Tumour Escape from CAR-T Cells

AbstractOver the past decade, CAR-T cells have emerged as one of the most powerful cellular immune therapy approaches in the battle against haematological malignancies. Nonetheless, similar to other immunotherapeutic approaches, tumour cells develop strategies to evade CAR-T cell therapy, often with the support of a highly immunosuppressive and protective tumour microenvironment. To date, antigen loss, immune dysfunction, exhaustion and (microenvironment-mediated) upregulation of antiapoptotic pathways have been identified as major modes of tumour escape from CAR-T cell therapy. This chapter will focus on our current understanding of these modes of immune escape from CAR-T cells

Minor H Antigen HA-1–specific Regulator and Effector CD8+ T Cells, and HA-1 Microchimerism, in Allograft Tolerance

The role of the hematopoietic lineage-restricted minor histocompatibility (H) antigen HA-1 in renal allograft tolerance was explored. We obtained peripheral blood samples from three recipients of histocompatibility leukocyte antigen (HLA)–matched, HA-1–mismatched renal transplants, one of which had discontinued immunosuppression >30 yr ago while sustaining normal kidney function. Peripheral blood mononuclear cells (PBMCs) were injected into the footpads of severe combined immunodeficiency mice to measure human delayed type hypersensitivity (DTH) responses. All three patients manifested regulated DTH responses to HA-1H peptide. By differential tetramer staining intensities, we observed two distinct minor H antigen HA-1–specific CD8+ T cell subsets. The one that stained dimly had the characteristics of a T regulatory (TR) cell and produced interleukin (IL) 10 and/or transforming growth factor (TGF) β. These HA-1–specific TR cells coexisted with bright tetramer-binding CD8+ T effector (TE) cells. The CD8+ TE cells mediated HA-1–specific DTH and produced interferon-γ. Suppression of these TE functions by TR cells was TGFβ, IL-10, and cytotoxic T lymphocyte–associated antigen 4 dependent. In addition, HA-1 microchimerism was detected in two recipients, primarily in the dendritic cell fraction of the PBMCs. This is the first demonstration of coexisting CD8+ memory TR and TE cells, both specific for the same HA-1 antigen, in the context of renal allograft tolerance

Toward targeting B cell cancers with CD4+ CTLs: identification of a CD19-encoded minor histocompatibility antigen using a novel genome-wide analysis

Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)–matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4+ T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19L-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19L-specific CD4+ T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8+ mHag-specific T cells. They also lysed CD19L-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19L-derived HLA class II–restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings

Combined CD28 and 4-1BB Costimulation Potentiates Affinity-tuned Chimeric Antigen Receptor-engineered T Cells

Purpose: Targeting nonspecific, tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CART) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However, decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here, we demonstrate the prime importance of the type of intracellular signaling on the function of lowaffinity CAR-T cells. Experimental Design: We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. Results: We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (K d < 1.9 × 10 -6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity, preservation of a central memory phenotype, and significantly improved in vivo antitumor function, while preserving their ability to discriminate target antigen density. Conclusions: A combinatorial costimulatory design allows the use of very low-affinity binding domains (K d < 1 mmol/L) for the construction of safe but also optimally effective CAR-T cells. Thus, very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs

Efficacy and safety of daratumumab combined with all-trans retinoic acid in relapsed/refractory multiple myeloma

The efficacy of daratumumab depends partially on CD38 expression on multiple myeloma (MM) cells. We have previously shown that all-trans retinoic acid (ATRA) upregulates CD38 expression and reverts daratumumab-resistance ex vivo. We therefore evaluated the optimal dose, efficacy, and safety of daratumumab combined with ATRA in patients with daratumumab-refractory MM in a phase 1/2 study (NCT02751255). In part A of the study, 63 patients were treated with daratumumab monotherapy. Fifty patients with daratumumabrefractory MM were subsequently enrolled in part B and treated with daratumumab (reintensified schedule) combined with ATRA until disease progression. The recommended phase 2 dose of ATRA in combination with daratumumab was defined as 45 mg/m2. At this dose, the overall response rate (ORR) was 5%, indicating that the primary endpoint (ORR $15%) was not met. However, most patients (66%) achieved at least stable disease. After a median follow-up of 43 months, the median progression-free survival (PFS) for all patients was 2.8 months. Patients who previously achieved at least a partial response or minimal response/stable disease with prior daratumumab monotherapy had a significantly longer PFS compared with patients who immediately progressed during daratumumab as single agent (median PFS 3.4 and 2.8 vs 1.3 months). The median overall survival was 19.1 months. The addition of ATRA did not increase the incidence of adverse events. Flow cytometric analysis revealed that ATRA temporarily increased CD38 expression on immune cell subsets. In conclusion, the addition of ATRA and reintensification of daratumumab had limited activity in patients with daratumumab-refractory MM, which may be explained by the transient upregulation of CD38 expression. This trial was registered at www.clinicaltrials.gov as #NCT02751255

