A Therapeutic Antigen-Presenting Cell-Targeting DNA Vaccine VB10.16 in HPV16-Positive High-Grade Cervical Intraepithelial Neoplasia: Results from a Phase I/IIa Trial.

PURPOSE: To evaluate the safety, immunogenicity and efficacy of a therapeutic DNA vaccine VB10.16, using a unique modular vaccine technology that is based on linking antigens to CCL3L1 targeting module, in women with HPV16-positive high-grade cervical intraepithelial neoplasia (CIN). PATIENTS AND METHODS: We conducted a first-in-human, open-label, phase I/IIa clinical trial of VB10.16 in subjects with confirmed HPV16-positive CIN 2/3. The primary endpoint was the proportion of participants with adverse events, including dose-limiting toxicities. Secondary outcome measures included measuring the E6/E7-specific cellular immune response. In the Expansion cohort HPV16 clearance, regression of CIN lesion size and grading were assessed during a 12-month follow-up period. RESULTS: A total of 34 women were enrolled: 16 in two dose cohorts and 18 in the expansion cohort. No serious adverse events or dose-limiting toxicities were observed, and none of the subjects discontinued treatment with VB10.16 due to an adverse event. Mild to moderate injection site reactions were the most commonly reported adverse event (79%). HPV16-specific T-cell responses were observed after vaccination in the majority of the subjects. In the expansion cohort, HPV16 clearance was seen in 8 of 17 evaluable subjects (47%). Reductions in lesion size were seen in 16 subjects (94%) and 10 subjects (59%) had regression to CIN 0/1. Correlation between strong IFNγ T-cell responses and lesion size reduction was statistically significant (P < 0.001). CONCLUSIONS: The novel therapeutic DNA vaccine VB10.16 was well tolerated and showed promising evidence of efficacy and strong HPV16-specific T-cell responses in subjects with high-grade CIN.

Antibody-mediated delivery of T-cell epitopes to antigen-presenting cells induce strong CD4 and CD8 T-cell responses.

Targeted delivery of antigen to antigen-presenting cells (APCs) enhances antigen presentation and thus, is a potent strategy for making more efficacious vaccines. This can be achieved by use of antibodies with specificity for endocytic surface molecules expressed on the APC. We aimed to compare two different antibody-antigen fusion modes in their ability to induce T-cell responses; first, exchange of immunoglobulin (Ig) constant domain loops with a T-cell epitope (Troybody), and second, fusion of T-cell epitope or whole antigen to the antibody C-terminus. Although both strategies are well-established, they have not previously been compared using the same system. We found that both antibody-antigen fusion modes led to presentation of the T-cell epitope. The strength of the T-cell responses varied, however, with the most efficient Troybody inducing CD4 T-cell proliferation and cytokine secretion at 10-100-fold lower concentration than the antibodies carrying antigen fused to the C-terminus, both in vitro and after intravenous injection in mice. Furthermore, we exchanged this loop with an MHCI-restricted T-cell epitope, and the resulting antibody enabled efficient cross-presentation to CD8 T cells in vivo. Targeting of antigen to APCs by use of such antibody-antigen fusions is thus an attractive vaccination strategy for increased activation of both CD4 and CD8 peptide-specific T cells.

Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement.

The manufacture of clinical grade cellular products for adoptive immunotherapy requires ex vivo culture and expansion of human T cells. One of the key components in manufacturing of T cell therapies is human serum (HS) or fetal bovine serum (FBS), which can potentially expose immunotherapy recipient to adventitious infectious pathogens and are thus considered as non-cGMP compliant for adoptive therapy. Here we describe a novel xeno-free serum replacement (SR) with defined components that can be reproducibly used for the production of clinical grade T-cell therapies in combination with several different cell culture media. Dynabeads CD3/CD28 Cell Therapy System (CTS)-activated or antigen-specific T cells expanded using the xeno-free SR, CTS Immune Cell SR, showed comparable growth kinetics observed with cell culture media supplemented with HS or FBS. Importantly the xeno-free SR supplemented medium supported the optimal expansion of T cells specific for subdominant tumour-associated antigens and promoted expansion of T cells with central memory T-cell phenotype, which is favourable for in vivo survival and persistence following adoptive transfer. Furthermore, T cells expanded using xeno-free SR medium were highly amenable to lentivirus-mediated gene transduction for potential application for gene-modified T cells. Taken together, the CTS Immune Cell SR provides a novel platform strategy for the manufacture of clinical grade adoptive cellular therapies.

