Augmenting CAR T Cell Functions with LIGHT.

Chimeric antigen receptor (CAR) T-cell therapy has resulted in remarkable clinical success in the treatment of B-cell malignancies. However, its clinical efficacy in solid tumors is limited, primarily by target antigen heterogeneity. To overcome antigen heterogeneity, we developed CAR T cells that overexpress LIGHT, a ligand of both LTßR on cancer cells and HVEM on immune cells. LIGHT-expressing CAR T cells displayed both antigen-directed cytotoxicity mediated by the CAR and antigen-independent killing mediated through the interaction of LIGHT with LTßR on cancer cells. Moreover, CAR T cells expressing LIGHT had immunostimulatory properties that improved the cells' proliferation and cytolytic profile. These data indicate that LIGHT-expressing CAR T cells may provide a way to eliminate antigen-negative tumor cells to prevent antigen-negative disease relapse.

Developing a membrane-proximal CD33-targeting CAR T cell.

BACKGROUND: CD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking. METHODS: We developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR T cell derived from a high-affinity binder obtained through membrane-proximal fragment immunization in humanized mice. RESULTS: We found that immunization exclusively with the membrane-proximal domain of CD33 is necessary for identification of membrane-proximal binders in humanized mice. Compared with clinically validated lintuzumab-based CAR T cells targeting distal CD33 epitopes, 3P14HLh28Z showed enhanced in vitro functionality as well as superior tumor control and increased overall survival in both low antigen density and clinically relevant patient-derived xenograft models. Increased activation and enhanced polyfunctionality led to enhanced efficacy. CONCLUSIONS: Showing for the first time that a membrane-proximal CAR is superior to a membrane-distal one in the setting of CD33 targeting, our results demonstrate the rationale for targeting membrane-proximal epitopes with high-affinity binders. We also demonstrate the importance of optimizing CAR T cells for functionality in settings of both low antigen density and clinically relevant patient-derived models.

Can a sperm selection technique improve embryo ploidy?

BACKGROUND: Spermatozoa with the highest motility retain a superior genomic integrity, and elevated sperm chromatin fragmentation (SCF) has been linked to a lower ability of the conceptus to develop and implant. Therefore, the utilization of a sperm selection method, such as microfluidic sperm selection (MFSS), is capable of reducing the SCF by yielding the most motile fraction of spermatozoa with the highest embryo developmental competence. What remains unclear, however, is the causal mechanism that links SCF to an impaired embryo development. OBJECTIVES: To identify a relationship between SCF and an unexpectedly high proportion of embryo aneuploidy, while addressing treatment options. MATERIALS AND METHODS: We identified couples with a high incidence of embryo aneuploidy in a previous intracytoplasmic sperm injection (ICSI) cycle with pre-implantation genetic testing for aneuploidy (PGT-A), utilizing spermatozoa selected by density gradient (DG). Terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) and neutral Comet assays were carried out on the semen specimens to assess total SCF and double-stranded DNA (dsDNA) fragmentation, respectively. These couples underwent subsequent ICSI/PGT-A cycles with MFSS. Total SCF and dsDNA fragmentation were compared between the two sperm selection methods. Embryo aneuploidy, implantation, clinical pregnancy, delivery, and pregnancy loss rates were compared between the couples' historical DG and subsequent MFSS cycles. RESULTS: In 57 couples undergoing 71 ICSI/PGT-A cycles, where DG sperm selection was carried out, a high incidence of aneuploid embryos (74.7%) resulted in poor implantation and no viable pregnancies. Testing for SCF, inclusive of dsDNA breaks, evidenced a SCF of 26.2% and dsDNA break of 3.6% in the raw specimen, that decreased to 18.0% (p < 0.001) and 3.1%, respectively, in the DG processed specimen. Following MFSS, total SCF and dsDNA fragmentation decreased to 1.9% and 0.3%, respectively (p < 0.001). The embryo euploidy rate remarkable improved from 25.3% in the DG cycles to 42.9% in the MFSS cycles (p < 0.001). The 6.7% implantation rate in the DG cycles increased to 65.5% in the MFSS cycles (p < 0.001). Similarly, the clinical pregnancy rate rose from 10.5% (DG) to 64.6% (MFSS), resulting in a 62.5% delivery rate (p < 0.001). DISCUSSION AND CONCLUSIONS: In couples with a relatively young female partner with a negative infertility workup, and a male partner with semen parameters adequate for ICSI, presenting with a high rate of embryo aneuploidy, an additional subtle male factor component may be the culprit. Thus, it is crucial to assess the SCF and test for the dsDNA breaks, which can eventually contribute to embryo chromosomal abnormalities. Given the inverse relationship between SCF and motility, a selection of the most motile gamete by MFSS enhanced the proportion of spermatozoa with an intact genome, contributing to the generation of more euploid embryos that are capable of implanting and yielding increased term pregnancies.

