Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model.

Improving CAR-T cell therapy for solid tumors requires a better understanding of CAR design and cellular composition. Here, we compared second-generation (BBζ and 28ζ) with third-generation (28BBζ) carbonic anhydrase IX (CAIX)-targeted CAR constructs and investigated the antitumor effect of CAR-T cells with different CD4/CD8 proportions in vitro and in vivo. The results demonstrated that BBζ exhibited superior efficacy compared with 28ζ and 28BBζ CAR-T cells in a clear-cell renal cell carcinoma (ccRCC) skrc-59 cell bearing NSG-SGM3 mouse model. The mice treated with a single dose of BBζ CD4/CD8 mixture (CAR4/8) showed complete tumor remission and remained tumor-free 72 days after CAR-T cells infusion. In the other CAR-T and control groups, tumor-infiltrating T cells were recovered and profiled. We found that BBζ CAR8 cells upregulated expression of major histocompatibility complex (MHC) class II and cytotoxicity-associated genes, while downregulating inhibitory immune checkpoint receptor genes and diminishing differentiation of regulatory T cells (Treg cells), leading to excellent therapeutic efficacy in vivo. Increased memory phenotype, elevated tumor infiltration, and decreased exhaustion genes were observed in the CD4/8 untransduced T (UNT) cells compared with CD8 alone, indicating that CD4/8 would be the favored cellular composition for CAR-T cell therapy with long-term persistence. In summary, these findings support that BBζ CAR4/8 cells are a highly potent, clinically translatable cell therapy for ccRCC.

The functional repertoire of rabbit antibodies and antibody discovery via next-generation sequencing.

To gain insight into the functional antibody repertoire of rabbits, the VH and VL repertoires of bone marrow (BM) and spleen (SP) of a naïve New Zealand White rabbit (NZW; Oryctolagus cuniculus) and that of lymphocytes collected from a NZW rabbit immunized (IM) with a 16-mer peptide were deep-sequenced. Two closely related genes, IGHV1S40 (VH1a3) and IGHV1S45 (VH4), were found to dominate (~90%) the VH repertoire of BM and SP, whereas, IGHV1S69 (VH1a1) contributed significantly (~40%) to IM. BM and SP antibodies recombined predominantly with IGHJ4. A significant proportion (~30%) of IM sequences recombined with IGHJ2. The VK repertoire was encoded by nine IGKV genes recombined with one IGKJ gene, IGKJ1. No significant bias in the VK repertoire of the BM, SP and IM samples was observed. The complementarity-determining region (CDR)-H3 and -L3 length distributions were similar in the three samples following a Gaussian curve with average length of 12.2 ± 2.4 and 11.1 ± 1.1 amino acids, respectively. The amino acid composition of the predominant CDR-H3 and -L3 loop lengths was similar to that of humans and mice, rich in Tyr, Gly, Ser and, in some specific positions, Asp. The average number of mutations along the IGHV/KV genes was similar in BM, SP and IM; close to 12 and 15 mutations for VH and VL, respectively. A monoclonal antibody specific for the peptide used as immunogen was obtained from the IM rabbit. The CDR-H3 sequence was found in 1,559 of 61,728 (2.5%) sequences, at position 10, in the rank order of the CDR-H3 frequencies. The CDR-L3 was found in 24 of 11,215 (0.2%) sequences, ranking 102. No match was found in the BM and SP samples, indicating positive selection for the hybridoma sequence. Altogether, these findings lay foundations for engineering of rabbit V regions to enhance their potential as therapeutics, i.e., design of strategies for selection of specific rabbit V regions from NGS data mining, humanization and design of libraries for affinity maturation campaigns.

Identification of critical amino acid residues for human iNOS functional activity.

Nitric oxide (NO) is a short-lived signaling molecule that mediates a variety of biological functions, including vascular homeostasis, neurotransmission, antimicrobial defense and antitumor activities. Three known NOS isoforms (eNOS, nNOS and iNOS) have been cloned and sequenced. Here, we show that upon expression in Escherichia coli using a novel expression vector, an iNOS sequence containing three mutations (A805D, F831S and L832P) within the iNOS reductase domain produced very little functionally active iNOS protein compared to the wild type (wt) iNOS. Each of these point mutations also was individually constructed into the wt iNOS sequence. The activity of the iNOS protein containing the A805D mutation was comparable to wt, while a drastic reduction in iNOS activity was observed for the F831S and L832P mutants. A comparison of the molecular models of the reductase domain of the wt and mutant iNOS revealed a reduced core packing density for the F831S and L832P mutations compared to wt. In addition, the modeling also suggests altered hydrogen bonding, van der Waals and hydrophobic interactions of these mutants.

Characterization of Gpr101 expression and G-protein coupling selectivity.

This report describes the identification and characterization of the murine orphan GPCR, Gpr101. Both human and murine genes were localized to chromosome X. Similar to its human ortholog, murine Gpr101 mRNA was detected predominantly in the brain within discrete nuclei. A knowledge-restricted hidden Markov model-based algorithm, capable of accurately predicting G-protein coupling selectivity, indicated that both human and murine GPR101 were likely coupled to Gs. This prediction was supported by the elevation of cyclic AMP levels and the activation of a cyclic AMP response element-luciferase reporter gene in HEK293 cells over-expressing human GPR101. Consistent with this, over-expression of human GPR101 in a yeast-based system yielded an elevated, agonist-independent reporter gene response in the presence of a yeast chimeric Galphas protein. These results indicate that GPR101 participates in a potentially wide range of activities in the CNS via modulation of cAMP levels.