Heavy Chain Variable Domains Research Articles

Asymmetrical forward and reverse developmental trajectories determine molecular programs of B cell antigen receptor editing.

During B lymphopoiesis, B cell progenitors progress through alternating and mutually exclusive stages of clonal expansion and immunoglobulin (Ig) gene rearrangements. Great diversity is generated through the stochastic recombination of Ig gene segments encoding heavy and light chain variable domains. However, this commonly generates autoreactivity. Receptor editing is the predominant tolerance mechanism for self-reactive B cells in the bone marrow (BM). B cell receptor editing rescues autoreactive B cells from negative selection through renewed light chain recombination first at Igκ then Igλ loci. Receptor editing depends on BM microenvironment cues and key transcription factors such as NF-κB, FOXO, and E2A. The specific BM factor required for receptor editing is unknown. Furthermore, how transcription factors coordinate these developmental programs to promote usage of the λ chain remains poorly defined. Therefore, we used two mouse models that recapitulate pathways by which Igλ light chain-positive B cells develop. The first has deleted J kappa (Jκ) genes and hence models Igλ expression resulting from failed Igκ recombination (Igκdel). The second models autoreactivity by ubiquitous expression of a single-chain chimeric anti-Igκ antibody (κ-mac). Here, we demonstrated that autoreactive B cells transit asymmetric forward and reverse developmental trajectories. This imparted a unique epigenetic landscape on small pre-B cells, which opened chromatin to transcription factors essential for Igλ recombination. The consequences of this asymmetric developmental path were both amplified and complemented by CXCR4 signaling. These findings reveal how intrinsic molecular programs integrate with extrinsic signals to drive receptor editing.

Engineering an autonomous VH domain to modulate intracellular pathways and to interrogate the eIF4F complex

An attractive approach to target intracellular macromolecular interfaces and to model putative drug interactions is to design small high-affinity proteins. Variable domains of the immunoglobulin heavy chain (VH domains) are ideal miniproteins, but their development has been restricted by poor intracellular stability and expression. Here we show that an autonomous and disufhide-free VH domain is suitable for intracellular studies and use it to construct a high-diversity phage display library. Using this library and affinity maturation techniques we identify VH domains with picomolar affinity against eIF4E, a protein commonly hyper-activated in cancer. We demonstrate that these molecules interact with eIF4E at the eIF4G binding site via a distinct structural pose. Intracellular overexpression of these miniproteins reduce cellular proliferation and expression of malignancy-related proteins in cancer cell lines. The linkage of high-diversity in vitro libraries with an intracellularly expressible miniprotein scaffold will facilitate the discovery of VH domains suitable for intracellular applications.

A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments

Single-chain variable fragments (scFvs), composed of variable domains of heavy and light chains of an antibody joined by a linker, share antigen binding capacity with their parental antibody. Due to intrinsically low solubility and stability, only two Escherichia coli-produced scFvs have been approved for therapy. Here we report that a 33-residue peptide, termed P17 tag, increases the solubility of multiple scFvs produced in Escherichia coli SHuffle strain by up to 11.6 fold. Hydrophilic sequence, especially charged residues, but not the predicted α-helical secondary structure of P17 tag, contribute to the solubility enhancement. Notably, the P17 tag elevates the thermostability of scFv as efficiently as intra-domain disulfide bonds. Moreover, a P17-tagged scFv targeting hepatitis B virus surface proteins shows over two-fold higher antigen-binding affinity and virus-neutralizing activity than the untagged version. These data strongly suggest a type I intramolecular chaperone-like activity of the P17 tag. Hence, the P17 tag could benefit the research, production, and application of scFv.

Development of a Humanized VHH Based Recombinant Antibody Targeting Claudin 18.2 Positive Cancers.

