Antigen Escape Research Articles

BSBM-14 MOLECULAR PROFILING OF CHECKPOINT LIGANDS AND KEY NK-ACTIVATION AND SUPPRESSIVE LIGANDS BY METASTATIC MELANOMA CELLS

Abstract Melanoma is an aggressive skin cancer with high propensity for brain metastasis. For advanced melanoma, immune checkpoint blockade (ICB) drugs, like pembrolizumab, have shown remarkable promise for progression-free survival. The ICB drugs have primarily targeted melanoma suppression of cytotoxic T-lymphocytes (CTLs). Earlier studies showed positive responses in up to 40% of cases, which has improved with discovery of new inhibitory ligands and combination therapy. However, not all patients respond, likely because of the capacity of cancer cells to regulate inhibitory and activating ligands, circumventing both immunomodulatory axes currently targeted by immunotherapies. In this study, we aim to characterize additional axes, prioritizing NK cells of the innate immune system. NK cells can mediate cytotoxic killing similar to CTLs but possess the advantages of germline-encoded receptors that recognize a broad range of ligands, thus, requiring relatively little priming and circumventing antigen escape. We have acquired up to ten patient-derived New Zealand Melanoma lines in collaboration with Auckland Cancer Society Research Centre, of which three are from brain metastases. We used NanoString technology to assess melanoma gene expression of immunoregulatory ligands governing NK activation, inhibition or both (via dual-functional ligands dictated by NK-receptors). The results showed expression of several inhibitory ligands but very few activating ligands. The expression trends were largely similar but varied in a select few ligands. Flow cytometry was used to characterize cell-surface expression of the immunoregulatory proteins. These were compared with the classical inhibitory checkpoint molecules evidencing that melanoma cells expressed more NK inhibitory molecules. We hypothesize that melanoma cells both express inhibitory ligands and suppress activation ligands, to hinder NK cell activity. Here we aim to identify novel immune axes leading towards expansion of ICB therapies. This will afford clinicians a personalized approach and access to combination immunotherapies, especially in combating refractory metastatic melanoma, which have poor prognoses.

Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment

T cell lymphoma (TCL) is a highly heterogeneous group of diseases with a poor prognosis and low 5-year overall survival rate. The current therapeutic regimens have relatively low efficacy rates. Clinical studies of single-target chimeric antigen receptor Tcell (CAR-T cell) therapy in T lymphocytes require large and multiple infusions, increasing the risks and cost of treatment; therefore, optimizing targeted therapy is a way to improve overall prognosis. Despite significant advances in bispecific CAR-T cell therapy to avoid antigen escape in treatment of B cell lymphoma, applying this strategy to TCL requires further investigation. Here, we constructed an alpaca nanobody (Nb) phage library and generated high-affinity and -specificity Nbs targeting CD30 and CD5, respectively. Based on multiple rounds of screening, bispecific NbCD30-CD5-CAR Tcells were constructed, and their superior anti-tumor effect against TCL was validated invitro and invivo. Our findings demonstrated that Nb-derived bispecific CAR-T cells significantly improved anti-tumor efficacy in TCL treatment compared with single-target CAR-T cells and bispecific single chain variable fragment (scFv)-derived CAR-T cells. Because Nbs are smaller and less immunogenic, the synergistic effect of Nb-based bispecific CAR-T cells may improve their safety and efficacy in future clinical applications.

Population dynamics of immunological synapse formation induced by bispecific T cell engagers predict clinical pharmacodynamics and treatment resistance.