Naive idiotope-specific B and T cells collaborate efficiently in the absence of dendritic cells.

Anti-idiotope (anti-Id) Abs have a role in therapy against B cell lymphomas, as inhibitors of pathogenic autoantibodies, and as surrogate Ags for immunization. Despite these observations, the mechanism by which Id(+) Ig generates anti-Id Abs is essentially unknown. To address this issue, we generated a double knock-in mouse that expresses V regions of a somatically mutated anti-Id mAb with intermediate affinity (affinity constant [Ka] = 0.77 × 10(7) M(-1)) for the myeloma protein M315. The anti-Id mice have normal peripheral B cell populations, and allelic exclusion is efficient. Anti-Id B cells from BCR knock-in mice, together with Id-specific CD4(+) T cells from previously established TCR-transgenic mice, enabled us to study Id-specific T cell-B cell collaboration by dilution of transferred cells into syngeneic BALB/c recipients. We show that previously unstimulated (naive) Id-specific B and T cells collaborate efficiently in vivo, even at low frequencies and in the presence of low amounts of Id(+) Ig, resulting in germinal center formation, plasma cell development, and secretion of isotype-switched anti-Id Abs. We further demonstrate that Id-specific T cell-B cell collaboration occurs readily in the absence of adjuvant and is not dependent on Id-presentation by dendritic cells. The results underscore the potency of anti-Id B cells in MHC class II-restricted presentation of Id(+) Ig and suggest that Id-specific T cell-B cell collaboration is of physiological relevance.

Recombinant antibodies for delivery of antigen: a single loop between beta-strands in the constant region can accommodate long, complex and tandem T cell epitopes.

Recombinant antibodies are increasingly used for efficient delivery of T cell epitopes to antigen-presenting cells (APCs), both for vaccination purposes and for immune modulation. We have previously shown that recombinant antibodies can accommodate single T cell epitopes inserted into loops between beta-strands in constant (C) domains. Such recombinant antibodies have in addition been equipped with variable regions that target APCs for increased delivery of C region T cell epitopes. We here show that loop 6 (loop FG) in C(H)1 of human gamma 3 can be exchanged with (i) long T cell epitopes up to 37 amino acids, (ii) epitopes with complex secondary structure such as gluten epitopes with a type II polyproline helical confirmation and (iii) two tandemly linked T cell epitopes. T cell responses increased with T cell epitope elongation, presumably due to a positive influence of flanking residues. Recombinant antibodies targeted to either CD14 on monocytes or HLA-DP on monocytes and dendritic cells gave similar results and were 2-4 logs more efficient at stimulating human T cells than were non-targeted controls. Thus, single loops in C regions of recombinant antibodies seem versatile and may be used for delivery of lengthy, complex and multiple T cell epitopes to human APCs.

Expression of SH2D2A in T-cells is regulated both at the transcriptional and translational level.

The T-cell specific adapter protein (TSAd) encoded by the SH2D2A gene is up-regulated in activated human CD4+ T-cells in a cAMP-dependent manner. Expression of SH2D2A is important for proper activation of T-cells. Here, we show that SH2D2A expression is regulated both at the transcriptional and translational level. cAMP signaling alone induces TSAd-mRNA expression but fails to induce increased TSAd protein levels. By contrast, TCR engagement provides signals for both TSAd transcription and translation. We further show that cAMP signaling can prime T-cells for a more prompt expression of TSAd protein upon TCR stimulation. Our study thus points to a novel mechanism for how cAMP signaling may modulate T-cell activation through transcriptional priming of resting cells.

Human receptors of innate immunity (CD14, TLR2) are promising targets for novel recombinant immunoglobulin-based vaccine candidates.

