Daratumumab in combination with urelumab to potentiate anti-myeloma activity in lymphocytedeficient mice reconstituted with human NK cells

Daratumumab is an anti-CD38 fully human IgG1 mAb approved for multiple myeloma treatment. One of the proposed mechanisms of action is the induction of antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells. NK cells acquire surface CD137 expression in the presence of solid-phase-attached daratumumab and when encountering a daratumumab-coated CD38+ tumor cell line. In this setting, addition of the agonist anti-CD137 mAb urelumab enhances NK-cell activation increasing CD25 expression and IFNɣ production. However, in vitro ADCC is not increased by the addition of urelumab both in 4h or 24h lasting experiments. To study urelumab-increased daratumumab-mediated ADCC activity in vivo, we set up a mouse model based on the intravenous administration of a luciferase-transfected multiple myeloma cell line of human origin, human NK cells and daratumumab to immuno-deficient NSG mice. In this model, intravenous administration of urelumab 24h after daratumumab delayed tumor growth and prolonged mice survival

Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration

Fractionated initial infusion and booster dose of ARI0002h, a humanised, BCMA-directed CAR T-cell therapy, for patients with relapsed or refractory multiple myeloma (CARTBCMA-HCB-01): a single-arm, multicentre, academic pilot study

[Background]: Chimeric antigen receptor (CAR) T-cell therapy is a promising option for patients with heavily treated multiple myeloma. Point-of-care manufacturing can increase the availability of these treatments worldwide. We aimed to assess the safety and activity of ARI0002h, a BCMA-targeted CAR T-cell therapy developed by academia, in patients with relapsed or refractory multiple myeloma.[Methods]: CARTBCMA-HCB-01 is a single-arm, multicentre study done in five academic centres in Spain. Eligible patients had relapsed or refractory multiple myeloma and were aged 18–75 years; with an Eastern Cooperative Oncology Group performance status of 0–2; two or more previous lines of therapy including a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody; refractoriness to the last line of therapy; and measurable disease according to the International Myeloma Working Group criteria. Patients received an initial fractionated infusion of 3 × 106 CAR T cells per kg bodyweight in three aliquots (0·3, 0·9, and 1·8 × 106 CAR-positive cells per kg intravenously on days 0, 3, and 7) and a non-fractionated booster dose of up to 3 × 106 CAR T cells per kg bodyweight, at least 100 days after the first infusion. The primary endpoints were overall response rate 100 days after first infusion and the proportion of patients developing cytokine-release syndrome or neurotoxic events in the first 30 days after receiving treatment. Here, we present an interim analysis of the ongoing trial; enrolment has ended. This study is registered with ClinicalTrials.gov, NCT04309981, and EudraCT, 2019-001472-11.[Findings]: Between June 2, 2020, and Feb 24, 2021, 44 patients were assessed for eligibility, of whom 35 (80%) were enrolled. 30 (86%) of 35 patients received ARI0002h (median age 61 years [IQR 53–65], 12 [40%] were female, and 18 [60%] were male). At the planned interim analysis (cutoff date Oct 20, 2021), with a median follow-up of 12·1 months (IQR 9·1–13·5), overall response during the first 100 days from infusion was 100%, including 24 (80%) of 30 patients with a very good partial response or better (15 [50%] with complete response, nine [30%] with very good partial response, and six [20%] with partial response). Cytokine-release syndrome was observed in 24 (80%) of 30 patients (all grade 1–2). No cases of neurotoxic events were observed. Persistent grade 3–4 cytopenias were observed in 20 (67%) patients. Infections were reported in 20 (67%) patients. Three patients died: one because of progression, one because of a head injury, and one due to COVID-19.[Interpretation]: ARI0002h administered in a fractioned manner with a booster dose after 3 months can provide deep and sustained responses in patients with relapsed or refractory multiple myeloma, with a low toxicity, especially in terms of neurological events, and with the possibility of a point-of-care approach.This work has been supported in part by grants from the Instituto de Salud Carlos III (cofunded by the EU), Spanish Ministry of Health (ICI19/00025, FIS PI18/00775, PI19/00669, and PI22/00647), complementary grant for CONCORD-023, RICORS-TERAV network (RD21/0017/0009 and RD21/0017/0019), Red de Terapia Celular TERCEL (RD16/0011/0005), Fondo Europeo de Desarrollo Regional (FEDER), 2017SGR00792 (AGAUR; Generalitat de Catalunya), Centro de Investigación Biomédica en Red de Cáncer CIBERONC (CB16/12/00369 and CB16/12/00489), La Caixa Foundation (CP042702/LCF/PR/GN18/50310007), Asociación Española Contra el Cancer (AECC) LABAE21971FERN, and Fundació Bosch I Aymerich support. AOC received funding from the resident grant Ajut Clínic-La Pedrera 2019, granted by Hospital Clínic de Barcelona.Peer reviewe