Effector T cells need to form immunological synapses (IS) with recognized target cells to elicit cytolytic effects. Facilitating IS formation is the principal pharmacological action of most T cell-based cancer immunotherapies. However, the dynamics of IS formation at the cell population level, the primary driver of the pharmacodynamics of many cancer immunotherapies, remains poorly defined. Using classic immunotherapy CD3/CD19 bispecific T cell engager (BiTE) as our model system, we integrate experimental and theoretical approaches to investigate the population dynamics of IS formation and their relevance to clinical pharmacodynamics and treatment resistance. Our models produce experimentally consistent predictions when defining IS formation as a series of spatiotemporally coordinated events driven by molecular and cellular interactions. The models predict tumor-killing pharmacodynamics in patients and reveal trajectories of tumor evolution across anatomical sites under BiTE immunotherapy. Our models highlight the bone marrow as a potential sanctuary site permitting tumor evolution and antigen escape. The models also suggest that optimal dosing regimens are a function of tumor growth, CD19 expression, and patient T cell abundance, which confer adequate tumor control with reduced disease evolution. This work has implications for developing more effective T cell-based cancer immunotherapies.

High frequency of Lamivudine and Telbivudine resistance mutations in hepatitis B virus isolates from human immunodeficiency virus co-infected patients on highly active antiretroviral therapy in Bucaramanga, Colombia.

Hepatitis B virus (HBV) antiviral Resistance-Associated Mutations (RAMs) in human immunodeficiency virus (HIV) coinfected patients undergoing highly active antiretroviral therapy (HAART) are complex and incompletely understood. We aimed to determine the prevalence of HBV coinfection, HBV genotypes, and RAMs in a cohort of people living with HIV (PLWH) in the northeastern region of Colombia. This cross-sectional study was carried out between February 2013 and February 2014. Virological, immunological and HAART data were collected from clinical records. In-house nested PCR and Sanger sequencing of the HBV pol gene were used to identify coinfections, genotypes, RAMs and HBV s antigen (HBsAg) escape mutants. Among 275 PLWH, HBV coinfection was confirmed in 32 patients (11.6%), of whom nine (28.2%) were HBsAg positive (active hepatitis B), and 23 (71.8%) were occult hepatitis B infections (OBI). All HBV sequences (n = 23) belonged to the genotype F3. Among HIV/HBV coinfections, 71.9% had CD4+ T cell counts above 200 cells/mm3 and 37.5% had undetectable HIV viral loads. The RAMs rtL80I, rtL180M, and rtM204V, which confer resistance to Lamivudine/Telbivudine and partially resistant to Entecavir, were found in all HBV isolates. An unknown rt236Y mutation to Tenofovir was also identified. Most patients under HAART received first-generation HBV antiviral therapy with a low genetic barrier to resistance. Antiviral Drug-associated Potential Vaccine-escape Mutations (ADAPVEMs) in the S gene were observed in all isolates ranging from 1-20 amino acid substitutions. However, no vaccine escape mutants were detected. In Conclusion, these findings highlight the importance of HBV molecular screening, antiviral resistance monitoring and new guidelines for PLWH to overcome RAMs and prevent HBV-related liver disease.

Challenges and solutions to superior chimeric antigen receptor-T design and deployment for B-cell lymphomas.

Chimeric antigen receptor-T (CAR-T) therapies represent a major breakthrough in cancer medicine, given the ex vivo-based technology that harnesses the power of one's own immune system. These therapeutics have demonstrated remarkable success for relapsed/refractory B-cell lymphomas. Although more than a decade has passed since the initial introduction of CAR-T therapeutics for patients with leukaemia and lymphoma, there is still significant debate as to where CAR-T therapeutics fit into the management paradigm, as consensus guidelines are limited. Competing interventions deployed in subsequent lines of therapy for aggressive lymphoma include novel targeted agents, bispecific antibodies, and time-honoured stem cell transplant. In this focused review, we discuss the major obstacles to advancing the therapeutic reach for CAR-T products in early lines of therapy. Such barriers include antigen escape, "cold" tumour microenvironments, host inflammation and CAR-T cell exhaustion. We highlight solutions including point-of-care CAR-T manufacturing and early T lymphopheresis. We review the evidence basis for early CAR-T deployment for B-cell lymphomas in light of the recent Food and Drug Administration (FDA) approval of three first-in-class anti-CD3/CD20 bispecific antibodies-mosunetuzumab, epcoritamab and glofitamab. We propose practical recommendations for 2024.