Experiments in mice have suggested that engagement of receptors of innate immunity has an adjuvant effect on adaptive immune responses. Such studies need to be extended to humans. We have here constructed recombinant scFv-based vaccine candidate proteins (vaccibodies) that target human TLR2 and CD14 for delivery of large antigens. Vaccibodies are homodimers, each chain consisting of scFv specific for surface molecules on antigen-presenting cells (APC), a homodimerization motif, and an antigenic unit. The TLR2- and CD14-specific vaccibodies bound their respective target receptors expressed on transfected CHO cells and PBMC. Large proteins such as paired mouse Ckappa-domains (229 aa) and fragment C of tetanus toxin (TetC, 451 aa) could be expressed as antigenic units with intact serological determinants detected by mAb or polyclonal antisera. In the presence of monocytes, TLR2- and CD14-specific vaccibodies having either Ckappa or TetC as antigenic unit were 100-10,000 more efficient at stimulating T cell clones in vitro compared to non-targeted vaccibodies expressing the same antigens. The results show that TLR2 and CD14 are efficient targets for delivery of antigen to APC for stimulation of HLA class II-restricted CD4(+) T cells. Thus, receptors of innate immunity should be further explored as targets for vaccines.

Delivery of antigen to CD40 induces protective immune responses against tumors.

Ligation of CD40 induces maturation of dendritic cells (DC) and could be a useful target for vaccines. In this study, we have constructed two types of Ab-based vaccine constructs that target mouse CD40. One type is a recombinant Ab with V regions specific for CD40 and has defined T cell epitopes inserted into its C region. The other type is a homodimer, each chain of which is composed of a targeting unit (single-chain fragment variable targeting CD40), a dimerization motif, and an antigenic unit. Such proteins bound CD40, stimulated maturation of DC, and enhanced primary and memory T cell responses. When delivered i.m. as naked DNA followed by electroporation, the vaccines induced T cell responses against MHC class II-restricted epitopes, Ab responses, and protection in two tumor models (myeloma and lymphoma). Two factors apparently contributed to these results: 1) agonistic ligation of CD40 and induction of DC maturation, and 2) delivery of Ag to APC and presentation on MHC class II molecules. These results highlight the importance of agonistic targeting of Ag to CD40 for induction of long-lasting and protective immune responses.

Human CD14 is an efficient target for recombinant immunoglobulin vaccine constructs that deliver T cell epitopes.

It has been shown in the mouse that recombinant immunoglobulin (Ig) molecules with T cell epitopes inserted into the constant domain (Troybodies) can target antigen-presenting cells (APC) for efficient delivery of T cell epitopes. Here, we have extended the Troybody concept to human applications. Moreover, we show that a receptor of innate immunity, CD14, which is a part of the lipopolysaccharide receptor complex on monocyte APC, is an efficient target. For construction of CD14-specific Troybodies, we used rearranged variable(diversity)joining regions cloned from the 3C10 mouse B cell hybridoma. As a model T cell epitope, amino acids 40-48 of mouse Ckappa, presented on human leukocyte antigen-DR4, were inserted into a loop connecting beta-strands in C(H)1 of human gamma3. In the presence of monocytes, CD14-specific Troybodies were >100 times as efficient as a nontargeting control antibody (Ab) at stimulating Ckappa(40-48)-specific/DR4-restricted T cells. Presentation was dependent on the conventional processing pathway for presentation on major histocompatibility complex (MHC) class II molecules. Enhanced presentation of the Ckappa epitope was most likely a result of increased loading of MHC class II molecules, as the CD14-specific monoclonal Ab 3C10 did not induce maturation of the APC. The results show that CD14, a receptor of innate immunity, may be a promising target of recombinant Ig-based vaccines for elicitation of T cell responses in humans.

Novel APC-like properties of human NK cells directly regulate T cell activation.

Initiation of the adaptive immune response is dependent on the priming of naive T cells by APCs. Proteomic analysis of unactivated and activated human NK cell membrane-enriched fractions demonstrated that activated NK cells can efficiently stimulate T cells, since they upregulate MHC class II molecules and multiple ligands for TCR costimulatory molecules. Furthermore, by manipulating antigen administration, we show that NK cells possess multiple independent unique pathways for antigen uptake. These results highlight NK cell-mediated cytotoxicity and specific ligand recognition by cell surface-activating receptors on NK cells as unique mechanisms for antigen capturing and presentation. In addition, we analyzed the T cell-activating potential of human NK cells derived from different clinical conditions, such as inflamed tonsils and noninfected and CMV-infected uterine decidual samples, and from transporter-associated processing antigen 2-deficient patients. This in vivo analysis revealed that proinflammatory, but not immune-suppressive, microenvironmental requirements can selectively dictate upregulation of T cell-activating molecules on NK cells. Taken together, these observations offer new and unexpected insights into the direct interactions between NK and T cells and suggest novel APC-like activating functions for human NK cells.

