Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

Urine Tenofovir Levels Strongly Correlate With Virologic Suppression in Patients With Human Immunodeficiency Virus on Tenofovir Alafenamide-Based Antiretroviral Therapy.

We found that urine tenofovir (TFV) levels >1500 ng/mL strongly predict virologic suppression among people with human immunodeficiency virus taking tenofovir alafenamide (odds ratio, 5.66; 95% confidence interval, 1.59-20.14; P = .007). This suggests an existing point-of-care assay developed for tenofovir disoproxil fumarate will support adherence monitoring for patients on all TFV-based antiretrovirals.

Masking terminal neo-epitopes of linear peptides through glycosylation favours immune responses towards core epitopes producing parental protein bound antibodies.

Glycosylation of hydrophobic peptides at one terminus effectively increases their water-solubility, and conjugation through the opposing end to a carrier protein, renders them more immunogenic. Moreover, the glycosylation minimizes antibody responses to potentially deleterious, non-productive terminal neo-epitope regions of the peptides, and consequently shifts peptide immunogenicity towards the core amino acid residues. As proof of concept, glycopeptide-protein conjugates related to influenza hemagglutinin (HA), neuraminidase (NA), and the dimerization loop region of human epidermal growth factor receptor 2 (Her2), demonstrated a favorable production of core peptide specific antibodies as determined by ELISA studies. Furthermore, glycosylated Her2 peptide conjugate antisera were also shown to recognize full length Her2 protein by ELISA and at the cell surface through flow cytometry analysis. In contrast, unmasked peptide conjugates generated significant antibody populations that were specific to the terminal neo-epitope of the peptide immunogen that are notably absent in parental proteins. Antibodies generated in this manner to peptides in the dimerization loop of Her2 are also functional as demonstrated by the growth inhibition of Her2 expressing SKBR3 carcinoma cells. This method provides a technique to tailor-make epitope-specific antibodies that may facilitate vaccine, therapeutic and diagnostic antibody development.

A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells.

Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, short-term co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced target-independent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.

Evaluation of recombinant adenovirus vectors and adjuvanted protein as a heterologous prime-boost strategy using HER2 as a model antigen.

Induction of strong antigen-specific cell-mediated and humoral responses are critical to developing a successful therapeutic vaccine. Herein, using HER2 as a model antigen, we aim to evaluate a therapeutic vaccine protocol that elicits anti-tumor antibody and cytotoxic T cells to HER2/neu antigen. Replication-competent (ΔPS AdV) and non-replicating recombinant adenoviral vectors (AdV) expressing a rat HER2/neu (ErbB2) oncogene, were generated and compared for four different doses and over four time points for their ability to induce antigen-specific T and B cell responses in mice. Although ΔPS AdV:Her2 vector was shown to induce more durable antigen-specific CD8+ T cell responses, overall, the AdV:Her2 vector induced broader T and B cell responses. Hence the AdV:Her2 vector was used to evaluate a heterologous prime-boost vaccination regimen using rat HER2 protein encapsulated in archaeosomes composed of a semi-synthetic glycolipid (sulfated S-lactosylarchaeol, SLA; and lactosylarchaeol, LA) (SLA/LA:HER2enc) or admixed with archaeosomes composed of SLA alone (SLA:HER2adm). We first tested AdV:Her2 using a prime-boost approach with SLA/LA:HER2enc, and thereafter evaluated a sub-optimal AdV:Her2 dose in a heterologous prime-boost approach with SLA:HER2adm. A single administration of AdV:Her2 alone induced strong cell-mediated immune responses, whereas SLA/LA:HER2enc alone induced strong antigen-specific IgG titers. In mice primed with a suboptimal dose of AdV:Her2, strong CD8+ T-cell responses were observed after a single dose which were not further augmented by protein boost. AdV:Her2 induced CD4+ specific T-cell responses were augmented by SLA:HER2adm. Homologous vaccination using SLA:HER2adm induced strong antigen-specific antibody responses. However, the overall magnitude of the responses was similar with three doses of SLA:HER2adm or Ad:HER2 prime followed by two doses of SLA:HER2adm. We demonstrate that AdV:Her2 is capable of inducing strong antigen-specific CD8+ T cell responses, even at a low dose, and that these responses can be broadened to include antigen-specific antibody responses by boosting with SLA adjuvanted proteins without compromising CD8 T cell responses elicited by AdV priming.