Claudin 18.2 (CLDN18.2), a tight junction (TJ) family protein controlling molecule exchange between cells, is frequently over-expressed in gastric cancer, pancreatic adenocarcinomas and in a fraction of non–small cell lung cancer cases. The tumor properties indicate that CLDN18.2 could be an attractive drug target for gastric and pancreatic cancers. In this study, we present effective strategies for developing anti-CLDN18.2 therapeutic candidates, based on variable domain of heavy chain of heavy chain antibodies (VHHs). CLDN18.2-specific VHHs were isolated by panning a phage display library from an alpaca immunized with a stable cell line highly expressing CLDN18.2. Humanized VHHs fused with human IgG1 Fc, as potential therapeutic candidates, exhibited desirable binding specificity and affinity to CLDN18.2. In vitro experiments showed that hu7v3-Fc was capable of eliciting both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) on CLDN18.2 positive tumor cells. In the mouse xenograft model, the anti-tumor efficacy of hu7v3-Fc was significantly more potent than Zolbetuximab, the benchmark anti-CLDN18.2 monoclonal antibody. Moreover, in vivo biodistribution using zirconium-89 (89Zr) labeled antibodies demonstrated that hu7v3-Fc (89Zr-hu7v3-Fc) exhibited a better tumor penetration and a faster tumor uptake than Zolbetuximab (89Zr-Zolbetuximab), which might be attributed to its smaller size and higher affinity. Taken together, anti-CDLN18.2 hu7v3-Fc is a promising therapeutic agent for human CLDN18.2 positive cancers. Furthermore, hu7v3 has emerged as a potential module for novel CLDN18.2 related therapeutics.

Abstract 2885: Generation of TCR-like fully human antibodies against HLA-antigen-peptide complexes using HLA transgenic RenMice

Abstract With the development of immunotherapy in recent years, therapeutic antibodies have become a major player combating cancer with the limitation of recognizing cell surface or secreted molecules. In contrast, the human T cell receptor (TCR) recognizes its cognate peptide antigen when presented on human leukocyte antigen (HLA) molecules, and thus detects a broader range of targets including intracellular proteins. Here we established an innovative strategy of generating fully human TCR mimic (TCRm) antibodies that can recognize antigen with a higher specificity and affinity. Previously, Biocytogen established RenMab™ and Renlite࣪ mice (RenMice). These humanized immunoglobulin mouse models generate fully human antibodies that carry human Ig heavy chain and human kappa light chain variable domains. Here, HLA genes were introduced into RenMice to build tolerance towards HLA. When immunized with HLA- peptide complexes, these mice generate antibodies that specifically target the HLA-Peptide complex, with no reactivity to HLA alone nor other peptide-HLA complexes. Compared to natural TCRs, these TCRm antibodies show higher affinity and specificity. Using optimized immunization methods and high-throughput screening, our antibody discovery platform generated fully human TCRm antibodies from HLA/RenMab™ and HLA/Renlite™ mice. Producing TCR-like binders with this unique TCRm technology can become a very powerful tool in the immunotherapy field. Citation Format: Jun Du, Nannan Wang, Li Hui, Ruixue Wang, Taolin Liu, Qingcong Lin. Generation of TCR-like fully human antibodies against HLA-antigen-peptide complexes using HLA transgenic RenMice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2885.

An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

Phage display strategy against outbreaks of unknown pathogens

The experience with SARS-CoV-2 that spread rapidly throughout the world makes us realize we need protocols to act quickly against unknown pathogens. The immune system protects recovering patients from any pathogen by producing antibodies against their immunogenic epitopes. Therefore, the strategy presented here is based on convalescent blood samples and a phage display platform for antibody and peptide drug discovery. Peptide libraries are screened against purified convalescent antibodies to identify immunogenic epitopes of the pathogen. Furthermore, the B cells of the recovery patients are used to amplify variable domains of antibody heavy and light chains expressed during the infection. These domains are cloned in a phagemid and produce free phage particles expressing the antibody fragments on their surfaces to select binders to pathogen immunogenic epitopes. These findings are essential in the identification of the unknown pathogen and the design of therapeutic molecules. In conclusion, this report describes a phage display strategy to combat outbreaks of unknown pathogens such as SARS-CoV-2.

