赤塚 美樹

Negative immunofixation electrophoresis (IFE) of serum and/or urine is a diagnostic marker for determining a complete response (CR) after immunotherapy for multiple myeloma (MM). However, residual therapeutic antibodies such as elotuzumab (IgG-κ), can compromise IFE evaluation when the affected immunoglobulins belong to the same IgG-κ subclass. We thus sought to develop a simple and rapid method to treat patient serum before IFE to distinguish the residual elotuzumab. Serum samples from patients receiving elotuzumab were treated with a predetermined amount of soluble signaling lymphocyte activation molecule F7 (SLAMF7) protein and then subjected to conventional IFE testing. We tested our method in samples from 12 patients. The IgG-κ band in IFE disappeared or shifted after elotuzumab treatment in four patients with no bone marrow minimal residual disease and normalized free light chain, whereas seven patients with any sign of residual MM showed a remaining IgG-κ band after treatment. One-hour incubation of samples with 6-9 molar excess soluble SLAMF7 before IFE was sufficient to distinguish residual elotuzumab in 11 of 12 samples. This simple method does not require special reagents, can be performed in most clinical laboratories, and enables differentiation between patients with a CR and those requiring further treatment.

Minor histocompatibility antigens (mHAgs) in allogeneic hematopoietic stem cell transplantation are highly immunogenic as they are foreign antigens and cause polymorphism between donors and recipients. Adoptive cell therapy with mHAg-specific T cells may be an effective option for therapy against recurring hematological malignancies following transplantation. Genetically modified T cells with T cell receptors (TCRs) specific to mHAgs have been developed, but formation of mispaired chimeric TCRs between endogenous and exogenous TCR chains may compromise their function. An alternative approach is the development of chimeric antigen receptor (CAR)-T cells with TCR-like specificity whose CAR transmembrane and intracellular domains do not compete with endogenous TCR for CD3 complexes and transmit their own activation signals. However, it has been shown that the recognition of low-density antigens by high-affinity CAR-T cells has poor sensitivity and specificity. This mini review focuses on the potential for and limitations of TCR-like CAR-T cells in targeting human leukocyte antigen-bound peptide antigens, based on their recognition mechanisms and their application in targeting mHAgs.

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that cytotoxic T lymphocytes (CTLs) are involved in the development of AA. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in AA. Therefore, HLA-B*4002 is thought to play a critical role in the autoantigen presentation by HSPCs to CTLs. We previously identified the T-cell receptor (TCR) sequences from bone marrow (BM) CD8+ T cells in two CsA-dependent AA patients possessing B4002-lacking leukocytes (Case 1, Espinoza et al, Blood Adv, 2018) and B5401-lacking leukocytes (Case 2, Elbadry et al, Haematologica, 2019) by single-cell T-cell receptor (TCR) sequencing. Identifying the TCRs specific to antigens presented by these HLA class I alleles should allow us to screen autoantigens in AA. [Method] We established B4002+ or B5401+ K562 cell lines expressing CD80 and CD137L for the screening of antigen-specific T cell responses. To identify ligands of the TCR, we transfected peripheral blood (PB) T cells with a retrovirus vector containing different TCR cDNA derived from BM T cells and examined their responses to B4002+CD80+CD137L+ or B5401+CD80+CD137L+ K562 cells. Specific responses of each TCR transfectant to K562 cells or iPSC-derived CD34+ cells were determined using an enzyme-linked immunosorbent assay for detecting IFN-γ. Deep TCR sequencing of a current PB sample taken from the same patients was performed to determine whether or not T cells with specific TCRs persisted after successful immunosuppressive therapy (IST). [Results] In Case 1, two TCR transfectants (TCR-K1 and TCR-K2 which were the third- and second-most frequent TCRs in the BM T cells, respectively) secreted greater IFN-γ levels (1730 pg/mL and 2157 pg/mL, respectively) in response to B4002+CD80+CD137L+ K562 cells than those secreted by the other six transfectants (710 to 1184 pg/mL, respectively). TCR-K1 and TCR-K2 did not respond to an A2402+ counterpart (Figure). Notably, deep TCR sequencing of a current PB sample taken from Case 1 nine years after BM sampling revealed the persistence of the TCR-K1 sequence, suggesting that TCR-K1 may be responsible for CsA dependency of this patient. Deep TCR sequencing of other three AA patients with B4002-lacking leukocytes revealed decreased diversity of the T cell repertoire in CD8+ T cells but failed to reveal the same TCR motifs as Case 1. In Case 2, two TCR transfectants (TCR-K3 and TCR-K4) showed a specific response to B5401+CD80+CD137L+ K562 cells. Furthermore, these 2 TCR transfectants secreted higher amounts of IFN-γ (1.7 and 2.0 folds for TCR-K3 and TCR-K4, respectively) in response to wild-type iPSC-derived CD34+ cells than to B5401(-) CD34+ cells. [Conclusions] Our results suggest that these TCR transfectants recognized some intrinsic antigens derived from K562 cells in a B4002 or B5401-restricted manner. These TCR transfectants are the ideal tools for screening libraries of cDNA expressed by B4002+ COS/293T cells to identify autoantigens in AA. Figure Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Chugai Pharmaceutical Co.,Ltd: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Kyowa Kirin: Honoraria; Novartis Pharma K.K: Honoraria.