Targeting Alloreactive Donor T-Cells to Hematopoietic System-Restricted Minor Histocompatibility Antigens to Dissect Graft-versus-Leukemia Effects from Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

The graft-versus-leukemia (GVL) effect of HLA-identical allogeneic stem cell transplantation is mainly mediated by alloreactive T-cells directed at the minor histocompatibility antigens (H ags) expressed on the leukemic cells of the recipient. Minor H ags are major histocompatibility complex-bound polymorphic peptides that are derived from intracellular proteins and that can show ubiquitous or hematopoietic system-restricted expression. Whereas ubiquitous minor H ags are involved both in the GVL effect and in graft-versus-host disease (GVHD), hematopoietic system-specific minor H ags expressed on leukemic cells are considered important targets for leukemia-specific cellular immunotherapy with a low risk of GVHD. This review will summarize the current knowledge of the immunobiology of minor H ags and discuss the advantages and drawbacks of cellular immunotherapy strategies that aim to separate the GVL effect from GVHD by targeting donor T-cells to hematopoietic system-specific minor H ags

Challenges for immunotherapy in multiple myeloma: Bone marrow microenvironment-mediated immune suppression and immune resistance

The power of immunotherapy in the battle of Multiple Myeloma (MM) started with allogeneic stem cell transplantation, and was rediscovered with immunomodulatory drugs and extended with the outstanding results achieved with targeted antibodies. Today, next to powerful antibodies Elotuzumab and Daratumumab, several T-cell-based immunotherapeutic approaches, such as bispecific antibodies and chimeric antigen receptor-transduced T-cells (CAR T-cells) are making their successful entry in the immunotherapy arena with highly promising results in clinical trials. Nonetheless, similar to what is observed in chemotherapy, MM appears capable to escape from immunotherapy, especially through tight interactions with the cells of the bone marrow microenvironment (BM-ME). This review will outline our current understanding on how BM-ME protects MM-cells from immunotherapy through immunosuppression and through induction of intrinsic resistance against cytotoxic effector mechanisms of T- and NK-cells

The Connection Between Minor H Antigens and Neoantigens and the Missing Link in Their Prediction

For hundreds of thousands of years, the human genome has extensively evolved, resulting in genetic variations in almost every gene. Immunological reflections of these genetic variations become clearly visible after an allogeneic stem cell transplantation (allo-SCT) as minor Histocompatibility (H) antigens. Minor H antigens are peptides cleaved from genetically encoded variable protein regions after which they are presented at the cell surface by HLA molecules. After allo-SCT with minor H antigen mismatches between donor and recipient, donor T cells recognize the minor H antigens of the recipient as foreign, evoking strong alloreactive immune responses. Studies in the late eighties have discovered that a subset of minor H antigens are encoded by hematopoietic system-specific genes. After allo-SCT, this subset is strictly expressed on the hematopoietic malignant cells and was therefore the first well-defined highly immunogenic group of tumor-specific antigens. In the last decade, neoantigens derived from genetic mutations in tumors have been identified as another group of immunogenic tumor-specific antigens. Therefore, hematopoietic minor H antigens and neoantigens are therapeutic equivalents. This review will connect our current knowledge about the immune biology and identification of minor H antigens and neoantigens leading to novel conclusions on their prediction

Characterization of the Toll-like receptor expression profile in human multiple myeloma cells.

Expression and function of Toll-like receptors (TLRs) in multiple myeloma (MM) has recently become the focus of several studies. Knowledge of expression and biology of these receptors in MM will provide us with a new insight into the role of an inflammatory environment in disease progression or pathogenesis of MM. However, to date a quite heterogeneous expression pattern of TLRs in MM particularly at gene level has been described while information on the TLR expression at the protein level is largely unavailable. In this study, we investigated the TLR expression in human myeloma cell lines (HMCLs) Fravel, L363, UM6, UM9, OPM1, OPM2, U266, RPMI 8226, XG1, and NCI H929 and primary cells from MM patients at both mRNA and protein level (western blot and flow cytometry). We found that all cell lines and primary cells expressed TLR1, TLR3, TLR4, TLR7, TLR8, and TLR9 mRNA and protein. TLR2 and TLR5 were expressed by the majority of HMCLs at mRNA but were not detectable at protein level, while primary samples showed a low level of TLR2, TLR3 and TLR5 protein expression. Our results indicate that MM cells express a broad range of TLRs with a degree of disparity between gene and protein expression pattern. The clear expression of TLRs in MM cells indicates a propensity for responding to tumor-induced inflammatory signals, which seem inevitable in the MM bone marrow environment