Modulation of Th17 and regulatory T-cell responses during murine pregnancy contributes to increased maternal susceptibility to Salmonella Typhimurium infection.

PROBLEM: Salmonella Typhimurium (S. Tm) infection in pregnant mice results in massive placental infection, fetal loss, and exacerbated systemic infection. The Th17 host response can aid control of S. Tm infection, whereas successful pregnancy correlates to a dampened inflammatory and enhanced regulatory T-cell (Treg) response. METHOD OF STUDY: Mice were infected systemically with S. Tm and tissue bacterial burden, splenic Th17 and Treg cell numbers, and serum cytokines were analyzed. Splenic and/or placental mRNA expression of IL-17A, RORγ-t, IL-10, and TNF was determined. The effects of in vivo CD25+ cell depletion and TLR4 blockade on the course of S. Tm infection and Th17 response were determined. RESULTS: Enhanced S. Tm burden in pregnant mice was associated with time-dependent increased serum inflammatory cytokines. In vivo, TLR4 blockade reduced splenic S. Tm burden, suggesting detrimental TLR4-mediated inflammation. However, the splenic and placental Th17 response was reduced in S. Tm-infected pregnant mice relative to non-pregnant controls. Alternatively, there was an increase in splenic Treg frequency in pregnant mice and depletion of this subset reduced bacterial burden and increased the Th17 response. CONCLUSION: Downregulation of Th17 cell responses by Tregs during pregnancy potentially contributes to exacerbation of S. Tm infection in pregnant mice.

Salmonella enterica serovar Typhimurium-induced placental inflammation and not bacterial burden correlates with pathology and fatal maternal disease.

Food-borne infections caused by Salmonella enterica species are increasing globally, and pregnancy poses a high risk. Pregnant mice rapidly succumb to S. enterica serovar Typhimurium infection. To determine the mechanisms involved, we addressed the role of inflammation and bacterial burden in causing placental and systemic disease. In vitro, choriocarcinoma cells were a highly conducive niche for intracellular S. Typhimurium proliferation. While infection of mice with S. Typhimurium wild-type (WT) and mutant (Delta aroA and Delta invA) strains led to profound pathogen proliferation and massive burden within placental cells, only the virulent WT S. Typhimurium infection evoked total fetal loss and adverse host outcome. This correlated with substantial placental expression of granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) and increased serum inflammatory cytokines/chemokines, such as G-CSF, IL-6, CCL1, and KC, evoked by WT S. Typhimurium infection. In contrast, infection with high doses of S. Typhimurium Delta aroA, despite causing massive placental infection, resulted in reduced inflammatory cellular and cytokine response. While S. Typhimurium WT bacteria were dispersed in large numbers across all regions of the placenta, including the deeper labyrinth trophoblast, S. Typhimurium Delta aroA bacteria localized primarily to the decidua. This correlated with the widespread placental necrosis accompanied by neutrophil infiltration evoked by the S. Typhimurium WT bacteria. Thus, the ability of Salmonella to localize to deeper layers of the placenta and the nature of inflammation triggered by the pathogen, rather than bacterial burden, profoundly influenced placental integrity and host survival.

Study on DBH genetic polymorphisms and plasma activity in attention deficit hyperactivity disorder patients from Eastern India.