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22.

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.

Production of Potyvirus-Derived Nanoparticles Decorated with a Nanobody in Biofactory Plants.

Viral nanoparticles (VNPs) have recently attracted attention for their use as building blocks for novel materials to support a range of functions of potential interest in nanotechnology and medicine. Viral capsids are ideal for presenting small epitopes by inserting them at an appropriate site on the selected coat protein (CP). VNPs presenting antibodies on their surfaces are considered highly promising tools for therapeutic and diagnostic purposes. Due to their size, nanobodies are an interesting alternative to classic antibodies for surface presentation. Nanobodies are the variable domains of heavy-chain (VHH) antibodies from animals belonging to the family Camelidae, which have several properties that make them attractive therapeutic molecules, such as their small size, simple structure, and high affinity and specificity. In this work, we have produced genetically encoded VNPs derived from two different potyviruses—the largest group of RNA viruses that infect plants—decorated with nanobodies. We have created a VNP derived from zucchini yellow mosaic virus (ZYMV) decorated with a nanobody against the green fluorescent protein (GFP) in zucchini (Cucurbita pepo) plants. As reported for other viruses, the expression of ZYMV-derived VNPs decorated with this nanobody was only made possible by including a picornavirus 2A splicing peptide between the fused proteins, which resulted in a mixed population of unmodified and decorated CPs. We have also produced tobacco etch virus (TEV)-derived VNPs in Nicotiana benthamiana plants decorated with the same nanobody against GFP. Strikingly, in this case, VNPs could be assembled by direct fusion of the nanobody to the viral CP with no 2A splicing involved, likely resulting in fully decorated VNPs. For both expression systems, correct assembly and purification of the recombinant VNPs was confirmed by transmission electron microscope; the functionality of the CP-fused nanobody was assessed by western blot and binding assays. In sum, here we report the production of genetically encoded plant-derived VNPs decorated with a nanobody. This system may be an attractive alternative for the sustainable production in plants of nanobody-containing nanomaterials for diagnostic and therapeutic purposes.

Functional Changes of Therapeutic Antibodies upon Exposure to Pro-Oxidative Agents.

Therapeutic monoclonal antibodies have exerted a transformative impact on clinical practice in last two decades. However, development of a therapeutic antibody remains a complex process. Various physiochemical and functional liabilities can compromise the production or the therapeutic efficacy of antibodies. One of these liabilities is the susceptibility to oxidation. In the present study, we portrayed an oxidation-dependent vulnerability of immunoglobulins that can be of concern for therapeutic antibodies. By using a library of 119 monoclonal IgG1 molecules, containing variable domain matching clinical-stage antibodies, we demonstrated that a substantial number of these molecules acquired antigen-binding polyreactivity upon exposure to ferrous ions. Statistical analyses revealed that the potential for induction of polyreactivity by the redox-active metal ions correlated with a higher number of somatic mutations in V genes encoding variable domains of heavy and light immunoglobulin chains. Moreover, the sensitive antibodies used with biased frequencies particular V gene families encoding variable domains of their light chains. Besides the exposure to ferrous ions the induction of polyreactivity of therapeutic antibodies occurred after contact with an unrelated pro-oxidative substance—hypochlorite ions. Our data also revealed that induction of polyreactivity by pro-oxidative agents did not impact the binding of antibodies to their cognate antigens. The results from this study may contribute for better selection of antibody therapeutics with suitable developability profiles.

