Development of novel humanized CD19/BAFFR bicistronic chimeric antigen receptor T cells with potent antitumor activity against B-cell lineage neoplasms.

Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy in treating advanced B-cell malignancies by targeting CD19, but antigen-negative relapses and immune responses triggered by murine-derived antibodies remain significant challenges, necessitating the development of novel humanized multitarget CAR-T therapies. Here, we engineered a second-generation 4-1BB-CD3ζ-based CAR construct incorporating humanized CD19 single-chain variable fragments (scFvs) and BAFFR single-variable domains on heavy chains (VHHs), also known as nanobodies. The resultant CAR-T cells, with different constructs, were functionally compared both in vitro and in vivo. We found that the optimal tandem and bicistronic (BI) structures retained respective antigen-binding abilities, and both demonstrated specific activation when stimulated with target cells. At the same time, BI CAR-T cells (BI CARs) exhibited stronger tumour-killing ability and better secretion of interleukin-2 and tumour necrosis factor-alpha than single-target CAR-T cells. Additionally, BI CARs showed less exhaustion phenotype upon repeated antigen stimulation and demonstrated more potent and persistent antitumor effects in mouse xenograft models. Overall, we developed a novel humanized CD19/BAFFR bicistronic CAR (BI CAR) based on a combination of scFv and VHH, which showed potent and sustained antitumor ability both in vitro and in vivo, including against tumours with CD19 or BAFFR deficiencies.

The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains.

T cell malignancies are a group of hematologic cancers with high recurrence and mortality rates. CD5 is highly expressed in ∼85% of T cell malignancies, although normal expression of CD5 is restricted to thymocytes, T cells, and B1 cells. However, CD5 expression on chimeric antigen receptor (CAR)-T cells leads to CAR-T cell fratricide. Once this limitation is overcome, CD5-targeting CAR-T therapy could be an attractive strategy to treat T cell malignancies. Here, we report the selection of novel CD5-targeting fully human heavy-chain variable (FHVH) domains for the development of a biepitopic CAR, termed FHVH3/VH1, containing FHVH1 and FHVH3, which were validated to bind different epitopes of the CD5 antigen. To prevent fratricide in CD5 CAR-T cells, we optimized the manufacturing procedures of a CRISPR-Cas9-based CD5 knockout (CD5KO) and lentiviral transduction of anti-CD5 CAR. In vitro and in vivo functional comparisons demonstrated that biepitopic CD5KO FHVH3/VH1 CAR-T cells exhibited enhanced and longer lasting efficacy; produced moderate levels of cytokine secretion; showed similar specificity profiles as either FHVH1, FHVH3, or the clinically tested H65; and is therefore suitable for further development.

Development and functional characterization of novel fully human anti-CD19 chimeric antigen receptors for T-cell therapy.

Impressive outcomes have been achieved by chimeric antigen receptor (CAR)-T cell therapy using murine-derived single-chain variable fragment (scFv) FMC63 specific for CD19 in patients with B cell malignancies. However, evidence suggests that human anti-mouse immune responses might be responsible for poor persistence and dysfunction of CAR-T cells, leading to poor outcomes or early tumor recurrence. Substituting a fully human scFv for murine-derived scFv may address this clinically relevant concern. In this study, we discovered two human anti-CD19 scFv candidates through an optimized protein/cell alternative panning strategy and evaluated their function in CAR-T cells and CD19/CD3 bispecific antibody formats. The two clones exhibited excellent cytotoxicity in CAR-T cells and bispecific antibodies in vitro compared with the benchmarks FMC63 CAR-T cells and blinatumomab. Furthermore, Clone 78-BBz CAR-T cells exhibited similar in vivo antitumor activity to FMC63-BBz CAR-T cells. Our results indicate that Clone 78-BBz CAR has excellent efficacy and safety profile and is a good candidate for clinical development.

T cells expressing CD5/CD7 bispecific chimeric antigen receptors with fully human heavy-chain-only domains mitigate tumor antigen escape.

Bispecific chimeric antigen receptor T-cell (CAR-T) therapies have shown promising results in clinical trials for advanced B-cell malignancies. However, it is challenging to broaden the success of bispecific CAR-T therapies to treat refractory/relapse (r/r) T-cell leukemia/lymphoma because targeting multiple T-cell-expressing antigens leads to exacerbated CAR-T cell fratricide and potential safety concerns. Fully human heavy chain variable (FHVH) antibodies that specifically target CD5 or CD7 were screened and constructed to CD5/CD7 bispecific CARs. A truncated Epidermal growth factor receptor were integrated into CAR constructs to address safety concerns. To tackle the fratricidal issue of CAR-T cells targeting T-cell-pan marker(s), CRISPR/Cas9-based CD5 and CD7 genes knockout were performed before lentiviral transduction of bispecific CARs. Functional comparison between different bispecific CAR structures: tandem CARs and dual CAR were performed in vitro and in vivo to determine the optimal construct suitable for addressing T-cell malignancy antigen escape in clinical setting. Knockout of CD5 and CD7 prevents fratricide of CD5/CD7 bispecific CAR-T cells, and FHVH-derived CD5/CD7 bispecific CAR-T cells demonstrate potent antitumor activity in vitro and in vivo. The fratricide-resistant FHVH-derived CD5/CD7 bispecific CAR-T cells have potent antitumor activity against T-cell malignancies, and tandem CARs are more effective than dual CAR in preventing tumor escape in heterogeneous leukemic cells. The meaningful clinical efficacy and safety of tandem CD5/CD7 CAR-T cells deserve to be explored urgently.

