Overexpression of an Engineered SerpinB9 Enhances Allogeneic T-Cell Persistence and Efficacy.

Allogeneic chimeric antigen receptor (CAR)-expressing T cells offer many advantages over autologous therapies, but their benefits are curtailed by graft-versus-host disease (GvHD) and elimination by recipient immune cells. Moreover, just as with autologous therapies, allogeneic CAR T cells are susceptible to activation-induced cell death (AICD) caused by chronic antigen exposure (CAE). Granzyme B (GzmB) and Fas/FasL-initiated, caspase-mediated apoptosis are key mechanisms of T-cell death caused by T/NK cell-mediated allorejection or CAE. We explored a protective strategy of engineering CAR T cells to overexpress variants of the GzmB-specific serine protease inhibitor, SerpinB9 (SB9), to improve allogeneic T-cell persistence and antitumor efficacy. We showed that the overexpression of an SB9 variant with broadened caspase specificity, SB9(CAS), not only significantly reduced rejection of allogeneic CAR T cells, but also increased their resistance to AICD and enabled them to thrive better under CAE, thus improving allogeneic T-cell persistence and antitumor activity in vitro and in vivo. In addition, while SB9(CAS)-overexpression improved the efficacy of allogeneic CAR T-cell therapy by conferring protection to cell death, we did not observe any autonomous growth and the engineered CAR T cells were still susceptible to an inducible suicide switch. Hence, SB9(CAS)-overexpression is a promising strategy that can strengthen current development of cell therapies, broadening their applications to address unmet medical needs.

B7-H3-Targeting Chimeric Antigen Receptors Epstein-Barr Virus-specific T Cells Provides a Tumor Agnostic Off-The-Shelf Therapy Against B7-H3-positive Solid Tumors.

Encouraged by the observations of significant B7-H3 protein overexpression in many human solid tumors compared to healthy tissues, we directed our focus towards targeting B7-H3 using chimeric antigen receptor (CAR) T cells. We utilized a nanobody as the B7-H3-targeting domain in our CAR construct to circumvent the stability issues associated with single-chain variable fragment-based domains. In efforts to expand patient access to CAR T-cell therapy, we engineered our nanobody-based CAR into human Epstein-Barr virus-specific T cells (EBVST), offering a readily available off-the-shelf treatment. B7H3.CAR-armored EBVSTs demonstrated potent in vitro and in vivo activities against multiple B7-H3-positive human tumor cell lines and patient-derived xenograft models. Murine T cells expressing a murine equivalent of our B7H3.CAR exhibited no life-threatening toxicities in immunocompetent mice bearing syngeneic tumors. Further in vitro evaluation revealed that while human T, B, and natural killer cells were unaffected by B7H3.CAR EBVSTs, monocytes were targeted because of upregulation of B7-H3. Such targeting of myeloid cells, which are key mediators of cytokine release syndrome (CRS), contributed to a low incidence of CRS in humanized mice after B7H3.CAR EBVST treatment. Notably, we showed that B7H3.CAR EBVSTs can target B7-H3-expressing myeloid-derived suppressor cells (MDSC), thereby mitigating MDSC-driven immune suppression. In summary, our data demonstrate that our nanobody-based B7H3.CAR EBVSTs are effective as an off-the-shelf therapy for B7-H3-positive solid tumors. These cells also offer an avenue to modulate the immunosuppressive tumor microenvironment, highlighting their promising clinical potential in targeting solid tumors. SIGNIFICANCE: Clinical application of EBVSTs armored with B7-H3-targeting CARs offer an attractive solution to translate off-the-shelf CAR T cells as therapy for solid tumors.

Myocardial ischemia by radionuclide imaging and long-term outcomes after kidney transplantation.