Identification and Characterization of Differentially Expressed IgM Transcripts of Channel Catfish Vaccinated with Antigens of Virulent Aeromonas hydrophila

Channel catfish (Ictalurus punctatus) is the top species produced in US aquaculture and motile Aeromonas septicemia, caused by virulent Aeromonas hydrophila (vAh), is one of the most severe diseases that afflict catfish farms. Previously, vaccination of fish with extracellular proteins (ECP) of vAh was shown to produce a robust antibody-mediated immune response against vAh infection. In this study, we analyzed IgM transcripts that were differentially expressed in the head kidney and liver of ECP-immunized and mock-immunized (control) fish with emphasis on a variable domain of heavy chain. Quantitative PCR analysis indicated that immunized fish produced significantly more IgM transcripts than control fish. Full-length IgM heavy chain cDNA was cloned, which encoded typical IgM peptide, including signal peptide, variable domain (VH), constant domain (CH), and carboxyl terminal peptide. Great sequence diversity was revealed in a VH segment, with the third complementarity diversity region (CDR3) being most variable. Using germline VH gene grouping method, variants (clones) of VH characterized in this study belonged to nine VH families. The most unique variants (approximately 49%) were found in the VH2 family. Vaccinated fish apparently had more unique variants than in the control fish. There were 62% and 79% of unique variants in the head kidney and liver of vaccinated fish, respectively, while 44% and 27% unique variants in the head kidney and liver of control fish, respectively. Among the unique variants in VH2 family, approximately 87% of them were found in vaccinated fish. Two-dimensional gel electrophoresis of semi-purified IgM protein confirmed that matured IgM protein was as variable as IgM transcripts identified in this study, with isoelectric points crossing from 6 to 10. Results of this study provided insight into the molecular and genetic basis of antibody diversity and enriched our knowledge of the complex interplay between antigens and antibodies in Ictalurid catfish.

B cell clonal expansion and mutation in the immunoglobulin heavy chain variable domain in response to Pfs230 and Pfs25 malaria vaccines

Malaria transmission-blocking vaccines induce antibodies that target Plasmodium in the mosquito vector. We recently reported that Pfs230 vaccine achieves activity superior to Pfs25 in humans. Here, we describe clonal expansion in the variable region of immunoglobulin heavy chains (VH) of antigen-specific single B cells collected from humans immunised with Pfs230D1-EPA or Pfs25-EPA conjugate vaccines formulated in Alhydrogel®. Based on studies of CD27+ memory B cells following Pfs230 vaccination, clonal expansion and somatic hypermutation was seen in four of five subjects. Pfs25 did not induce sufficient CD27+ cells for sorting; based instead on CD19+ Pfs25-reactive B cells, clonal expansion was only seen in two of five subjects. Clonal expansions and mutations in Pfs230-specific single B cells combined with the enhanced activity of Pfs230 antibodies by complement, might justify the outstanding activity of Pfs230D1 as a TBV candidate.

Isolation and Characterization of Human Monoclonal Antibodies to Pneumococcal Capsular Polysaccharide 3.

ABSTRACTThe current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human ST3 pneumococcal capsular polysaccharide (PPS3) antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven PPS3-specific human monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination in vitro, and four mediated both activities. Two humAbs, namely, C10 and C27, that used the same variable heavy (VH) and light (VL) chain domains (VH3-9*01/VL2-14*03) both altered ST3 gene expression in vitro; however, C10 had fewer VL somatic mutations, higher PPS3 affinity, and promoted in vitro ST3 opsonophagocytic and agglutinating activity, whereas C27 did not. In C57BL/6 mice, both humAbs reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. After performing VL swaps, C10VH/C27VL exhibited reduced ST3 binding and agglutination, but C27VH/C10VL binding was unchanged. However, both humAbs lost the ability to reduce colonization in vivo when their light chains were replaced. Our findings associate the ability of PPS3-specific humAbs to reduce colonization with ST3 agglutination and opsonophagocytic activity, and reveal an unexpected role for the VL in their functional activity in vitro and in vivo. These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3.IMPORTANCE Despite the global success of vaccination with pneumococcal conjugate vaccines, serotype 3 (ST3) pneumococcus remains a leading cause of morbidity and mortality. In comparison to other vaccine-included serotypes, the ST3 pneumococcal capsular polysaccharide (PPS3) induces a weaker opsonophagocytic response, which is considered a correlate of vaccine efficacy. Previous studies of mouse PPS3 monoclonal antibodies identified ST3 agglutination as a correlate of reduced ST3 nasopharyngeal colonization in mice; however, neither the agglutinating ability of human vaccine-elicited PPS3 antibodies nor their ability to prevent experimental murine nasopharyngeal colonization has been studied. We generated and analyzed the functional and in vivo efficacy of human vaccine-elicited PPS3 monoclonal antibodies and found that ST3 agglutination associated with antibody affinity, protection in vivo, and limited somatic mutations in the light chain variable region. These findings provide new insights that may inform the development of antibody-based therapies and next-generation vaccines for ST3.