<title>Key Points</title> T cells expressing a CAR consisting of scFv #213 targeting WT1 peptide/HLA-A*2402 complex killed HLA-A*2402+ WT1+ tumor cell lines. The therapeutic efficacy of #213 scFv CAR-T cells was shown to be enhanced by DC vaccine in a murine xenograft model.

Selective graft-versus-tumor (GVT) reactivity with minimal risk of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation is thought to be induced by targeting minor histocompatibility (H) antigens (Ags) expressed only on patients’ hematopoietic cells. Among HLA-A* 02:01 positive patients, minor H Ags such as HA-1 and HA-2 have been shown to be associated with anti-tumor responses with minimal GVHD and explored for application to adoptive immunotherapy. Because preparation of Ag-specific cytotoxic T cell clones (CTLs) or lines for adoptive immunotherapy is labor-intensive and time consuming, the genetic transfer of T-cell receptors (TCRs) directed toward target Ags into T lymphocytes has been used to efficiently generate anti-tumor T cells without the need for in vitro induction and expansion. Alternatively, T cells could be gene-modified with a chimeric antigen receptor (CAR) harnessing a single chain antibody moiety (scFv). The conventional CAR strategy has the limitation of only targeting cell surface Ags on target cells. One possible way to attain intracellular Ag targeting with a CAR is to generate a TCR-like monoclonal antibody (mAb) as a source of scFv. In this study, we sought to generate highly specific mAbs specific for HA-1H minor H Ag by immunizing mice with tetramerized recombinant HLA-A2 incorporating HA-1H minor H Ag peptides and β2-microglobulin (HA-1H/HLA-A2). We hypothesized that the use of HLA-A2 transgenic mice, which should be tolerant to human HLA-A2, would facilitate efficient induction of mAbs specific for peptides presented on HLA-A2. Phage libraries were generated from splenic B cells and screened by panning for clones reactive to plate-bound HA-1H/HLA-A2 in the presence of free MAGEA4/HLA-A2 for competition. Candidate scFv encoded by obtained phage clones were transformed to scFv tetrameric Ab form or introduced into T cells as CAR coupled to CD28 transmembrane and CD3ζ domains (CD28-ζ). A total of 144 clones were randomly selected from 8.1×108 clones that had been recovered after the third panning. Among 144 clones, 18 (12.5%) showed preferential binding to HA-1/HLA-A2, 137 showed similar binding to both pMHC complexes, and 7 showed reactivity to neither of them. One of 18 scFv Abs, clone #131, demonstrated high affinity (KD = 8.34nM) for the HA-1H/HLA-A2 complex. Primary human CD8 T cells transduced with #131 scFv-CD28-ζ were stained with HA-1H/HLA-A2 tetramers as strongly as a CTL clone, EH6, specific for endogenously HLA-A2- and HA-1H-positive cells. Unexpectedly, however, #131 scFv-CD28-ζ CAR-T cells required ∼100-fold higher Ag density when pulsed exogenously to exert cytotoxicity than did the cognate EH6-CTL. In addition, mAb blocking experiments demonstrated that #131 scFv-CD28-ζCAR-T cells were less sensitive to CD8 blockade when they were completely blocked with HA-1H/HLA-A2 tetramer. These data suggest that T cells with higher affinity antigen receptors than TCRs (average KD ranging between 1μM∼100μM) are less able to recognize low density peptide/MHC antigens as reported in the case of affinity-matured TCR or CAR, and that CD8+ CAR-T cells may not be necessarily CD8-dependent possibly due to failure to form complexes with CD3. <sec> <title>Disclosures:</title> No relevant conflicts of interest to declare. </sec>