Variable Lymphocyte Receptors Research Articles

Screening and Characterization of Sialic Acid-Binding Variable Lymphocyte Receptors from Hagfish

Sialic acid is a diverse group of monosaccharides often found on the termini of N- and O-linked glycans as well as being components of glycoconjugates. Hypersialylation has been associated with the progression of chronic inflammation-mediated diseases such as cardiovascular disease and cancer. Given its role in infection and disease-related processes, sialic acid is a promising target for therapeutic approaches that utilize carbohydrate-binding molecules. In this study, we screened for sialic acid-recognizing variable lymphocyte receptors (VLRBs) or ccombodies from inshore hagfish (Eptatretus burgeri) using a synthetic Neu5Ac-glycoconjugate as an antigen in immunoassay. Resulting ccombodies, 2D8, 5G11, 4A1, and 5F8 were further characterized in terms of their binding activity and specificity. A competitive ELISA using free haptens showed strong inhibition using either N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). The half-maximal inhibitory concentrations (IC50) for Neu5Ac ranged from 7.02 to 17.06 mM, with candidates 4A1 and 5G11 requiring the least and highest amounts, respectively. IC50 values for Neu5Gc ranged from 8.12 to 13.91 mM, for 4A1 and 5G11, respectively. Candidate ccombodies also detected naturally occurring sialic acid from known sialoglycoproteins using a dot blot assay. Neu5Gc-5G11 and Neu5Ac-2D8 yielded the strongest and weakest docking interactions with affinity values of −5.9 kcal/mol and −4.9 kcal/mol, respectively. Hydrogen bonding and hydrophobic interactions were predicted to be the predominant noncovalent forces observed between the ccombodies and sialic acid. This study demonstrates that glycan-binding VLRBs from hagfish hold promise in augmenting the glycobiologists’ toolkit in investigating the roles of glycans in human and animal health and disease.

Passive immunoprophylaxis with Ccombodies against Vibrio parahaemolyticus in Pacific white shrimp (Penaeus vannamei)

The Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (AHPND) in shrimp secretes toxins A and B (PirAVp/PirBVp). These toxins have been implicated in pathogenesis and are targets for developing anti-AHPND therapeutics or prophylactics that include passive immunization. We have previously reported that Ccombodies (recombinant hagfish variable lymphocyte receptor B antibodies; VLRB) targeting PirBVp conferred protection against V. parahaemolyticus in shrimp when administered as a feed supplement. In this study, we screened a phage-displayed library of engineered VLRBs for PirAVp-targeting Ccombodies that were mass-produced in a bacterial expression system. We then introduced these Ccombodies into the diet of Pacific white shrimp (Penaeus vannamei) over a seven-day period. Subsequently, the shrimp were exposed to a challenge with V. parahaemolyticus. Mortality rates were then observed and recorded over the following seven days. Administering shrimp feed supplemented with Ccombodies at a high dose (100 mg per 100 g feed) reduced mortality in recipient animals (2.96-5.19%) statistically similar to mock-challenged control (1.48%), but significantly different from the Ccombody-deficient control (74.81%). This suggests that the Ccombodies provided strong protection against the bacterium. Feeding shrimp with a median dose (10 mg/100 g feed) gave statistically comparable low mortality (5.93-6.67%) as the high dose. Reducing the Ccombody dose to 1 mg/100 g feed showed variable effects. Ccombody A2 showed mortality (11.85%) significantly lower than that of the Ccombody-deficient group (74.81%), suggesting that it can effectively protect against the bacterial challenge at a low dose. Our results demonstrate the ability of the phage-displayed VLRB library to generate antigen-specific Ccombodies rapidly and simply, with the expression of high protein levels in bacteria. The protective effect provided by these Ccombodies aligns with our earlier results, strongly supporting the use of VLRB antibodies as a substitute for IgY in passive immunoprophylaxis against AHPND in shrimp.

High throughput long-read sequencing of circulating lymphocytes of the evolutionarily distant sea lamprey reveals diversity and common elements of the variable lymphocyte receptor B (VLRB) repertoire.

