Autologous chimeric antigen receptor (CAR) T cells have changed the therapeutic landscape in haematological malignancies. Nevertheless, the use of allogeneic CAR T cells from donors has many potential advantages over autologous approaches, such as the immediate availability of cryopreserved batches for patient treatment, possible standardization of the CAR-T cell product, time for multiple cell modifications, redosing or combination of CAR T cells directed against different targets, and decreased cost using an industrialized process. However, allogeneic CAR T cells may cause life-threatening graft-versus-host disease and may be rapidly eliminated by the host immune system. The development of next-generation allogeneic CAR T cells to address these issues is an active area of research. In this Review, we analyse the different sources of T cells for optimal allogeneic CAR-T cell therapy and describe the different technological approaches, mainly based on gene editing, to produce allogeneic CAR T cells with limited potential for graft-versus-host disease. These improved allogeneic CAR-T cell products will pave the way for further breakthroughs in the treatment of cancer. The use of allogeneic chimeric antigen receptor T cells from donors has many potential advantages over autologous approaches, such as immediate availability, standardization and the possibility of redosing or combination. This Review analyses the different sources of T cells and technological approaches to produce optimal allogeneic chimeric antigen receptor T cells with limited potential for graft-versus-host disease and increased persistence.

'Off-the-shelf' allogeneic CAR T cells: development and challenges.

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression

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HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells

Progress in adoptive T-cell therapy for cancer and infectious diseases is hampered by the lack of readily available, antigen-specific, human T lymphocytes. Pluripotent stem cells could provide an unlimited source of T lymphocytes, but the therapeutic potential of human pluripotent stem cell-derived lymphoid cells generated to date remains uncertain. Here we combine induced pluripotent stem cell (iPSC) and chimeric antigen receptor (CAR) technologies to generate human T cells targeted to CD19, an antigen expressed by malignant B cells, in tissue culture. These iPSC-derived, CAR-expressing T cells display a phenotype resembling that of innate γδ T cells. Similar to CAR-transduced, peripheral blood γδ T cells, the iPSC-derived T cells potently inhibit tumor growth in a xenograft model. This approach of generating therapeutic human T cells 'in the dish' may be useful for cancer immunotherapy and other medical applications.

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Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy

Genetic and epigenetic variations in iPSCs: potential causes and implications for application.

https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/242420/1/yigak04494.pdf

Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility.

Generating an iPSC bank for HLA-matched tissue transplantation based on known donor and recipient HLA types.

How human genetic variation contributes to vaccine immunogenicity and effectiveness is unclear, particularly in infants from Africa. We undertook genome-wide association analyses of eight vaccine antibody responses in 2,499 infants from three African countries and identified significant associations across the human leukocyte antigen (HLA) locus for five antigens spanning pertussis, diphtheria and hepatitis B vaccines. Using high-resolution HLA typing in 1,706 individuals from 11 African populations we constructed a continental imputation resource to fine-map signals of association across the class II HLA observing genetic variation explaining up to 10% of the observed variance in antibody responses. Using follicular helper T-cell assays, in silico binding, and immune cell eQTL datasets we find evidence of HLA-DRB1 expression correlating with serological response and inferred protection from pertussis following vaccination. This work improves our understanding of molecular mechanisms underlying HLA associations that should support vaccine design and development across Africa with wider global relevance.

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High-resolution African HLA resource uncovers HLA-DRB1 expression effects underlying vaccine response


 
 
 Antibody characterisation is fundamental in transplantation and infectious diseases, but current immunoassays cannot determine two fundamental antibody properties, affinity (KD) and concentration ([Ab]). We aimed to overcome these limitations to allow in-depth profiling of antibodies directly in sera and provide insights into clinical translation.
 
 
 
 Using a microfluidic diffusional sizing-based strategy, we developed microfluidic antibody affinity profiling (MAAP), a novel in-solution immunoassay that simultaneously determines KD and [Ab] directly in serum. MAAP was developed and validated using the HLA-alloantibody system and applied in HLA Ab-incompatible (HLAi) transplantation and in anti-SARS-CoV-2 immunity.
 
 
 
 MAAP enabled quantification (KD and [Ab]) of alloantibody-HLA interactions in both purified and alloantibody-spiked sera. We demonstrated that transplant single-HLA-bead (SAB) immunoassays were avidity and [Ab]-dependent, cellular immunoassays (flow-cytometry and complement-dependent-cytotoxicity) were KD and [Ab]-dependent, antibody-mediated cytotoxicity was proportional to antibody-HLA KD, and micromolar antibody-HLA interactions were functionally insignificant despite high SAB signal. In HLAi transplants, MAAP differentiated clinically significant donor-specific-alloantibodies (leading to rejection) from those tolerated despite similar SAB assay output and provided insights into memory re-activation and immune-monitoring post-transplantation. In SARS-CoV-2, MAAP showed wide variation in anti-RBD antibody KD in convalescent sera (n=34), good correlation with serum neutralisation capacity (p<0.001), and evidence of affinity maturation 3-months post-infection (despite [Ab] reduction). In vaccinated sera (n=17), anti-RBD antibody KD was significantly weaker against Omicron than wild-type (p<0.001), providing insights into variant immune-escape strategies.
 
 
 
 This work outlines a path towards in-depth antibody profiling and demonstrates the importance of antibody abundance and affinity in clinically relevant humoral immunity.


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Sarah Peacock

Papers

Generating an iPSC bank for HLA-matched tissue transplantation based on known donor and recipient HLA types.

High-resolution African HLA resource uncovers HLA-DRB1 expression effects underlying vaccine response

O010 A novel microfluidic immunoassay for in-solution quantification of alloantibody affinity and concentration in transplantation and beyond