Detection of breast cancer-related antigens through cDNA phage-displayed protein microarray.

High-throughput molecular profiling techniques are helpful in the diagnosis of multifactorial disease. In this study, a cDNA-phage-displayed protein microarray using phage particles spotted directly onto it as sensors was used to detect related antigens in breast tumor sera. cDNA sequences from 17 positive clones were determined, which included some sequences encoding known breast cancer-related antigens and proteins related to other diseases, as well as proteins with unknown functions. Our results not only provide some useful information for breast cancer research, but also suggest that the strategy used here would be efficient to search for disease-related proteins and other functional target proteins.

Association of Fcgamma receptor type 2A and 3A genotypes with rheumatoid arthritis in Chinese population.

AIM: SNPs of FcγRs were implicated in pathogenesis of rheumatoid arthritis (RA) and treatment efficacy of TNF inhibitors (TNFi). This study aims to investigate the associations of FcγRIIa and FcγRIIIa genotypes with autoantibody production and treatment response to TNFi in Chinese patients with RA. PATIENTS & METHODS: FcγRIIa and FcγRIIIa polymorphisms were genotyped in 158 RA patients. Response to TNFi was evaluated in 18 patients at 3 and 6 months after treatment. RESULTS: FcγRIIa-131H allele was significantly increased in autoantibody-negative RA patients. FcγRIIa-131H/H+H/R was closely associated with differences in 28-joint disease activity score in patients at months 3 and 6 of TNFi treatment. CONCLUSION: FcγRIIa-131H allele may have a protective role in autoantibody production and might be a biomarker for predicting good response to TNFi in Chinese RA patients.

Investigation on recombinant hirudin via oral route.

The possibility for oral administration of peptide recombinant hirudin variant (rHV2-K47) as an anticoagulant agent was evaluated in several aspects. The proteolytic properties of rHV2-K47 and its stability during storage were examined by in vitro experiments. Radiolabeled rHV2-K47 was infused into the duodenum of rats and rHV2-K47 absorbed into serum was shown to be intact by electrophoresis pattern. The in vivo coagulation time of blood from mouse was prolonged significantly after oral administration of rHV2-K47. The bioavailability (F) of rHV2-K47 via oral route reached 10.11% in comparison with intravenous administration as gold standard. All the results suggested that rHV2-K47 could be delivered successfully via the oral route.

Targeting the intracellular WT1 oncogene product with a therapeutic human antibody.

The Wilms tumor 1 (WT1) oncoprotein is an intracellular, oncogenic transcription factor that is overexpressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8+ T cell human leukocyte antigen (HLA)-A0201 epitope, is a validated target for T cell-based immunotherapy. Using phage display technology, we discovered a fully human "T cell receptor-like" monoclonal antibody (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A0201 complex. ESK1 bound to several leukemia and solid tumor cell lines and primary leukemia cells, in a WT1- and HLA-A0201-restricted manner, with high avidity [dissociation constant (Kd)=0.1 nM]. ESK1 mediated antibody-dependent human effector cell cytotoxicity in vitro. Low doses of naked ESK1 antibody cleared established, disseminated, human acute lymphocytic leukemia and Philadelphia chromosome-positive leukemia in nonobese diabetic/severe combined immunodeficient γc-/- (NSG) mouse models. At therapeutic doses, no toxicity was seen in HLA-A0201 transgenic mice. ESK1 is a potential therapeutic agent for a wide range of cancers overexpressing the WT1 oncoprotein. This finding also provides preclinical validation for the strategy of developing therapeutic mAbs targeting intracellular oncogenic proteins.

The study on venom proteins of Lapemis hardwickii by cDNA phage display.

In this study, a cDNA T7 phage display library was constructed from sea snake Lapemis hardwickii venom gland mRNA to analyze the components in venom and find new toxins. All the venom gland cDNA-encoding proteins, including housekeeping proteins and venom proteins were expressed on the surface of bacteriophage T7. This library was then panned with rabbit anti-sea snake venom IgG. Phage particles displaying venom-components with interaction with the antibodies were enriched. Thus, phage-carrying venom proteins, such as short chain neurotoxin, long chain neurotoxin, PLA2-like toxin, and c-type lectin-like protein were found, some of them were never reported previously. Four different short chain neurotoxins (SNT-1, 2, 3, 4) were cloned and expressed in Escherichia coli. The LD(50) and analgesic activities of their purified forms were determined. Their structure-function relationship were studied with the aid of a toxin-nAChR complex model constructed by ourselves. Among them, SNT-4 was a new neurotoxin identified in this study and showed potential as pain killer. These results prove that cDNA phage display technique has great advantage than traditional cDNA library method because of the linkage between phenotype and genotype of phage, which provided an effective means to study unknown proteins with target functions.