Dysfunctions in the norepinephric pathway have been speculated in the etiology of attention deficit hyperactivity disorder (ADHD), a common problem for children. Synthesis of norepinephrine from dopamine is catalyzed by the enzyme dopamine beta-hydroxylase and numerous polymorphisms in the DBH gene have been found to exert their direct influence on the enzyme activity independently. In the present study association of ADHD with four genetic polymorphisms, DBH-STR, rs1611115, rs1108580, and rs2519152, was examined in subjects belonging to eastern India. ADHD subjects (n = 111) were recruited following DSM-IV criteria. Peripheral blood samples were collected from nuclear families with ADHD probands. A group of ethnically matched healthy volunteers (n = 130) was also recruited. Genomic DNA was analyzed by PCR amplification followed by restriction digestion and genotyping. Data obtained were subjected to both family-based as well as population-based statistical analyses. Plasma DbetaH activity was measured using a photometric assay and its correlation with the genetic polymorphisms was analyzed using analysis of variance. Case-control analysis revealed no significant differences in allelic frequencies; however, significant paternal over-transmission (P = 0.02) of the rs2519152 'G' allele to ADHD probands was noticed. A haplotype, composed of 12R-C-G-G, also showed biased transmission. Strong correlation was observed between enzyme activity and rs1611115, rs1108580, and rs2519152 (P = 1.51E-6, 0.04, and 0.003, respectively). The present study hints toward the fact that DBH gene polymorphisms have some role in the etiology of ADHD in eastern Indian population and their study could be useful for therapeutic intervention.

Correlation between cystathionine beta synthase gene polymorphisms, plasma homocysteine and idiopathic mental retardation in Indian individuals from Kolkata.

Deficiency in cystathionine beta synthase (CBS) enzyme sometimes leads to hyperhomocysteinemia/homocystinuria, conditions often associated with mental retardation (MR). In this investigation, association of idiopathic MR (IMR) with six CBS gene polymorphisms and fasting total plasma homocysteine (plHcy) was explored. Nuclear families with IMR probands (N=180) and control subjects (N=106) were recruited. Genomic DNA was subjected to PCR amplification and RFLP analysis. plHcy was measured by enzyme immunoassay. Data obtained was subjected to statistical analyses. Linkage disequilibrium between polymorphic sites was computed. T833C/844ins68 polymorphism revealed significant maternal transmission in IMR cases. The 31bpVNTR 21 repeat allele was significantly higher in male IMR cases as compared to sex-matched controls (P=0.004). A significant difference was also noticed in genotype frequencies of male IMR cases (P=0.005). Four other sites, G919A, C1105T, G1316A and G1330A, were not polymorphic in the studied population. While no significant contribution of any particular genotype was observed, plHcy level was significantly higher in male IMR cases as compared to sex-matched controls (P=0.0001). The data presented here is probably indicative of a higher risk of IMR in male subjects in association with two CBS polymorphisms and mild elevation in plHcy concentration.

Screening of rural children in West Bengal for fragile-X syndrome.

BACKGROUND & OBJECTIVE: Screening for Fragile X syndrome (FRAXA), the most common genetic cause for mental retardation (MR), has mostly been carried out among MR patients. The present study was conducted to find out prevalence of FRAXA amongst children residing in the rural areas of West Bengal. METHODS: Demographic details including age, sex, nutritional status as well as birth, medical, and developmental histories, were collected amongst rural children (n=38,803) of West Bengal, India, over three years (2004-2007). Based on the records of scholastic backwardness, 179 children were short-listed and examined by a team of experts comprising of child psychiatrist, clinical psychologist, paediatrician and special educator. Blood samples were collected and molecular and cytogenetic studies were performed for identification of CGG repeats and determination of FMR1 gene promoter methylation. RESULTS: Of the selected 179 children, six were diagnosed as Down syndrome, one as cerebral palsy and 140 as non-syndromic MR. These 140 children with MR were grouped as mild (56), moderate (60), and severely (4) retarded based on IQ; children <5 yr were grouped as developmental delay (20). FRAXA was not detected in any of these children (frequency being 0% with 0-.02% confidence interval). Prevalence of MR was found to be low (about 4/1000 children). Down syndrome also had a lower frequency (0.15/1000 children). INTERPRETATION & CONCLUSION: The data obtained in the present study indicated that familial disorders like FRAXA were less frequent in the studied population.

Screening for methylenetetrahydrofolate reductase C677T and A1298C polymorphisms in Indian patients with idiopathic mental retardation.