Design and in Vitro Evaluation of a CAR-T Prototype (ARI-0003) Targeting CD123 for Acute Myeloid Leukemia

IntroductionThe outcome of patients with Acute Myeloid Leukemia (AML) is still dismal, especially in patients with a relapsed or refractory (R/R) disease. In these patients, innovative treatment strategies must be considered in order to improve survival. Chimeric Antigen Receptor T-cell (CART) immunotherapy has demonstrated its efficacy and safety in diverse B-cell neoplasms and therefore is being investigated in other hematological malignancies. In AML, CART development is challenging due to the absence of a universal target antigen across AML subtypes, as well as on-target/off-tumor toxicity in healthy tissues. Among all explored AML antigens, CD123 seems to be a safe antigen to target, given its expresion in a high proportion of bulk and leukemic AML stem cells and with a higher level compared to normal hematopoietic progenitors cells. Herein we detail the generation and the in vitro assays of a CD123 CAR-T model (ARI-0003) for AML.MethodsWe developed an anti-CD123 antibody-secreting hybridoma and thereafter identified the variable domains of heavy and light chains of the immunoglobulin to create the scFv domain. The CAR (ARI-0003) genetic sequence was designed and cloned into a pCCL plasmid, and together with a VSV-G, RRE and REV plasmids we transfected HEK 293T cells to obtain 3 rd generation lentivirus (Figure 1A). T-lymphocytes were obtained from healthy volunteers and were transduced with lentiviral vectors to generate CAR-T cells. CAR expression in infected T-cells was monitored with flow cytometry using anti-F(ab) 2 antibodies. To test in vitro activity against AML, non-transduced T-lymphocytes (NT) and CAR-T cells were incubated with AML cell lines and AML primary samples from diverse genetic subtypes, and cytotoxicity was evaluated by flow cytometry at 24 and 48 hours using CD123 and CD33 antibodies.ResultsAt the time of co-incubation of target cells and lymphocytes, the mean scFv expression of ARI-0003 in CAR-T cells was 37% (range, 20-57). Cytotoxicity assays with AML cell lines (THP-1, Kasumi1) showed higher target cell mortality with CAR-T cells, compared to NT cells (mean percentage of alive cells at 48h, obtained with an effector-target cell ratio of 1:1, of 0 and 1% [CAR] vs 45 and 57 [NT] in THP-1 and Kasumi1, respectively; Figure 1B-C). This differential cytotoxic activity was maintained using distinct effector ratio, being statistically significant in higher ratios (namely, 1:1, 1:2 and 1:4). Moreover, cytotoxicity induced by CAR-T cells was higher at 48 hours compared to 24 hours. To demonstrate antigen specificity of ARI-0003 CAR-T cells, CD123 negative cell lines (Raji, 697) were used, and target cell mortality was not statistically significant between CAR-T and NT in CD123-negative cell lines.Finally, ARI-0003 CAR-T efficacy was tested against 8 primary AML samples with a distinct genetic risk, including 5 unfavorable cases according to the European LeukemiaNet classification and 3 AML samples refractory to multiple high-intensity regimens (Figure 1F). Interestingly, a significant cytotoxic effect was observed against all 8 samples after incubation with ARI-0003 CAR-T cells at higher E:T ratio (p < 0.05 for ratios 1:1, 1:2 and 1:4; Figure D-E).ConclusionsIn vitro assays showed that immunotherapy with CAR-T cells ARI0003 against CD123 could be an effective approach for R/R AML. In vivo experiments are needed in order to confirm these results before translating this therapy to clinical phases. Moreover, on-target/off-tumor toxicity induced by ARI-0003 CAR-T needs to be explored, especially myelotoxicity due to target antigen expression in hematopoietic precursor cells.Figure legendA) Structure of the ARI0003 CAR. B) Cytotoxicity of CAR-T cells against cell lines (normalized to background toxicity observed with NT cells). C) Flow cytometry of THP1 cells against NT and CAR-T cells. D) Cytotoxicity of CAR-T cells against AML primary samples (normalized to NT percentage). E) Absolut number of remaining AML cells after 48h of coincubation with CAR or NT cells. F) Characteristics of AML primary samples used for in vitro assays. [Display omitted] DisclosuresDiaz-Beyá: Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Esteve: Abbvie: Consultancy; Astellas: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Jazz: Consultancy; Novartis: Research Funding.