Engineered promiscuous T helper peptides for the induction of immune responses

Following recognition of antigens by T helper (Th) lymphocytes, T cell help is elicited to induce humoral and cellular immune responses. These antigens are presented as short peptides, T helper peptides (THP), bound to MHC class II molecules. Since both endogenous THP (from antigens of interest) or exogenous THP (not encompassed by the sequence of the antigen of interest) are able to elicit T cell help, we decided to engineer promiscuous exogenous THP capable of binding to several HLA-DR molecules, in order to cover an important proportion of the human population. Some of these exogenous THP were able to bind to all seven HLA-DR molecules tested and were immunogenic in vivo in HLA-DR4 transgenic mice. Among them, peptides p37, p62 and p45 elicited Th1 cytokine profiles in vivo, providing help for the induction of potent CTL responses. Finally, in vitro stimulation assays carried out using human cells, showed that these peptides could induce T cell responses using cells obtained from individuals with a broad spectrum of HLA-DR molecules. Thus, engineered exogenous THP may be a valuable tool for the induction of immune responses in a large proportion of human population

Engineered promiscuous T helper peptides for the induction of immune responses

Following recognition of antigens by T helper (Th) lymphocytes, T cell help is elicited to induce humoral and cellular immune responses. These antigens are presented as short peptides, T helper peptides (THP), bound to MHC class II molecules. Since both endogenous THP (from antigens of interest) or exogenous THP (not encompassed by the sequence of the antigen of interest) are able to elicit T cell help, we decided to engineer promiscuous exogenous THP capable of binding to several HLA-DR molecules, in order to cover an important proportion of the human population. Some of these exogenous THP were able to bind to all seven HLA-DR molecules tested and were immunogenic in vivo in HLA-DR4 transgenic mice. Among them, peptides p37, p62 and p45 elicited Th1 cytokine profiles in vivo, providing help for the induction of potent CTL responses. Finally, in vitro stimulation assays carried out using human cells, showed that these peptides could induce T cell responses using cells obtained from individuals with a broad spectrum of HLA-DR molecules. Thus, engineered exogenous THP may be a valuable tool for the induction of immune responses in a large proportion of human population

Combined immunization with adjuvant molecules poly(I:C) and anti-CD40 plus a tumor antigen has potent prophylactic and therapeutic antitumor effects

The low immunogenicity of malignant cells is one of the causes responsible for the lack of antitumor immune responses. Thus, development of new therapeutic strategies aimed at enhancing presentation of tumor antigens to T cells is a main goal of cancer immunotherapy. With this aim, we studied the efficacy of administering adjuvants poly(I:C) and agonistic anti-CD40 antibody plus a tumor antigen. Joint intravenous immunization with these adjuvants and a model tumor antigen (ovalbumin) was able to synergistically induce potent and long lasting antitumor T-cell responses. These responses protected against challenge with E.G7-OVA tumor cells in prophylactic short- and long-term vaccination. In a therapeutic setting, repeated intratumor administration of adjuvants plus antigen was able to reject established tumors in all treated animals, leading in some cases to the rejection of both locally treated and untreated tumors. Antitumor immune responses induced by these protocols were mediated not only by T-cells but also by NK cells. In conclusion, combined administration of adjuvants poly(I:C) and anti-CD40 plus a tumor antigen is an efficient strategy for prophylactic and therapeutic antitumor vaccination

Hepatitis C virus structural proteins impair dendritic cell maturation and inhibit in vivo induction of cellular immune responses

Hepatitis C virus (HCV) chronic infection is characterized by low or undetectable cellular immune responses against HCV antigens. Some studies have suggested that HCV proteins manipulate the immune system by suppressing the specific antiviral T-cell immunity. We have previously reported that the expression of HCV core and E1 proteins (CE1) in dendritic cells (DC) impairs their ability to prime T cells in vitro. We show here that immunization of mice with immature DC transduced with an adenovirus encoding HCV core and E1 antigens (AdCE1) induced lower CD4(+)- and CD8(+)-T-cell responses than immunization with DC transduced with an adenovirus encoding NS3 (AdNS3). However, no differences in the strength of the immune response were detected when animals were immunized with mature DC subsequently transduced with AdCE1 or AdNS3. According to these findings, we observed that the expression of CE1 in DC inhibited the maturation caused by tumor necrosis factor alpha or CD40L but not that induced by lipopolysaccharide. Blockade of DC maturation by CE1 was manifested by a lower expression of maturation surface markers and was associated with a reduced ability of AdCE1-transduced DC to activate CD4(+)- and CD8(+)-T-cell responses in vivo. Our results suggest that HCV CE1 proteins modulate T-cell responses by decreasing the stimulatory ability of DC in vivo via inhibition of their physiological maturation pathways. These findings are relevant for the design of therapeutic vaccination strategies in HCV-infected patients