PURPOSE: We examined the incidence of myocardial ischemia (MI) in kidney transplant recipients (KTR) using myocardial perfusion imaging (MPI), and its association with long-term outcomes after transplantation. METHODS: A retrospective observational study was conducted of asymptomatic KTRs who underwent post-transplant MPI screening for MI, as defined by moderate to severe myocardial perfusion defects, post-stress myocardial stunning or balanced ischemia. A composite outcome of all-cause mortality, graft loss, and major adverse cardiovascular events (MACE) was examined over minimum 5 years. RESULTS: We studied 135 KTRs who underwent 226 MPIs, with follow-up duration of 10 (7-13) years. 110 (81%) patients had normal MPIs, 11 (8%) had mild perfusion defects, and 14 (10%) had MI. Correspondingly, composite outcome developed in 6%, 27%, and 43% (p = 0.04), and MACE occurred in 7%, 0%, and 21% (p = 0.11), of the respective subgroups. Twenty-six patients developed the composite outcome after 5 (3-7) years post-transplantation, including 11 patients with MACE. On multivariate analysis, MI, higher low-density lipoprotein levels, and proteinuria > 0.3 g/day independently predicted the composite outcome; only MI predicted MACE (all p < 0.05). Ninety-one patients had two serial MPIs, which increased the positive predictive value for MACE from 17 to 25%. Absence of MI had negative predictive value of 83% for MACE and 93% for the composite outcome. CONCLUSION: MI that is detected early post-kidney transplantation predicts long-term mortality, graft loss, and MACE in KTRs, with excellent negative but poor positive predictive values.

Targeting mutant p53-expressing tumours with a T cell receptor-like antibody specific for a wild-type antigen.

Accumulation of mutant p53 proteins is frequently found in a wide range of cancers. While conventional antibodies fail to target intracellular proteins, proteosomal degradation results in the presentation of p53-derived peptides on the tumour cell surface by class I molecules of the major histocompatibility complex (MHC). Elevated levels of such p53-derived peptide-MHCs on tumour cells potentially differentiate them from healthy tissues. Here, we report the engineering of an affinity-matured human antibody, P1C1TM, specific for the unmutated p53125-134 peptide in complex with the HLA-A24 class I MHC molecule. We show that P1C1TM distinguishes between mutant and wild-type p53 expressing HLA-A24+ cells, and mediates antibody dependent cellular cytotoxicity of mutant p53 expressing cells in vitro. Furthermore, we show that cytotoxic PNU-159682-P1C1TM drug conjugates specifically inhibit growth of mutant p53 expressing cells in vitro and in vivo. Hence, p53-associated peptide-MHCs are attractive targets for the immunotherapy against mutant p53 expressing tumours.

Defining the structural basis for human alloantibody binding to human leukocyte antigen allele HLA-A*11:01.

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope-epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses.

Enhancing Antigen Cross-Presentation in Human Monocyte-Derived Dendritic Cells by Recruiting the Intracellular Fc Receptor TRIM21.

Suboptimal immune responses to pathogens contribute to chronic infections. One way to improve immune responses is to boost Ag presentation. In this study, we investigate the potential of the tripartite motif-containing 21 (TRIM21) pathway. TRIM21 is a ubiquitously expressed cytosolic protein that recognizes the Fc region of Abs. When Abs that are bound to pathogens enter the cell as immune complexes, binding of TRIM21 to Fc initiates downstream inflammatory signaling and targets the immune complexes for proteasomal degradation. In APCs, peptides generated by proteasomes are loaded onto MHC class I molecules to stimulate CD8 T cell responses, which are crucial for effective immunity to pathogens. We hypothesized that increasing the affinity between immune complexes and TRIM21 might markedly improve CD8 T cell responses to Ags processed by the TRIM21 pathway. Using phage display technology, we engineered the human IgG1 Fc to increase its affinity for TRIM21 by 100-fold. Adenovirus immune complexes with the engineered Fc induced greater maturation of human dendritic cells (DC) than immune complexes with unmodified Fc and stimulated increased Ag-specific CD8 T cell proliferation and IFN-γ release in cocultures of DC-PBMC. Thus, by increasing the affinity between Fc and TRIM21, Ags from immune complexes undergo enhanced cross-presentation on DC, leading to greater CD8 T cell responses. Our study reveals an approach that could potentially be used in vaccines to increase cytotoxic T cell responses against Ags that are targeted or delivered by Fc-modified Abs.

Nephrectomy-induced reduced renal function and the health-related quality of life of living kidney donors.