Assessing male gamete genome integrity to ameliorate poor assisted reproductive technology clinical outcome.

OBJECTIVE: To assess the role of evaluating sperm chromatin fragmentation (SCF) as a tool to guide treatment in couples who achieved unexpectedly poor clinical outcomes after intracytoplasmic sperm injection (ICSI). DESIGN: We identified couples with an unexpectedly suboptimal clinical outcome after ICSI who were then screened for SCF. Consequently, the same couples were counseled to undergo a subsequent ICSI cycle using either ejaculates processed by microfluidic sperm selection (MFSS) or spermatozoa retrieved from the testis, and clinical outcomes were compared between history and treatment cycles. To confirm the sole effect of a compromised male gamete, we compared the ICSI outcome in cycles where male gametes with abnormal SCF were used to inseminate autologous and donor oocytes. Finally, to eliminate an eventual confounding female factor component, we compared the clinical outcome of ICSI cycles using sibling donor oocytes injected with spermatozoa with normal or abnormal SCF. SETTING: Academic reproductive medicine center point of care. PATIENT(S): The patient population consisted of 76 couples with reproductively healthy and relatively young female partners and male partners with compromised semen parameters, but suitable for ICSI. In a subanalysis, we identified 67 couples with abnormal SCF who underwent ICSI cycle(s) with donor oocytes. Furthermore, we identified 29 couples, 12 with normal SCF and 17 with abnormal, uncorrected SCF, and 7 couples with abnormal, corrected SCF vs. a control, who used sibling donor oocytes for their ICSI cycle(s). INTERVENTION(S): For couples who resulted in surprisingly low clinical outcomes after ICSI, despite semen parameters adequate for ICSI and a normal female infertility evaluation, a SCF assessment was performed on the semen specimen using the terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labeling (TUNEL) assay. The couples then underwent a subsequent ICSI cycle with spermatozoa processed by MFSS or surgically retrieved. Moreover, cycles with donor oocytes were used to confirm the sole contribution of the male gamete. MAIN OUTCOME MEASURE(S): Clinical outcomes, such as fertilization, embryo implantation, clinical pregnancy, delivery, and pregnancy loss rates were compared between history and treatment cycle(s) using ejaculated spermatozoa selected by MFSS or from a testicular biopsy, taking into consideration the level of SCF. In a subanalysis, we reported the clinical outcomes of 67 patients who used donor oocytes and compared them with cycles where their own oocytes were used. Furthermore, we compared the ICSI clinical outcomes between cycles using sibling donor oocytes injected with low or high SCF with or without sperm intervention aimed at correcting, or alleviating the degree of SCF. RESULT(S): In a total of 168 cycles, 76 couples had in a prior cycle a 67.1% fertilization rate, and clinical pregnancy and pregnancy loss rates of 16.6% and 52.3%, respectively. After testing for SCF, the DNA fragmentation rate was 21.6%. This led to a subsequent ICSI cycle with MFSS or testicular sperm extraction, resulting in clinical pregnancy and delivery rates of 39.2%, and 37.3%, respectively. The embryo implantation rate increased to 23.5%, whereas the pregnancy loss rate decreased to 5% in the treatment cycle. This was particularly significant in the moderate SCF group, reaching embryo implantation, clinical pregnancy, and delivery rates of 24.3%, 40.4%, and 36.2%, respectively, and reducing the pregnancy loss rate to 10.5% in post-sperm treatment cycles. In 67 patients with high SCF who used donor oocytes, a significantly higher fertilization rate of 78.1% and embryo implantation rate of 29.1% were reported, compared with those in couples also with an elevated SCF who used their own. Interestingly, the clinical pregnancy and delivery rates only increased slightly from 28.0%-36.1% and from 23.7%-29.2%, respectively. To further control for a female factor, we observed couples who shared sibling donor oocytes, 17 with normal SCF and 12 with abnormal (uncorrected) SCF. Interestingly, the abnormal SCF group had impaired fertilization (69.3%), embryo implantation (15.0%), and delivery (15.4%) rates. For an additional 15 couples who split their donor oocytes, 8 had normal SCF, and although 7 couples originally had abnormal SCF, 4 used microfluidic processing, 2 used testicular spermatozoa, and 1 used donor spermatozoa to alleviate the degree of SCF, resulting in comparable clinical outcomes with the normal SCF group. CONCLUSION(S): A superimposed male factor component may explain the disappointing ICSI outcome in some couples despite reproductively healthy female partners. Therefore, it may be useful to screen couples for SCF to guide treatment options and maximize chances of a successful pregnancy. The improved, but suboptimal pregnancy and delivery outcomes observed in couples using donor oocytes confirmed the exclusive detrimental role that the male gamete exerted on embryo development despite the presence of putative oocyte repair mechanisms.