T-cell redirecting bispecific and trispecific antibodies in multiple myeloma beyond BCMA.

B-cell maturation antigen (BCMA)-directed T-cell immunotherapies, such as chimeric antigen receptor T-cells (CAR T-cells) and bispecific antibodies (BsAbs) have markedly improved the survival of triple-class refractory multiple myeloma (MM). However, the majority of patients still develops disease progression, underlining the need for new agents for these patients. Novel T-cell redirecting BsAbs targeting alternative tumor-associated antigens have shown great promise in heavily pretreated MM, including patients previously exposed to BCMA-directed therapies. This includes the G-protein-coupled receptor class 5 member D (GPRC5D)-targeting BsAbs talquetamab and forimtamig, as well as the Fc receptor-homolog 5 (FcRH5)-targeting BsAb cevostamab. Toxicity associated with these BsAbs includes cytokine-release syndrome, cytopenias, and infections. In addition, GPRC5D-targeting BsAbs are associated with specific 'on target/off tumor' toxicities including rash, nail disorders, and dysgeusia. Trispecifc antibodies targeting two different MM-associated antigens to prevent antigen escape are in early clinical development, as well as trispecific antibodies (TsAbs) that provide an additional co-stimulatory signal to T-cells to prevent their exhaustion. Various T-cell redirecting BsAbs are in advanced stages of clinical development with promising activity and a manageable toxicity profile. Ongoing studies are evaluating combination strategies, fixed-duration treatment, and use of BsAbs in earlier lines of therapy. TsAbs hold great promise for the future.

FLT3-directed UniCAR T-cell therapy of acute myeloid leukaemia.

Adaptor chimeric antigen receptor (CAR) T-cell therapy offers solutions for improved safety and antigen escape, which represent main obstacles for the clinical translation of CAR T-cell therapy in myeloid malignancies. The adaptor CAR T-cell platform 'UniCAR' is currently under early clinical investigation. Recently, the first proof of concept of a well-tolerated, rapidly switchable, CD123-directed UniCAR T-cell product treating patients with acute myeloid leukaemia (AML) was reported. Relapsed and refractory AML is prone to high plasticity under therapy pressure targeting one single tumour antigen. Thus, targeting of multiple tumour antigens seems to be required to achieve durable anti-tumour responses, underlining the need to further design alternative AML-specific target modules (TM) for the UniCAR platform. We here present the preclinical development of a novel FMS-like tyrosine kinase 3 (FLT3)-directed UniCAR T-cell therapy, which is highly effective for in vitro killing of both AML cell lines and primary AML samples. Furthermore, we show in vivo functionality in a murine xenograft model. PET analyses further demonstrate a short serum half-life of FLT3 TMs, which will enable a rapid on/off switch of UniCAR T cells. Overall, the presented preclinical data encourage the further development and clinical translation of FLT3-specific UniCAR T cells for the therapy of AML.

The Emerging Role of Engineering Immune Cells in Cancer Treatments

Cell-based immunotherapy has become one of the forefronts of cancer treatments and led to significant clinical success in multiple refractory/recurrent hematological malignancies. Compared with other conventional treatment approaches, engineered immune cells are considered “living factories” that are able to continually produce anti-tumor factors and have the potential to mediate long-lasting therapeutic benefits following a single application. The intrinsic ability to expand and respond in portion to needs encompasses this technology a greater and more transformative potential to enable a more effective anti-tumor response with less off-target toxicity. Nevertheless, there are still some significant barriers to successfully applying cell-based therapy to treat solid tumors. Five main challenges include restricted trafficking and infiltration, antigen escape and heterogeneity, suboptimal persistence, immunosuppressive tumor microenvironment (TME), and potentially severe side effects and immune-related toxicities. The technological advancement of various biomolecular tools and genetic engineering strategies provides exciting opportunities to address these limitations. In addition, combination therapy that incorporates other treatment modalities within the treatment regimen of cell-based strategy also creates therapeutic synergies that can greatly improve the clinical success of the therapy. This review introduces current observed challenges in treating cancers, with an emphasis on solid malignancies, and discusses some potential engineering solutions that have shown promising results in recent preclinical studies.