OBJECTIVE: To evaluate the health impact of nephrectomy on living kidney donors (LKDs) by comparing the health-related quality of life (HrQOL) scores measured by Short Form-36 (SF36) between those with and without postdonation renal function impairment (PRFI). METHODS: Eighty-two LKDs (47 females, mean age=50.2±11.2 years) were prospectively recruited to participate in a SF-36 HrQOL survey. Chart review, individual baseline, and postoperative renal function (eGFR) was determined using the Modification of Diet in Renal Disease formula. PRFI was defined as eGFR<60 mL/min/1.73 m2 or proteinuria. Mean SF-36 domain scores were compared between those with and without PRFI. RESULTS: After a median follow-up of 5.7 years, the prevalence of postdonation comorbidities was 29.3% (n=24) PRFI, 25.6% (n=21) hypertension, 6.1% (n=5) diabetes, and 3.7% (n=3) heart disease, and no LKDs developed end-stage renal disease. Mean eGFR before and after donor nephrectomy was 95.5±23.4 and 71.0±17.3 mL/min/1.73 m2 (P<.01). Mean SF-36 scores of LKDs were not significantly different between those with and without PRFI in all the domains (all P>.05). Similarly, the proportion of LKDs with PRFI did not differ significantly between the patients with SF-36 domain scores above and below the published reference values. CONCLUSION: Nephrectomy-induced PRFI may not have a significant impact on the HrQOL of the LKD population with a low proportion of other major comorbidities such as diabetes and ischemic heart disease.

A novel human anti-interleukin-1ß neutralizing monoclonal antibody showing in vivo efficacy.

The pro-inflammatory cytokine interleukin (IL)-1ß is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1ß have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1ß with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a>30-fold increased affinity to human IL-1ß compared with its parent antibody. This anti-human IL-1ß IgG also cross-reacts with mouse and monkey IL-1ß, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1ß pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1ß monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal.

Cdk5-mediated phosphorylation of delta-catenin regulates its localization and GluR2-mediated synaptic activity.

Cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation plays an important role in proper synaptic function and transmission. Loss of Cdk5 activity results in abnormal development of the nervous system accompanied by massive disruptions in cortical migration and lamination, therefore impacting synaptic activity. The Cdk5 activator p35 associates with delta-catenin, the synaptic adherens junction protein that serves as part of the anchorage complex of AMPA receptor at the postsynaptic membrane. However, the implications of Cdk5-mediated phosphorylation of delta-catenin have not been fully elucidated. Here we show that Cdk5-mediated phosphorylation of delta-catenin regulates its subcellular localization accompanied by changes in dendritic morphogenesis and synaptic activity. We identified two Cdk5 phosphorylation sites in mouse delta-catenin, serines 300 and 357, and report that loss of Cdk5 phosphorylation of delta-catenin increased its localization to the membrane. Furthermore, mutations of the serines 300 and 357 to alanines to mimic nonphosphorylated delta-catenin resulted in increased dendritic protrusions accompanied by increased AMPA receptor subunit GluR2 localization at the membrane. Consistent with these observations, loss of Cdk5 phosphorylation of delta-catenin increased the AMPA/NMDA ratio. This study reveals how Cdk5 phosphorylation of the synaptic mediator protein delta-catenin can alter its localization at the synapse to impact neuronal synaptic activity.

Human leukocyte antigen crossmatch testing is important for liver retransplantation.

Although human leukocyte antigen (HLA) crossmatching is often thought to be unnecessary for liver transplants (LTs), we provide evidence that for retransplants, it is essential. Sera from 139 retransplant patients who had received livers from deceased donors were retrospectively analyzed with single antigen beads on a Luminex platform for HLA antibodies. Each patient received at least 2 transplants and was followed up for at least 6 months from the second LT, which was deemed to have failed if the patient had a third LT or died. Second LT survival was calculated from the date of the second LT to the date of the third LT or death. Our study cohort consisted of 118 adult patients (> or = 18 years old) as well as 21 pediatric patients (<18 years old). Class I HLA antibodies were associated with significantly poorer regraft survival in adults [survival differences of 21.3% (P = 0.046), 22.1% (P = 0.042), and 23.7% (P = 0.033) at 1, 3, and 5 years, respectively]; however, the presence of these antibodies was not associated with significant survival differences in the pediatric population. A univariate analysis of the effect of class I antibodies on second LT survival in adults showed a hazard ratio of 2.0 (95% confidence interval = 1.0-3.8, P = 0.028). Graft survival in patients with and without HLA antibodies or class II antibodies was similar. Because class I antibodies have a deleterious effect on liver regraft survival, crossmatch testing should be performed before liver retransplantation.

Graft survival trends in kidney transplants: an analysis of the UNOS database.