Treatment of Patients with T Cells Expressing a Fully-Human Anti-BCMA CAR with a Heavy-Chain Antigen-Recognition Domain Caused High Rates of Sustained Complete Responses and Relatively Mild Toxicity

Multiple myeloma (MM) is a malignancy of plasma cells that is nearly always incurable. T cells expressing chimeric antigen receptors (CAR) that target B-cell maturation antigen (BCMA) can recognize and eliminate MM. The murine or other non-human sequences in the single-chain variable fragments (scFv) of many anti-BCMA CARs can elicit recipient immune responses against CAR T cells. We constructed a CAR incorporating an anti-BCMA fully-human heavy-chain variable domain designated FHVH33. FHVH33 lacks the light chain, the artificial linker sequence, and the 2 linker-associated junctions of a scFv, so FHVH33 is smaller than a scFv and is likely to be less immunogenic. The FHVH33-containing CAR utilized in this clinical trial also incorporated a CD8a hinge and transmembrane domain, a 4-1BB domain, and a CD3z domain. The CAR was designated FHVH33-CD8BBZ and was encoded by a gamma-retroviral vector. T cells expressing FHVH33-CD8BBZ were designated FHVH33-T. The FHVH33-T production process was initiated with unsorted peripheral blood mononuclear cells and took 7 days. The treatment protocol was 300 mg/m 2 of cyclophosphamide and 30 mg/m 2 of fludarabine on days -5 to -3 followed by infusion of FHVH33-T on day 0.Twenty-five patients received FHVH33-T infusions. Median age of the treated patients was 62 (range 39-73). Patients received a median of 6 prior lines of therapy (range 3-10). Five dose levels were assessed (Table). Dose level 4, 6x10 6 CAR + T cells/kg was identified as the maximum feasible dose after considering efficacy and manufacturing factors.Twenty-three of 25 patients (92%) obtained objective responses (OR) of partial response (PR) or better. Seventeen patients (68%) attained a best response of stringent complete response (sCR) or very good partial response (VGPR). Thirteen patients have ongoing responses. To date, the median duration of response is 50 weeks for the highest two dose levels. At present, the overall median progression free survival (PFS) is 78 weeks; as responses are ongoing in 13 patients (52%), PFS will likely improve. Nine of 25 patients had extramedullary plasmacytomas at baseline; patients with extramedullary plasmacytomas at baseline were less likely to achieve sCR (P=0.011).All 25 treated patients were evaluable for toxicity. Eighteen patients had grade 1 or 2 cytokine-release syndrome (CRS), and 6 patients had grade 3 CRS. One patient had no CRS. No patients had grade 4 CRS. Five patients received tocilizumab and 4 patients received corticosteroids for CRS. Two of twenty-five patients had grade 3 neurological toxicity possibly attributable to FHVH33-T. No patient had grade 4 neurologic toxicity attributable to CAR T cells. One patient died of influenza pneumonia.We assessed blood CAR+ cells by quantitative PCR. The median peak blood CAR+ cell level was 126.5 cells/µl (range 3-1071 cells/µl), and the median time post-infusion of peak blood CAR + cell levels was 10.5 days (range 7-14). Peak CAR T-cell level was not associated with obtaining a sCR. In contrast, blood CAR+ T cell levels at both 1 and 2 months after infusion were statistically higher for patients obtaining sCR. For the 1-month time-point, blood CAR+ cell levels in cells/mL were 20 for sCR patients and 4 for not sCR patients (P=0.04). Pretreatment serum BCMA was not statistically different when patients obtaining or not obtaining sCR were compared (median serum BCMA in pg/mL: sCR patients 86,243; not sCR patients 261,675, P=0.20). We assessed cell-surface BCMA expression level on MM cells by antibody binding capacity (ABC) flow cytometry. Cell-surface BCMA expression level was not statistically different in sCR versus not sCR patients (median ABC in sites/cell: sCR patients 844; not sCR patients 535, P=0.29). Patients with MM expressing low levels of BCMA obtained durable responses of greater than 2 years duration, which suggests that FHVH33-T can recognize low levels of cell-surface BCMA. Eight patients had extramedullary plasmacytomas at relapse; 4 patients had plasmacytomas biopsied. Two of the biopsied plasmacytomas were BCMA+, and two were BCMA-negative by immunohistochemistry.FHVH33-CD8BBZ CAR T cells caused relatively mild toxicity and a high rate of sCRs in patients with relapsed MM including MM with low cell-surface BCMA expression. [Display omitted] DisclosuresBrudno: Kyverna Therapeutics: Membership on an entity's Board of Directors or advisory committees. Lam: Kite, a Gilead Company: Patents & Royalties. Kochenderfer: Kite, a Gilead Company: Patents & Royalties: on anti-CD19 CARs, Research Funding; Bristol Myers Squibb: Research Funding.