Natural Products for the Immunotherapy of Glioma.

Glioma immunotherapy has attracted increasing attention since the immune system plays a vital role in suppressing tumor growth. Immunotherapy strategies are already being tested in clinical trials, such as immune checkpoint inhibitors (ICIs), vaccines, chimeric antigen receptor T-cell (CAR-T cell) therapy, and virus therapy. However, the clinical application of these immunotherapies is limited due to their tremendous side effects and slight efficacy caused by glioma heterogeneity, antigen escape, and the presence of glioma immunosuppressive microenvironment (GIME). Natural products have emerged as a promising and safe strategy for glioma therapy since most of them possess excellent antitumor effects and immunoregulatory properties by reversing GIME. This review summarizes the status of current immunotherapy strategies for glioma, including their obstacles. Then we discuss the recent advancement of natural products for glioma immunotherapy. Additionally, perspectives on the challenges and opportunities of natural compounds for modulating the glioma microenvironment are also illustrated.

Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors

Although adoptive T cell therapy provides the T cell pool needed for immediate tumor debulking, the infused T cells generally have a narrow repertoire for antigen recognition and limited ability for long-term protection. Here, we present a hydrogel that locally delivers adoptively transferred T cells to the tumor site while recruiting and activating host antigen-presenting cells with GMCSF or FLT3L and CpG, respectively. T cells alone loaded into these localized cell depots provided significantly better control of subcutaneous B16-F10 tumors than T cells delivered through direct peritumoral injection or intravenous infusion. T cell delivery combined with biomaterial-driven accumulation and activation of host immune cells prolonged the activation of the delivered T cells, minimized host T cell exhaustion, and enabled long-term tumor control. These findings highlight how this integrated approach provide both immediate tumor debulking and long-term protection against solid tumors, including against tumor antigen escape.

Phase 1b multicenter study to evaluate CHM 1101 in patients with recurrent or progressive glioblastoma.

TPS2086 Background: Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor. More than 300,000 new cases are diagnosed globally with over 250,000 deaths each year (Sung H, et al. CA Cancer J Clin. 2021). Patients with recurrent GBM have a poor prognosis, with limited treatment options and a median survival of less than 1 year (Gallego. Curr Oncol, 2015). While prior attempts to treat GBM with chimeric antigen receptor (CAR) T-cells have been limited by tumor heterogeneity, chlorotoxin (CLTX)-directed CAR T-cells in mice demonstrated broad anti-tumor activity and prolonged survival with no off-tumor toxicity or antigen escape (Wang, et al. Sci Transl Med, 2020). CLTX, a 36–amino acid peptide identified in scorpion venom, selectively binds to malignant glioma cells through matrix metalloproteinase-2 (MMP2) and clinical administration of CLTX-based biologics has been well tolerated in patients (Mamelak, et al. J Clin Oncol, 2005, Patil, et al. Neurosurgery, 2019). CHM 1101 is the first CAR T to utilize CLTX as its tumor targeting domain for autologous CAR T-cell therapy. A single-center first-in-human phase 1 study of CHM 1101 in patients with recurrent GBM is ongoing. Methods: Clinical Trial NCT05627323 is a phase 1b, multi-center study of CHM 1101 in adult subjects with MMP2+ recurrent or progressive GBM after standard therapy. After leukapheresis and tumor resection, pts will receive CHM 1101 at Dose Level 1 (240 × 106 CAR T cells) or Dose Level 2 (440 x 106d CAR T-cells), divided across 3 once-weekly intracranial (intracavitary and intraventricular) administrations; after disease assessment at 28 days, additional doses of CHM 1101 can be administered on a weekly schedule. Eligible subjects have a prior histologically confirmed diagnosis of a grade 4 GBM, a grade 2 or 3 malignant glioma with radiographic progression consistent with a grade 4 GBM (IDH wild type), grade 4 diffuse astrocytoma (IDH mutant), or a unifocal relapse of GBM. MMP2+ tumor expression is confirmed by IHC (≥20% moderate/high MMP2 score [2+ or 3+]). The primary endpoint is safety; key secondary endpoints include feasibility, progression-free survival, overall survival, response rate (RANO criteria), and cellular kinetics. Recruitment is ongoing. Clinical trial information: NCT05627323 .

