HM). In a mouse FRI model, phages were recovered from all tissues within all treatment groups receiving dual PA-HM. Moreover, animals that received the dual PA-HM either with or without systemic antibiotics had less incidence of phage resistance and less serum neutralization compared to phages in saline. The dual PA-HM could reduce bacterial load in soft tissue when combined with systemic antibiotics, although the infection was not eradicated. The use of alginate microbeads and injectable hydrogel for controlled release of phages and antibiotics, leads to the reduced development of phage resistance and lower exposure to the adaptive immune system, which highlights the translational potential of the dual PA-HM. However, further optimization of phage therapy and its delivery system is necessary to achieve higher bacterial killing activity in vivo in the future.

Photo-crosslinked composite hydrogels with silver-deposited polymeric carbon nitride for boosting antibacterial activity

Rational design of antibacterial hydrogels is an important link to promote the multi-scenario application of this promising soft material. Herein, acrylamide and sodium alginate were selected as mixed monomers, silver-deposited polymeric carbon nitride nanosheets were used as a photoinitiator, pre-gelation was achieved under UV irradiation, and then a composite hydrogel was obtained through calcium ion crosslinking. The composite hydrogels exhibit good mechanical stability, structural stability in water and degradability under simulated physiological conditions. The composite hydrogels have the activity of killing Escherichia coli with or without light. The visible light response and surface plasmon resonance effect of the silver-deposited polymeric carbon nitride boosted the photocatalytic antibacterial activity of the composite hydrogels, and the bactericidal rate of the optimal sample reached 95.5% after 120 min of visible light irradiation. This work provides a constructive and green roadmap for the design of composite dual-network photoresponsive antibacterial hydrogels.