Application of phage display technology for the production of antibodies against Streptococcus suis serotype 2.

Streptococcus suis (S. suis) serotype 2 infection is a problem in the swine industry and responsible for most cases of human infection worldwide. Since current multiplex PCR cannot differentiate between serotypes 2 and 1/2, then serotype-specific antibodies (Abs) are required for serotype identification to confirm infection by serotype 2. This study aimed to generate Abs specific to S. suis serotype 2 by phage display from a human heavy chain variable domain (VH) antibody library. For biopanning, whole cells of S. suis serotype 2 were used as the target antigen. With increasing selection stringency, we could select the VH Abs that specifically bound to a S. suis serotype 2 surface antigen, which was identified as the capsular polysaccharide (CPS). From ELISA analysis, the specific phage clone 47B3 VH with the highest binding activity to S. suis serotype 2 was selected and shown to have no cross-reactivity with S. suis serotypes 1/2, 1, and 14 that shared a common epitope with serotype 2 and occasionally cause infections in human. Moreover, no cross-reactivity with other bacteria that can be found in septic blood specimens was also observed. Then, 47B3 VH was successfully expressed as soluble 47B3 VH in E. coli TG1. The soluble 47B3 VH crude extract was further tested for its binding ability in a dose-dependent ELISA assay. The results indicated that the activity of phage clone 47B3 was still retained even when the Ab occurred in the soluble form. A quellung reaction demonstrated that the soluble 47B3 VH Ab could show bioactivity by differentiation between S. suis serotypes 2 and 1/2. Thus, it will be beneficial to use this VH Ab in the diagnosis of disease or discrimination of S. suis serotypes Furthermore, the results described here could motivate the use of phage display VH platform to produce serotyping antibodies.