First results from the RedirecTT-1 study with teclistamab (tec) + talquetamab (tal) simultaneously targeting BCMA and GPRC5D in patients (pts) with relapsed/refractory multiple myeloma (RRMM).

8002 Background: Tec is the first BCMA-directed bispecific antibody approved for the treatment of triple-class exposed RRMM. Tal, a bispecific antibody targeting the novel myeloma antigen GPRC5D, has shown promising efficacy in pts with RRMM. Simultaneously targeting 2 validated myeloma target antigens, using tec + tal in combination may lead to improved outcomes by overcoming resistance mechanisms, such as antigen escape. Here, we report the first results from the phase 1b RedirecTT-1 trial (NCT04586426) in pts with RRMM. Methods: Enrolled pts had MM per International Myeloma Working Group 2016 criteria; were RR or intolerant to the last line of therapy (LOT); were exposed to a proteasome inhibitor, immunomodulatory drug, and anti-CD38 therapy; and had measurable disease. The primary objectives are to evaluate safety and to identify a recommended phase 2 regimen (RP2R) for the combination. Responses were investigator assessed. AEs were graded per CTCAE v5.0. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded per ASTCT criteria. Results: As of Dec 12, 2022, 63 pts received tec + tal. Median (range) age was 67 y (39–81); median (range) prior LOT was 5 (1–11); 33% (15/45) had high-risk cytogenetics; 78% (49/63) were triple-class refractory; 63% (40/63) were penta-drug exposed; and 43% (27/63) had extramedullary disease (EMD; all bone independent). Median (range) duration of follow-up was 14.4 mos (0.5–21.9). The most common treatment-emergent AEs were CRS (81%; grade [gr] 3, 3%, no gr 4), neutropenia (76%; gr 3/4, 75%), and anemia (60%; gr 3/4, 43%). Dose-limiting toxicities (DLTs) were reported at dose level 1 (gr 3 herpetic stomatitis) and dose level 3 (gr 3 AST/ALT elevation). One ICANS event was reported at dose level 3. No DLTs were reported at the RP2R. Across all dose levels, overall response rate (ORR) was 84% (52/62) among all evaluable pts and 73% (19/26) among evaluable pts with EMD; rate of CR or better (≥CR) was 34% (21/62) and 31% (8/26), respectively. At the RP2R, ORR was 92% (12/13) among all evaluable pts and 83% (5/6) among evaluable pts with EMD; rate of ≥CR was 31% (4/13) and 33% (2/6), respectively. Median duration of response has not been reached. Updated data, with 19 additional pts at the RP2R, will be presented. Conclusions: In this first combination study of a BCMA- and GPRC5D-targeted bispecific antibody, tec + tal at the RP2R has a manageable safety profile consistent with each of the monotherapies. A 92% ORR was observed in pts with advanced RRMM at the RP2R, and an ORR of 83% was achieved in pts with EMD, a high-risk population with unmet need, supporting further evaluation of the combination. Clinical trial information: NCT04586426 .

Preclinical Evidence of an Allogeneic Dual CD20xCD22 CAR to Target a Broad Spectrum of Patients with B-cell Malignancies.

Despite the remarkable success of autologous chimeric antigen receptor (CAR) T cells, some patients relapse due to tumor antigen escape and low or uneven antigen expression, among other mechanisms. Therapeutic options after relapse are limited, emphasizing the need to optimize current approaches. In addition, there is a need to develop allogeneic "off-the-shelf" therapies from healthy donors that are readily available at the time of treatment decision and can overcome limitations of current autologous approaches. To address both challenges simultaneously, we generated a CD20xCD22 dual allogeneic CAR T cell. Herein, we demonstrate that allogeneic CD20x22 CAR T cells display robust, sustained and dose-dependent activity in vitro and in vivo, while efficiently targeting primary B-cell non-Hodgkin lymphoma (B-NHL) samples with heterogeneous levels of CD22 and CD20. Altogether, we provide preclinical proof-of-concept data for an allogeneic dual CAR T cell to overcome current mechanisms of resistance to CAR T-cell therapies in B-NHL, while providing a potential alternative to CD19 targeting.

