High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

Second-generation anti-carcinoembryonic antigen designer T cells resist activation-induced cell death, proliferate on tumor contact, secrete cytokines, and exhibit superior antitumor activity in vivo: a preclinical evaluation.

PURPOSE: This report describes the development and preclinical qualification tests of second-generation anti-carcinoembryonic (CEA) designer T cells for use in human trials. EXPERIMENTAL DESIGN: The progenitor first-generation immunoglobulin-T-cell receptor (IgTCR) that transmits Signal 1-only effectively mediated chimeric immune receptor (CIR)-directed cytotoxicity, but expressor T cells succumbed to activation-induced cell death (AICD). The second-generation CIR (termed "Tandem" for two signals) was designed to transmit TCR Signal 1 and CD28 Signal 2 to render T cells resistant to AICD and provide prolonged antitumor effect in vivo. RESULTS: A CIR was created that combines portions of CD28, TCRzeta, and a single chain antibody domain (sFv) specific for CEA into a single molecule (IgCD28TCR). As designed, the gene-modified Tandem T cells exhibit the new property of being resistant to AICD, showing instead an accelerated proliferation on tumor contact. Tandem T cells are more potent than first generation in targeting and lysing CEA+ tumor. Tandem T cells secrete high levels of interleukin-2 and IFNgamma on tumor contact that first-generation T cells lacked, but secretion was exhaustible, suggesting a need for interleukin-2 supplementation in therapy even for these second-generation agents. Finally, second-generation T cells were more effective in suppressing tumor in animal models. CONCLUSION: An advanced generation of anti-CEA designer T cells is described with features that promise a more potent and enduring antitumor immune response in vivo. These preclinical data qualify the human use of this agent that is currently undergoing trial in patients with CEA+ cancers.

Induction of CD4-independent E7-specific CD8+ memory response by heat shock fusion protein.

Infection with human papillomavirus type 16 (HPV16) is strongly associated with a number of disease states, of which cervical and anal cancers represent the most drastic endpoints. Induction of T-cell-mediated immunity, particularly cytotoxic T lymphocytes (CTL), is important in eradication of HPV-induced lesions. Studies have shown that heat shock protein fusion proteins are capable of inducing potent antigen-specific CTL activity in experimental animal models. In addition, E7-expressing tumors in C57BL/6 mice can be eradicated by treatment with HspE7, an Hsp fusion protein composed of Mycobacterium bovis BCG Hsp65 linked to E7 protein of HPV16. More importantly, HspE7 has also displayed significant clinical benefit in phase II clinical trials for the immunotherapy of HPV-related diseases. To delineate the mechanisms underlying the therapeutic effects of HspE7, we investigated the capability of HspE7 to induce antigen-specific protective immunity. Here, we demonstrate that HspE7 primes potent E7-specific CD8(+) T cells with cytolytic and cytokine secretion activities. These CD8(+) T cells can differentiate into memory T cells with effector functions in the absence of CD4(+) T-cell help. The HspE7-induced memory CD8(+) T cells persist for at least 17 weeks and confer protection against E7-positive murine tumor cell challenge. These results indicate that HspE7 is a promising immunotherapeutic agent for treating HPV-related disease. Moreover, the ability of HspE7 to induce memory CD8(+) T cells in the absence of CD4(+) help indicates that HspE7 fusion protein may have activity in individuals with compromised CD4(+) functions, such as those with invasive cancer and/or human immunodeficiency virus infection.

The lymphoid cell surface receptor NTB-A: a novel monoclonal antibody target for leukaemia and lymphoma therapeutics.

NTB-A is a CD2-related cell surface protein expressed primarily on lymphoid cells including B-lymphocytes from chronic lymphocytic leukaemia (CLL) and lymphoma patients. We have generated a series of monoclonal antibodies (mAbs) against NTB-A and assessed their therapeutic potential for CLL. Selective mAbs to NTB-A were further tested in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicty (ADCC) assays in cell lines and B lymphocytes freshly isolated from CLL patients. While lower levels of NTB-A were detected in T and natural killer (NK) cells, CDC activity was demonstrated primarily in B cells isolated from CLL patients and B lymphoma cell lines. Knockdown of NTB-A by small interfering RNA in target cells results in lower cytotoxicity, demonstrating the specificity of the mAbs. Furthermore, anti NTB-A mAbs demonstrated anti-tumour activity against CA46 human lymphoma xenografts in nude mice and against systemically disseminated Raji human lymphoma cells in severe combined immunodeficient mice. Taken together, these results demonstrate NTB-A as a potential new target for immunotherapy of leukaemia and lymphomas.