Antibody-mediated delivery of antigen to chemokine receptors on antigen-presenting cells results in enhanced CD4+ T cell responses.

Here, we have investigated if targeting of T cell epitopes to chemokine receptors results in improved CD4+ T cell responses. Mouse monoclonal antibodies (mAb) with kappaL chains were targeted to various chemokine receptors expressed on human monocytes or immature dendritic cells (DC), and proliferation of cloned human, DR4-restricted CD4+ T cells specific for mouse Ckappa(40-48) was measured. When using monocytes as antigen-presenting cells, mAb specific for CCR1, CCR2, CCR5, and CXCR4 were 100-10,000-fold more efficient at inducing T cell proliferation when compared to isotype-matched control mAb on a per molecule basis. Targeting of immature DC was less effective and was only seen with anti-CCR1 and anti-CXCR4 mAb. Anti-chemokine receptors mAb required to be processed by the conventional endosomal MHC class II presentation pathway. The mAb did not induce signaling through the chemokine receptors as they failed to induce mobilization of cytosolic Ca2+ and actin polymerization. They also failed to induce APC maturation. The results strongly suggest that chemokine receptors channel antigen into the endocytic pathway for presentation on MHC class II molecules. Targeting T cell epitopes to chemokine receptors by recombinant antibody should be a useful vaccine strategy for the induction of strong CD4+ T cell responses.

Cutting edge: link between innate and adaptive immunity: Toll-like receptor 2 internalizes antigen for presentation to CD4+ T cells and could be an efficient vaccine target.

An ideal vaccine for induction of CD4(+) T cell responses should induce local inflammation, maturation of APC, and peptide loading of MHC class II molecules. Ligation of Toll-like receptor (TLR) 2 provides the first two of these three criteria. We have studied whether targeting of TLR2 results in loading of MHC class II molecules and enhancement of CD4(+) T cell responses. To dissociate MHC class II presentation from APC maturation, we have used an antagonistic, mouse anti-human TLR2 mAb (TL2.1) as ligand and measured proliferation of a mouse Ckappa-specific human CD4(+) T cell clone. TL2.1 mAb was 100-1000 times more efficiently presented by APC compared with isotype-matched control mAb. Moreover, TL2.1 mAb was internalized into endosomes and processed by the conventional MHC class II pathway. This novel function of TLR2 represents a link between innate and adaptive immunity and indicates that TLR2 could be a promising target for vaccines.

A mouse C kappa-specific T cell clone indicates that DC-SIGN is an efficient target for antibody-mediated delivery of T cell epitopes for MHC class II presentation.

In vaccine development, a major objective is to induce strong, specific T cell responses. This might be obtained by targeting antigen to cell surface molecules that efficiently channel the antigen into endocytic compartments for loading of MHC molecules. Antibodies have been used to deliver antigen; however, it is important to define optimal targets on antigen-presenting cells (APC) for efficient delivery. For this purpose, we have established a T cell readout that can be used to screen large numbers of different mAb for their ability to load MHC class II molecules. The novel human CD4+ T cell clone is specific for mouse Ig C kappa (40-48) and restricted by HLA-DR4 (DRA1,B1*0401). DR4 apparently presents both mouse and human C kappa 40-48, but there is no cross-reaction at the T cell level. B cells from DR4 transgenic mice spontaneously process and present the mouse C kappa peptide. The mouse C kappa -specific T cell readout was used to demonstrate that mouse mAb specific for human dendritic cell (DC)-specific ICAM-grabbing non-integrin (DC-SIGN), a novel DC-specific molecule, were 10- to 1000-fold more potent at inducing kappa-specific human CD4+ T cell proliferation compared to control mAb. Consistent with this finding, DC-SIGN-specific mAb were rapidly internalized upon binding and found in intracellular vesicles. These results strongly argue that DC-SIGN-specific mAb are channeled into the MHC class II presentation pathway. Thus, DC-SIGN could be an efficient target for antibody-mediated delivery of T cell epitopes in vaccine development.