Loss of Human Leukocyte Antigen and Immune Escape in Head and Neck Cancer.

Cancer cells evade recognition by the immune system to survive. Head and neck squamous cell carcinoma (HNSCC) is characterized by high levels of immune infiltration and mutation-associated neoantigens; therefore, immune evasion is likely to be an important mechanism in HNSCC tumorigenesis and progression. A commonly employed mechanism of immune evasion is downregulation of human leukocyte antigen (HLA) or loss of heterozygosity (LOH) in tumor cells. The objective of this study was to integrate multi-dimensional genomic and transcriptomic data from HNSCC tumors to better understand the clinical and immunologic implications of HLA LOH. Cross-sectional integrated clinical and genomic analysis. Whole-exome sequencing and RNA-sequencing data from 522 tumors profiled in The Cancer Genome Atlas HNSCC cohort were analyzed and integrated with secondary analyses including immune cell deconvolution data. Associations were analyzed with categorical hypothesis testing and multivariable logistic and Cox regression. HLA LOH was a prevalent event that was identified in 53% of HNSCC tumors; in many cases, more than one class I HLA gene was targeted for LOH. HLA LOH was more common in advanced-stage tumors. Tumors with somatic HLA LOH had tumor microenvironments defined by decreased lymphocyte and T cell infiltration. HLA LOH is one of the most prevalent genetic alterations in HNSCC, and is associated with a cold immune microenvironment, suggesting that HLA LOH is a means of immune evasion. It may have value as a predictive biomarker or potential as a cancer cell-specific therapeutic target. 3 Laryngoscope, 134:160-165, 2024.

Bicistronic CAR-T cells targeting CD123 and CLL1 for AML to reduce the risk of antigen escape

PurposeAcute myeloid leukemia (AML) is a highly heterogeneous neoplastic disease with a poor prognosis that relapses even after its treatment with chimeric antigen receptor (CAR)-T cells targeting a single antigen. CD123 and CLL1 are expressed in most AML blasts and leukemia stem cells, and their low expression in normal hematopoietic stem cells makes them ideal targets for CAR-T. In this study, we tested the hypothesis that a new bicistronic CAR targeting CD123 and CLL1 can enhance antigenic coverage and prevent antigen escape and subsequent recurrence of AML. MethodsCD123 and CLL1 expressions were evaluated on AML cell lines and blasts. Then, in addition to concentrating on CD123 and CLL1, we introduced the marker/suicide gene RQR8 with a bicistronic CAR. Xenograft models of disseminated AML and in vitro coculture models were used to assess the anti-leukemia efficacy of CAR-T cells. The hematopoietic toxicity of CAR-T cells was evaluated in vitro by colony cell formation assays. It was demonstrated in vitro that the combination of rituximab and NK cells caused RQR8-mediated clearance of 123CL CAR-T cells. ResultsWe have successfully established bicistronic 123CL CAR-T cells that can target CD123 and CLL1. 123CL CAR-T cells effectively cleared AML cell lines and blasts. They also demonstrated appreciable anti-AML activity in animal transplant models. Moreover, 123CL CAR-T cells can be eliminated in an emergency by a natural safety switch and don't target hematopoietic stem cells. ConclusionsThe bicistronic CAR-T cells targeting CD123 and CLL1 may be a useful and secure method for treating AML.

Combination of CAR‑T cell therapy and radiotherapy: Opportunities and challenges in solid tumors (Review).