IGFL: A secreted family with conserved cysteine residues and similarities to the IGF superfamily.

We have discovered a family of small secreted proteins in Homo sapiens and Mus musculus. The IGF-like (IGFL) genes encode proteins of approximately 100 amino acids that contain 11 conserved cysteine residues at fixed positions, including two CC motifs. In H. sapiens, the family is composed of four genes and two pseudogenes that are referred as IGFL1 to IGFL4 and IGFL1P1 and IGFL1P2, respectively. Human IGFL genes are clustered together on chromosome 19 within a 35-kb interval. M. musculus has a single IGFL family member that is located on chromosome 7. Further, evolutionary analysis shows a lack of direct orthology between any of the four human members and the mouse gene. This relationship between the mouse and the human family members suggests that the multiple members in the human complement have arisen from recent duplication events that appear limited to the primate lineage. Structural considerations and sequence comparisons would suggest that IGFL proteins are distantly related to the IGF superfamily of growth factors. IGFL mRNAs display specific expression patterns; they are expressed in fetal tissues, breast, and prostate, and in many cancers as well, and this pattern is consistent with that of the IGF family members.

Mitogenic influence of human R-spondin1 on the intestinal epithelium.

Several described growth factors influence the proliferation and regeneration of the intestinal epithelium. Using a transgenic mouse model, we identified a human gene, R-spondin1, with potent and specific proliferative effects on intestinal crypt cells. Human R-spondin1 (hRSpo1) is a thrombospondin domain-containing protein expressed in enteroendocrine cells as well as in epithelial cells in various tissues. Upon injection into mice, the protein induced rapid onset of crypt cell proliferation involving beta-catenin stabilization, possibly by a process that is distinct from the canonical Wnt-mediated signaling pathway. The protein also displayed efficacy in a model of chemotherapy-induced intestinal mucositis and may have therapeutic application in gastrointestinal diseases.

PAQR proteins: a novel membrane receptor family defined by an ancient 7-transmembrane pass motif.

An emerging series of papers has identified new receptor proteins that predict seven-transmembrane pass topologies. We have consolidated this family to 11 human genes and have named the family PAQR, after two of the initially described ligands (progestin and adipoQ receptors). This protein family has ancient evolutionary roots, with identified homologs found in eubacteria. To date, published data indicate that the prokaryotic members of this family appear to encode hemolysin-type proteins, while in eukaryotes, PAQR proteins encode functional receptors with a broad range of apparent ligand specificities. We provide the complete human and mouse complement of this family, suggest a conserved structure/topology with invariant intracellular amino acid residues, and have measured mRNA expression levels for these genes across a range of human tissues.

The complete complement of C1q-domain-containing proteins in Homo sapiens.

The C-terminal domains of the A, B, C chains of C1q subcomponent of C1 complex represent a common structural motif, the C1q domain, that is found in a diverse range of proteins. We analyzed the human genome for the complete complement of this family and have identified a total of 31 independent gene sequences. The predominant organization of C1q-domain-containing (C1qDC) proteins includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 15 highly conserved residues within the C1q domain, among which 8 are invariant within the human gene set and these are predicted to cluster within the hydrophobic core of the protein. We suggest a 3-subfamily classification based on sequence homology. For some C1qDC-encoding genes, strict orthology has been retained throughout vertebrate evolution and these examples suggest a highly specific functional role for C1qDC proteins that has been under significant selective pressure. Alternatively, individual species have co-opted C1qDC proteins for roles that are highly specific to their biology, suggesting an evolutionary strategy of gene duplication and functional diversification. A more extensive analysis of the evolutionary relationship of C1qDC proteins reveals an ancient rooting, with clear members found in eubacterial species. Curiously, we have been unable to identify C1qDC-encoding genes in many eukaryotic genomcs, such as Sacchromyces cerivisae and C. elegans, suggesting that the retention or loss of this gene family throughout evolution has been sporadic.