Helper cell-independent antitumor activity of potent CD8+ T cell epitope peptide vaccines is dependent upon CD40L

Peptide vaccines derived from CD8+ T-cell epitopes have shown variable efficacy in cancer patients. Thus, some peptide vaccines are capable of activating CD8+ T-cell responses, even in the absence of CD4+ T-cell epitopes or dendritic cell (DC)-activating adjuvants. However, the mechanisms underlying the clinical activity of these potent peptides are poorly understood. Using CT26 and ovalbumin-expressing B16 murine allograft tumor models, we found that the antitumor effect of helper cell-independent CD8 T-cell peptide vaccines is inhibited by the blockade of CD40 ligand (CD40L) in vivo. Furthermore, in vitro stimulation with antigenic peptides of cells derived from immunized mice induced the expression of CD40L on the surface of CD8+ T cells and fostered DC maturation, an effect that was partially inhibited by CD40L-blocking antibodies. Interestingly, CD40L blockade also inhibited CD8+ T-cell responses, even in the presence of fully mature DCs, suggesting a role for CD40L not only in promoting DC maturation but also in mediating CD8+ T-cell co-stimulation. Importantly, these potent peptides share features with bona fide CD4 epitopes, since they foster responses against less immunogenic CD8+ T-cell epitopes in a CD40L-dependent manner. The analysis of peptides used for the vaccination of cancer patients in clinical trials showed that these peptides also induce the expression of CD40L on the surface of CD8+ T cells. Taken together, these results suggest that CD40L expression induced by potent CD8+ T-cell epitopes can activate antitumor CD8+ T-cell responses, potentially amplifying the immunological responses to less immunogenic CD8+ T-cell epitopes and bypassing the requirement for CD4+ helper T cells in vaccination protocols

Monocyte-derived dendritic cells from HCV-infected patients transduced with an adenovirus expressing NS3 are functional when stimulated with the TLR3 ligand poly(I:C)

Dendritic cells (DC) transfected with an adenovirus encoding hepatitis C virus (HCV) NS3 protein (AdNS3) induce potent antiviral immune responses when used to immunize mice. However, in HCV infected patients, controversial results have been reported regarding the functional properties of monocyte-derived DC (MoDC), a cell population commonly used in DC vaccination protocols. Thus, with the aim of future vaccination studies we decided to characterize MoDC from HCV patients transfected with AdNS3 and stimulated with the TLR3 ligand poly(I:C). Phenotypic and functional properties of these cells were compared with those from MoDC obtained from uninfected individuals. PCR analysis showed that HCV RNA was negative in MoDC from patients after the culture period. Also, phenotypic analysis of these cells showed lower expression of CD80, CD86, and CD40, but similar expression of HLA-DR molecules as compared to MoDC from uninfected individuals. Functional assays of MoDC obtained from patients and controls showed a similar ability to activate allogeneic lymphocytes or to produce IL-12 and IL-10, although lower IFN-alpha levels were produced by cells from HCV patients after poly(I:C) stimulation. Moreover, both groups of MoDC induced similar profiles of IFN-gamma and IL-5 after stimulation of allogeneic T-cells. Finally, migration assays did not reveal any difference in their ability to respond to CCL21 chemokine. In conclusion, MoDC from HCV patients are functional after transduction with AdNS3 and stimulation with poly(I:C). These findings suggest that these cells may be useful for therapeutic vaccination in chronic HCV infection

Induction of potent and long-lasting CD4 and CD8 T-cell responses against hepatitis C virus by immunization with viral antigens plus poly(I:C) and anti-CD40

Development of vaccination strategies against hepatitis C virus (HCV) is of paramount importance. With this aim, we tested the ability of dendritic cell-activating reagents polyinosinic-polycytidylic acid (poly(I:C)) and anti-CD40, as adjuvants to induce T-cell responses against HCV. Immunization of mice with these adjuvants induced dendritic cell maturation in vivo. Also, joint administration of poly(I:C) and anti-CD40 plus HCV antigens had a synergistic effect on the induction of anti-HCV T-cell responses. CD4 responses displayed a Th1 cytokine profile, and CD8 responses could be induced by immunization with a minimal CD8 epitope. Addition of a low amount of NS3 protein (as a source of Th epitopes) to the immunization mixture enhanced CD8 responses, whereas immunization with higher doses of NS3 induced both CD4 and CD8 responses. Surprisingly, immunization with NS3 protein but not with CD8 epitopes was able to induce CD8 responses and able to recognize cells expressing HCV antigens endogenously. Moreover, immunization with these adjuvants activated NK cells, which in turn helped to induce Th1 responses. Finally, this combined immunization protocol afforded long-lasting T-cell responses, suggesting that this strategy may prove to be useful in vaccination and/or treatment of HCV infection