Chimeric antigen receptor (CAR) T cell therapy has emerged as a new and breakthrough cancer immunotherapy. Although CAR-T cell therapy has made significant progress clinically in patients with refractory or drug-resistant hematological malignancies, there are numerous challenges in its application to solid tumor therapy, including antigen escape, severe toxic reactions, abnormal vascularization, tumor hypoxia, insufficient infiltration of CAR-T cells and immunosuppression. As a conventional mode of anti-tumor therapy, radiotherapy has shown promising effects in combination with CAR-T cell therapy by enhancing the specific immunity of endogenous target antigens, which promoted the infiltration and expansion of CAR-T cells and improved the hypoxic tumor microenvironment. This review focuses on the obstacles to the application of CAR-T technology in solid tumor therapy, the potential opportunities and challenges of combined radiotherapy and CAR-T cell therapy, and the review of recent literature to evaluate the best combination for the treatment of solid tumors.

CAR T Cell Therapy: Evolutions and Emergent Improvements

One of the latest developments in the field of anti-cancer immunotherapy is chimeric antigen receptor (CAR) T cell therapy, which is considered magnificent and impressive owing to its superior response rates and efficacy. It has been proven to exhibit great efficacy in curing patients with B cell lymphoma and leukemia, and sometimes multiple myeloma as well. Nevertheless, despite the significant outcomes it has displayed, several limitations and restrictions still deeply restrain CAR T cell therapy from being widely used throughout the world. Particular challenges associated with T cells range from severe cytokine-related toxicities, the on-target off-tumor effect, and antigen escape, to inferior CAR T cell trafficking and immunosuppressive tumor microenvironment. To address the related problems, investigators learn from the evolutions of CAR design and incessantly attempt to modify and ameliorate CAR constructs and launch it to the wider extent of solid tumors and malignancies. A large array of methods and strategies have been adopted by investigators to fulfill this goal, ameliorating the efficacy, security, and applicability of CAR constructs. In this review, we will concentrate on the evolutions of CAR designs, and several limitations CAR T cell therapy recently faces, along with some innovative strategies come up by researchers to tackle them and explore the clinical benefits of this therapy against various cancers.

Editing Mutations of GABRB3 Using CRISPR-Cas9 as a Potential Treatment for Epilepsy and Autism Spectrum Disorders

The treatment of a variety of recurrent or resistant hematologic malignancies with chimeric antigen receptor T cell (CAR- T) cell therapy has seen significant success in recent years. The current CAR- T cell therapy approach is not without flaws, nevertheless, and there are still several issues with clinical treatment, including antigen escape, significant toxicity, and sus-ceptibility to drug-resistant recurrence. This paper introduces the structural development and characteristics of CAR-T cells, reviews the limitations of CAR- T cell therapy, including an-tigen escape, toxicity, CAR-T cell depletion and drug-resistant relapse after treatment, and summarizes the related improvement and optimization strategies. The paper concludes that CAR-T cell immunotherapy has brought new hope to patients with hematologic malignancies, making a cure for refractory and recurrent hematologic malignancies possible. Although CAR-T cell therapy still has many challenges at present, such as immunogenicity, drug re-sistance, and toxicity.

Structural and Research Advances in CAR-T Cell Therapy

The treatment of a variety of recurrent or resistant hematologic malignancies with chimeric antigen receptor T cell (CAR- T) cell therapy has seen significant success in recent years. The current CAR- T cell therapy approach is not without flaws, nevertheless, and there are still several issues with clinical treatment, including antigen escape, significant toxicity, and sus-ceptibility to drug-resistant recurrence. This paper introduces the structural development and characteristics of CAR-T cells, reviews the limitations of CAR- T cell therapy, including an-tigen escape, toxicity, CAR-T cell depletion and drug-resistant relapse after treatment, and summarizes the related improvement and optimization strategies. The paper concludes that CAR-T cell immunotherapy has brought new hope to patients with hematologic malignancies, making a cure for refractory and recurrent hematologic malignancies possible. Although CAR-T cell therapy still has many challenges at present, such as immunogenicity, drug re-sistance, and toxicity.