Although genetic, nutritional, and environmental factors have been found to aggravate mental retardation in approximately 1% of individuals, no cause is known till date. In this study, two genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR), C677T (rs#1801133) and A1298C (rs#1801131), have been investigated in idiopathic mental retardation (IMR) subjects. Significantly higher frequency of the C677 allele was observed in IMR (n = 155; chi(2) = 5.5; P = 0.019) and moderate IMR (n = 67; chi(2) = 6.16; P = 0.013) groups as compared to controls (n = 126); for A1298C, no significant difference was noticed. TDT analysis revealed preferential transmission of C677 allele to a small group of mild IMR probands (chi(2) = 5.545; P = 0.018). Higher frequency of CA haplotype was also noticed in IMR cases as compared to controls (chi(2) = 6.28; P = 0.012). We infer from the present investigation that these polymorphisms are not contributing to the aetiology of IMR in this population since both case-control and family-based analysis revealed no significant transmission of the mutated allele.

Lack of association between down syndrome and polymorphisms in dopamine receptor D4 and serotonin transporter genes.

Down Syndrome (DS) patients suffer from cognitive dysfunction, depression, hyperactivity, irritability etc. Dopamine (DA) and serotonin (5HT) are known to control cognitive and behavioral attributes. An increased number of the DA receptor 4 (DRD4) is detected in brain regions primarily involved in cognition. Impairments in executive function have also been reported with depletion in 5HT. A variable number of tandem repeat (VNTR) in the exon 3 of DRD4 and an insertion/deletion polymorphism in the promoter region of 5HT transporter (5HTTLPR) have been found to be associated with different neurobehavioral disorders; however, association of these polymorphisms with DS has never been explored. The present family-based analysis on DS revealed significant over-transmission of a DRD4 VNTR allele which encodes for D4 receptor with average activity. No association was noticed for the 5HTTLPR. We may conclude that these genetic polymorphisms are not contributing to the neuromotor and cognitive dysfunctions observed in DS.

MAOA promoter polymorphism and attention deficit hyperactivity disorder (ADHD) in indian children.

Attention deficit hyperactivity disorder (ADHD) is a highly disabling, early onset childhood neurobehavioral disorder with a higher occurrence in boys as compared to girls. Pharmacological and molecular genetic studies have revealed the influence of dopaminergic and serotonergic systems in the etiology of the disorder. Monoamine oxidase A (MAOA) is a mitochondrial enzyme that regulates the dopaminergic signals in the pre-synaptic region. Polymorphism in the promoter region of the MAOA gene, which comprises of 30 bp repeats with repeat number varying between 2.5, 3.5, 4.5, and 5.5, has been shown to be associated with various neurobehavioral disorders including ADHD. This is the first study on Indian ADHD cases to validate an association between transmission of MAOA promoter polymorphism and risk of ADHD. We have analyzed the MAOA promoter polymorphism in a group of ADHD probands, their parents and ethnically matched controls by UNPHASED. Our findings indicate significant difference in the frequency of 3.5 repeat allele (P = 0.02) between cases and controls and preferential transmission of the short allele (3.5 repeat) from mothers to male ADHD probands (P = 0.005). We conclude that the short 3.5 repeat allele of the MAOA-u VNTR is probably associated with ADHD in our population and could be the reason for making boys prone to ADHD as compared to girls.

Association of dopamine D4 receptor (DRD4) polymorphisms with attention deficit hyperactivity disorder in Indian population.

Attention deficit hyperactivity disorder (ADHD) is a childhood onset neurobehavioral disorder. Several studies worldwide have implicated a possible association between ADHD and transmission of different polymorphisms of the dopamine D4 receptor gene (DRD4) in different ethnic groups. However, this is the first report on the transmission of different polymorphisms of DRD4 in Indian subjects. Association of 5' flanking 120-bp duplication, exon 1 12-bp duplication, and exon 3 48-bp variable numbers of tandem repeats (VNTR) were analyzed in 50 ADHD cases. Haplotype-based haplotype relative risk (HHRR) analysis and transmission disequilibrium test (TDT) were carried out to ascertain the association of these polymorphisms with the disorder. Linkage disequilibria (LD) between the polymorphisms were calculated using EH+ and 2LD programs. Our preliminary data showed lack of association between ADHD and transmission of the 5' flanking 120-bp duplication and exon 1 12-bp duplication. But, the transmissions of 6 and 7 repeat alleles of exon 3 48-bp VNTR showed significant association with ADHD. We have also examined the haplotype frequencies and biased transmission of one haplotype was observed in ADHD probands. LD analysis showed very strong disequilibrium between exon 1 12-bp duplication and exon 3 48-bp VNTR. Strong LD was also observed between the 5' flanking 120-bp duplication and exon 1 12-bp duplication. The observed association between higher repeat alleles of exon 3 48-bp VNTR and Indian ADHD children is consistent with some of the earlier reports.