We have summarized data on 92,636 living donor (LD) and 170,587 deceased donor (DD) kidney transplants reported to the OPTN/UNOS registry from January 1988 to December 2008. The increase in number of kidney transplants performed annually is mainly due to the increase in number of LD. The majority of LD, DD and their respective recipients, come from the 18-50 age group. Sibling donors made up the majority of LD, though there was a greater proportion of donations from unrelated donors than from spouses/partners. Graft survival has been significantly better in the 18-50 age group, compared with younger and older recipients, likely due to their better physical condition and lower number of co-morbidities. Asians appear to have superior graft survival, compared with Whites, Hispanics and Blacks. Part of the reason could be due to a larger proportion of them having an ideal BMI at transplant, as well as a smaller proportion having pre-transplant diabetes mellitus with its associated medical complications, leading to a detrimental effect on graft survival. Furthermore, a greater proportion of Asians had at least a college education compared with recipients of other ethnic groups, suggesting they could have a better understanding of the lifestyle modifications required after kidney transplantation, which would give rise to better compliance and hence better outcomes post-transplant. Delayed graft function (DGF), the absence of urine production in the first 24 hours posttransplant, as well as a lack of decline in serum creatinine by at least 25% in the first week posttransplant were all associated with poor short- and long-term graft survival. An early acute rejection (AR) episode, which could be antedated by any of the above, also leads to poorer short- and long-term outcomes. It is of note that the majority of recipients who underwent re-transplants had a higher PRA compared with first transplants. With their higher immunological risk, it is unsurprising that a significantly higher number had early AR episodes, and hence poorer short- and long-term graft outcomes.

An analysis of liver transplant survival rates from the UNOS registry.

The annual number of liver transplants increased from 1,678 in 1988 to 6,139 in 2006 and appeared to decline slightly in 2007. Living donor (LD) liver transplants (most from biologically unrelated donors) increased from less than 100 each year prior to 1999 to 522 in 2001 and have slowly declined since. In 2007, 265 LD liver transplants were performed. Most of the increased activity in liver transplantation has been in the adult population. About 500 pediatric liver transplants are performed each year and the number has remained roughly constant since 1990. Pediatric LD liver recipients were significantly younger than deceased donor (DD) liver recipients. Unadjusted graft and patient survival rates were 61% and 71% at 7 years, respectively, for 2,375 LD recipients and 58% and 66%, respectively, for 71,686 DD recipients during 1988-2007. Five-year graft survival rates improved by 9% comparing transplants done in 1988-1993 to those done in 2002-2004. Adult female patients and recipients of a male donor graft had better survival rates, whereas recipient and donor gender in the pediatric age group showed no significant association with survival rates. Implementation of the MELD/PELD scoring system resulted in more patients with severe liver failure receiving DD grafts. Nevertheless, recipients transplanted from 2002 onwards showed improved graft survival. Comparing patients with different levels of MELD/PELD scores however, showed that the cohort with the highest score (> 30) had the worst survival, suggesting that improved survival rates since 2002 would be more likely attributed to the great progress in medical science.

Altered prion protein glycosylation in the aging mouse brain.

The normal cellular prion protein (PrP(C)) is a glycoprotein with two highly conserved potential N-linked glycosylation sites. All prion diseases, whether inherited, infectious or sporadic, are believed to share the same pathogenic mechanism that is based on the conversion of the normal cellular prion protein (PrP(C)) to the pathogenic scrapie prion protein (PrP(Sc)). However, the clinical and histopathological presentations of prion diseases are heterogeneous, depending not only on the strains of PrP(Sc) but also on the mechanism of diseases, such as age-related sporadic vs. infectious prion diseases. Accumulated evidence suggests that N-linked glycans on PrP(C) are important in disease phenotype. A better understanding of the nature of the N-linked glycans on PrP(C) during the normal aging process may provide new insights into the roles that N-linked glycans play in the pathogenesis of prion diseases. By using a panel of 19 lectins in an antibody-lectin enzyme-linked immunosorbent assay (ELISA), we found that the lectin binding profiles of PrP(C) alter significantly during aging. There is an increasing prevalence of complex oligosaccharides on the aging PrP(C), which are features of PrP(Sc). Taken together, this study suggests a link between the glycosylation patterns on PrP(C) during aging and PrP(Sc).