Systematic Engineering of Optimized Autonomous Heavy-Chain Variable Domains

Autonomous heavy-chain variable (VH) domains are the smallest functional antibody fragments, and they possess unique features, including small size and convex paratopes, which provide enhanced targeting of concave epitopes that are difficult to access with larger conventional antibodies. However, human VH domains have evolved to fold and function with a light chain partner, and alone, they typically suffer from low stability and high aggregation propensity. Development of autonomous human VH domains, in which aggregation propensity is reduced without compromising antigen recognition, has proven challenging. Here, we used an autonomous human VH domain as a scaffold to construct phage-displayed synthetic libraries in which aspartate was systematically incorporated at different paratope positions. In selections, the library yielded many anti-EphA1 receptor VH domains, which were characterized in detail. Structural analyses of a parental anti-EphA1 VH domain and an improved variant provided insights into the effects of aspartate and other substitutions on preventing aggregation while retaining function. Our naïve libraries and in vitro selection procedures offer a systematic approach to generating highly functional autonomous human VH domains that resist aggregation and could be used for basic research and biomedical applications.

Fecal multi-omics analysis reveals diverse molecular alterations of gut ecosystem in COVID-19 patients

Gut ecosystem has profound effects on host physiology and health. Gastrointestinal (GI) symptoms were frequently observed in patients with COVID-19. Compared with other organs, gut antiviral response can result in more complicated immune responses because of the interactions between the gut microbiota and host immunity. However, there are still large knowledge gaps in the impact of COVID-19 on gut molecular profiles and commensal microbiome, hindering our comprehensive understanding of the pathogenesis of SARS-CoV-2 and the treatment of COVID-19. We performed longitudinal stool multi-omics profiling to systemically investigate the molecular phenomics alterations of gut ecosystem in COVID-19. Gut proteomes of COVID-19 were characterized by disturbed immune, proteolysis and redox homeostasis. The expression and glycosylation of proteins involved in neutrophil degranulation and migration were suppressed, while those of proteases were upregulated. The variable domains of Ig heavy chains were downregulated and the overall glycosylation of IgA heavy chain constant regions, IgGFc-binding protein, and J chain were suppressed with glycan-specific variations. There was a reduction of beneficial gut bacteria and an enrichment of bacteria derived deleterious metabolites potentially associated with multiple types of diseases (such as ethyl glucuronide). The reduction of Ig heave chain variable domains may contribute to the increase of some Bacteroidetes species. Many bacteria ceramide lipids with a C17-sphingoid based were downregulated in COVID-19. In many cases, the gut phenome did not restore two months after symptom onset. Our study indicates widely disturbed gut molecular profiles which may play a role in the development of symptoms in COVID-19. Our findings also emphasis the need for ongoing investigation of the long-term gut molecular and microbial alterations during COVID-19 recovery process. Considering the gut ecosystem as a potential target could offer a valuable approach in managing the disease.

Enhancement of heterologous protein production in Corynebacterium glutamicum via atmospheric and room temperature plasma mutagenesis and high-throughput screening

Atmospheric and room temperature plasma (ARTP) is a new and efficient mutation breeding technique. In this study, we discuss a strategy combining ARTP mutagenesis and high-throughput screening to engineer Corynebacterium glutamicum towards high yield production of heterologous proteins. First, three target strains, MC2, MA8, and MA6, were screened from the mutant library with enhanced green fluorescent protein (EGFP) as the reporter protein, and their growth stability and the influence of heterologous protein production were verified. Second, genes encoding three high-value medicinal proteins (glycoprotein D, gD; endoxylanase, XynA; and variable domain of heavy chain of heavy-chain antibody, VHH) were expressed in the mutagenized strain, which confirmed its applicability for an increased biosynthesis of other heterologous proteins. During the large-scale fermentation of C. glutamicum for VHH production, the fermentation characteristics of the best mutant MA6 were verified. Compared to the original strain, the yield of VHH obtained with strain MA6 was increased by nearly 91 % to approximately 862 mg/L. Finally, through systematic genome analysis mutations in five genes were obtained. These genes code for putative proteases or are potentially related to the bacterial restriction repair systems. These findings will help to obtain optimized chassis cells and provide a direction for in-depth research on genetic targets that can increase protein production.

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.