Abstract CT023: Phase 1 trial of CD19/CD20 bispecific chimeric antigen receptor-engineered naïve/memory T cells for relapsed or refractory non-Hodgkin lymphoma

Abstract Despite excellent responses to anti-CD19 chimeric antigen receptor (CAR)-T cell therapy in patients with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (NHL), more than half of patients will relapse due to poor CAR-T persistence or CD19 antigen escape. Engineering naïve/memory T (TN/MEM) cells with a bispecific anti-CD19/CD20 CAR could improve outcomes by mitigating these limitations. Here we report an update of the first-in-human phase 1 clinical trial with TN/MEM cells expressing a bispecific anti-CD19/CD20 CAR (CART19/20) for patients with R/R NHL (NCT04007029). Eligible patients were ≥18 years old with R/R diffuse large B-cell lymphoma (DLBCL) or primary mediastinal B-cell lymphoma (PMBCL) after ≥2 prior lines of therapy, or with mantle-cell lymphoma (MCL), follicular lymphoma (FL), or CLL/SLL after ≥3 prior lines of therapy. Prior CAR-T cell therapy was excluded, but other forms of CD19- or CD20-targeted therapies were allowed. Autologous leukocytes were obtained by leukapheresis and sorted for CD14-/CD25-/CD62L+ TN/MEM cells, followed by lentiviral transduction of the bispecific CD19/CD20 CAR. Bridging therapy after leukapheresis was allowed. Lymphodepletion chemotherapy with fludarabine 30 mg/m2/day and cyclophosphamide 500 mg/m2/day was administered for 3 days from Day −5 to Day −3 prior to CAR-T cell infusion. The primary endpoint was safety and the secondary endpoints were ORR, progression free survival (PFS), overall survival (OS), and CART19/20 transgene persistence. As of December 19, 2022, 11 patients have been treated with CART19/20 cells (7 DLBCL, including 4 transformed FL and 1 PMBCL; 3 FL; and 1 MCL). Patients were enrolled at two dose levels, including 50 × 106 CAR+ cells (8 patients) and 200 × 106 CAR+ cells (3 patients). The median age was 58 (range: 34 to 70). All patients had stage IV disease and 9 of 11 patients received bridging therapy. Six patients had grade-1 cytokine release syndrome (CRS), without any occurrence of higher-grade CRS. Neurotoxicity was not observed. Ten of the 11 patients achieved an objective response (91% ORR), with 8 patients (73%) achieving complete response (CR). One patient with FL relapsed at 18 months, received a second CART19/20 cell infusion (112 × 106 CAR+ cells), and re-achieved a CR that has persisted for >6 months at last follow-up. With a median follow-up of 20.9 months (range: 3.4 - 37 months), 7 patients remain in CR with a median PFS of 18.2 months (95% CI 3.4 months - not estimable) and the median OS was not reached. This phase 1 study demonstrates robust safety and tolerability of CART19/20 T-cells in patients with R/R NHL, without occurrence of severe CRS or neurotoxicity. CART19/20 cells, utilizing a bispecific CAR and TN/MEM cells, may be an effective strategy to overcome the challenges of poor CAR T-cell persistence and antigen escape. Citation Format: Benjamin R. Puliafito, Christopher Walthers, Brenda Ji, Sanaz N. Ghafouri, Jacob Naparstek, Jacqueline Trent, Jia M. Chen, Mobina Roshandell, Caitlin Harris, Mobina Khericha, Thomas Schweppe, Beata Berent-Maoz, Stanley B. Gosliner, Amr Almaktari, Melanie Ayala Ceja, Martin S. Allen-Auerbach, Jonathan Said, Karla Nawaly, Monica Mead, Sven de Vos, Patricia A. Young, Caspian Oliai, Gary J. Schiller, John M. Timmerman, Antoni Ribas, Yvonne Y. Chen, Sarah M. Larson. Phase 1 trial of CD19/CD20 bispecific chimeric antigen receptor-engineered naïve/memory T cells for relapsed or refractory non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT023.