TAFA: a novel secreted family with conserved cysteine residues and restricted expression in the brain.

We have discovered a family of small secreted proteins in Homo sapiens and Mus musculus using a novel database searching strategy. The family is composed of five highly homologous genes referred to as TAFA-1 to -5. The TAFA genes encode proteins of approximately 100 amino acids that contain conserved cysteine residues at fixed positions. TAFA-1 to -4 are more closely related to each other than to TAFA-5, in which a conserved motif including CC in TAFA-1 to -4 is not present. In H. sapiens, TAFA-3 has two isoforms formed by alternative splicing. Sequence homology analyses reveal that TAFA proteins appear distantly related to MIP-1alpha, a member of the CC-chemokine family. TAFA mRNAs are highly expressed in specific brain regions, with little expression seen in other tissues.

Generating potent Th1/Tc1 T cell adoptive immunotherapy doses using human IL-12: Harnessing the immunomodulatory potential of IL-12 without the in vivo-associated toxicity.

Interleukin (IL)-12 is a cytokine originally identified from medium conditioned by an Epstein-Barr virus transformed cell line. IL-12 has been shown to increase IFN-gamma secretion from NK and T cells, significantly enhance cytolytic activity in both of these cell types, and promote the development of Th1/Tc1 immune responses. These properties make IL-12 an attractive candidate for the development of various clinical protocols ranging from the treatment of viral diseases to tumor immunotherapy. The initial attempts to use IL-12 in the treatment of tumors demonstrated toxicity at potentially therapeutic doses. To circumvent the toxicity associated with IL-12 administration, the authors have developed an adoptive immunotherapy protocol that uses IL-12 for a brief period during ex vivo T cell activation. They show that IL-12 conditioning may be achieved without altering the growth characteristics of the in vitro expanding T cells. T cells generated in the presence of IL-12 show a shift to a Th1/Tc1 dominant phenotype. The resultant cells are more potent killers in vitro and in vivo as assessed by CTL assays and tumor regression. The ability to harness the potent Th1/Tc1 generating potential of IL-12 while avoiding its associated in vivo toxicity has the potential to benefit a large number of clinical trial protocols using adoptive transfer of T cells specific for tumors, viruses, or intracellular pathogens.

Adenoviral-mediated gene transfer of lymphotactin to the lungs of mice and rats results in infiltration and direct accumulation of CD4+, CD8+, and NK cells.

Chemokines are small 8-12-kDa chemotactic cytokines that were initially characterized for their ability to control leukocyte trafficking and, to a lesser extent, leukocyte function. Lymphotactin was first described as a T lymphocyte-specific chemotactic factor. However, it has since been shown to also be a potent attractant for natural killer (NK) cells. The chemotactic properties of lymphotactin suggested from in vitro data prompted us to study the in vivo activity of this chemokine. We constructed an adenovirus vector expressing murine lymphotactin (Ad mLym) and used this construct to overexpress lymphotactin in the lungs of both mice and rats, with similar outcomes. In brief, the accumulation of CD4(+) and CD8(+) T cells and NK cells surprisingly demonstrated slow kinetics, uncharacteristic of the chemoattractant potential seen with other chemokines. Lymphocyte accumulation in the lung was not evident prior to 24 h after gene transfer and reached a peak by day 7 in mice and day 14 in rats. Interestingly, the cellular infiltrate recruited to the lung by lymphotactin was a heterogeneous mixture of lymphocytes, monocytes, and neutrophils. Administration of Ad mLym to BALB/c SCID mice demonstrated that the presence of monocytes and neutrophils in the bronchoalveolar lavage (BAL) of wild-type BALB/c mice was likely due to the action of lymphotactin on lymphocytes. These findings extend the previous in vitro findings on the activity of lymphotactin and provide a model for studying the local effects of overexpressing chemokines in various tissues in vivo.