Cystathionine beta-synthase T833C/844INS68 polymorphism: a family-based study on mentally retarded children.

BACKGROUND: Cystathionine beta-synthase (CBS) mediates conversion of homocysteine to cystathionine and deficiency in enzyme activity may lead to hyperhomocysteinemia/homocystinuria, which are often associated with mental retardation (MR). A large number of polymorphisms have been reported in the CBS gene, some of which impair its activity and among these, a T833C polymorphism in cis with a 68 bp insertion at 844 in the exon 8 is found to be associated with mild hyperhomocysteinemia in different ethnic groups. METHODS: The present study is aimed at investigating the association between T833C/844ins68 polymorphism and MR. One hundred and ninety MR cases were recruited after psychometric evaluation. Hundred and thirty-eight control subjects, two hundred and sixty-seven parents of MR probands and thirty cardiovascular disorder (CVD) patients were included for comparison. Peripheral blood was collected after obtaining informed written consent. The T833C/844ins68 polymorphism was investigated by PCR amplification of genomic DNA and restriction fragment length polymorphism analysis, followed by statistical analysis. RESULTS: The genotypic distribution of the polymorphism was within the Hardy-Weinberg equilibrium. A slightly increased genotypic frequency was observed in the Indian control population as compared to other Asian populations. Both haplotype-based haplotype relative risk analysis and transmission disequilibrium test reveled lack of association of the T833C/844ins68 polymorphism with MR; nevertheless, the relative risk calculated was higher (>1) and in a limited number of informative MR families, preferential transmission of the double mutant from heterozygous mothers to the MR probands was noticed (chi2 = 4.00, P < 0.05). CONCLUSION: This is the first molecular genetic study of CBS gene dealing with T833C/844ins68 double mutation in MR subjects. Our preliminary data indicate lack of association between T833C/844ins68 polymorphism with MR. However, higher relative risk and biased transmission of the double mutation from heterozygous mothers to MR probands are indicative of a risk of association between this polymorphism with mental retardation.

Analysis of polymorphisms in the dopamine beta hydroxylase gene: association with attention deficit hyperactivity disorder in Indian children.

OBJECTIVE: To study the association of Attention Deficit Hyperactivity Disorder (ADHD) and polymorphism in the dopamine beta hydroxylase (DBH) gene in Indian ADHD cases. SUBJECTS: Forty one ADHD cases were diagnosed as per the DSM-IV-TR criteria and evaluated by Conners Parents and Teachers Rating Scale and Wechslers Intelligence Scale for Children. METHODS: Genomic DNA was amplified for exon 2 *444g/a and intron 5 (Taq I) polymorphism in the DBH gene followed by restriction fragment length polymorphism (RFLP) analysis. Haplotype-based haplotype relative risk (HHRR) was analyzed to ascertain the transmission pattern of these two polymorphisms in ADHD cases. Linkage disequilibrium (LD) between the two polymorphisms was calculated using EH+ and 2LD programs. RESULTS: In the limited number of samples analyzed, a slight increase in transmission of the 444a allele in ADHD subjects was observed for DBH 444g/a. The intron 5 (Taq I) polymorphism showed no significant association with ADHD in these cases. Strong disequilibrium was observed between DBH444g/a and intron 5 (Taq I) polymorphism. CONCLUSION: This is the first molecular genetic study on ADHD in Indian subjects exploring transmission of polymorphisms in the DBH gene. Preliminary investigation shows a trend towards association between the transmission of DBH444a allele and ADHD. No association was noticed between transmission of intron 5 (Taq I) polymorphism and ADHD in the Indian subjects. Presence of strong LD may point towards co-segregation of these two polymorphisms more often than expected.