The leucine-rich repeat-based variable lymphocyte receptor B (VLRB) antibody system of jawless vertebrates is capable of generating an antibody repertoire equal to or exceeding the diversity of antibody repertoires of jawed vertebrates. Unlike immunoglobulin-based immune repertoires, the VLRB repertoire diversity is characterized by variable lengths of VLRB encoding transcripts, rendering conventional immunoreceptor repertoire sequencing approaches unsuitable for VLRB repertoire sequencing. Here we demonstrate that long-read single-molecule real-time (SMRT) sequencing (PacBio) approaches permit the efficient large-scale assessment of the VLRB repertoire. We present a computational pipeline for sequence data processing and provide the first repertoire-based analysis of VLRB protein characteristics including properties of its subunits and regions of diversity within each structural leucine-rich repeat subunit. Our study provides a template to explore changes in the VLRB repertoire during immune responses and to establish large scale VLRB repertoire databases for computational approaches aimed at isolating monoclonal VLRB reagents for biomedical research and clinical applications.

Design of targeted antiviral polypeptides specific to SARS-CoV-2. Challenges and prospects

The COVID-19 epidemic demanded the rapid development of high-affinity molecules of different types aimed at a single target, the S-protein of SARS-CoV-2. The simultaneous development and testing of such molecules provide a unique opportunity to compare the features of biotechnological platforms for creating therapeutic proteins. This review considers classical antibodies, variable lymphocyte receptors, single-domain antibodies, and artificial scaffolds (DARPins, affibodies, VH), that are compared in terms of affinity, neutralizing activity, size and compatibility with different delivery methods. It can be concluded that all platforms used have produced high-affinity proteins that specifically bind to the coronavirus S-protein. The highest affinity of the targeting molecules with the virus protein was achieved by developing classical antibodies, nanobodies and by combining several binding modules into multivalent constructs with high avidity. Based on the results of <i>in vivo</i> experiments, it can be concluded that a high affinity of the therapeutic protein for the surface antigens of SARS-CoV-2 is a necessary but not sufficient condition for suppression of COVID-19 due to the peculiarities of the biology of this virus. The experience gained in the development of therapeutic agents against coronavirus will be useful for design of effective targeted drugs for the treatment of known and new viral infections.<br>The bibliography includes 126 references.

A Hybrid Design for the Functional Assay of LvLRRm (Protein Containing LRR Domain) of the White Leg Shrimp, Litopenaeus vannamei

In the crustacean immune system, leucine-rich repeat (LRR) is one of the major structures for recognizing pathogen-associated molecular patterns (PAMPs). LRR domain-containing proteins belong to the LRR family, which is a large group of proteins with more than 6000 genes in the database. They are involved in very diverse physiological functions, mainly by interacting with other proteins. In a previous study, the LvLRRm, a transmembrane protein containing only LRR domain, was identified in the white leg shrimp, Litopenaeus vannamei. Its versatile role in performing multiple immunomodulation activities has been reported. However, there is still a lack of research on its efficient function at the protein level. To investigate its interactions with other proteins, we applied a convenient method called the ‘Hybrid LRR technique’ to produce a recombinant LvLRRm. The LvLRRm and hagfish’s variable lymphocyte receptors (VLRs) fragments were fused to the conserved LxxLxLxxN motif while retaining the β-strand. In addition, we established interactions between hybrid proteins and the flagellin of Salmonella typhimurium by performing surface plasmon resonance (SPR) analysis. The results of the SPR analysis demonstrated notable affinity for both LvLRRm and hybrid proteins towards Salmonella flagellin. The designed LvLRRm hybrid proteins bring insight for universal applications without losing protein functions.

Pattern recognition receptors involved in the immune system of hagfish (Eptatretus burgeri)

The initial defense against invading pathogenic microbes is the activation of innate immunity by binding of pattern recognition receptors (PRRs) to pathogen associated molecular patterns (PAMPs). To explain the action of PRRs from hagfish, one of the extant jawless vertebrates, we purified the GlcNAc recognition complex (GRC) from serum using GlcNAc-agarose. The GRC comprises four proteins of varying molecular masses: 19 kDa, 26 kDa, 27 kDa, and 31 kDa. Exposure of Escherichia coli to the GRC led to the phagocytic activation of macrophages, revealing the opsonic function of the GRC. The GRC in serum formed a large complex with a molecular mass of approximately 1200 kDa. The GRC bound to Escherichia coli but not to rabbit red blood cells, despite both having GlcNAc on their surface. These structural and binding properties are similar to those of mannose-binding lectin (MBL). The amino acid sequence of a portion of the 31 kDa protein in the GRC matched the amino acid sequence of variable lymphocyte receptor (VLR)-B in some place. According to the Western blot analysis, the 31 kDa protein was recognized by the anti-hagfish VLR-B antiserum. Based on the results, it appears that the GRC functions as a PRR like MBL and that its 31 kDa protein has a structure similar to that of VLR-B.

Evolution of two distinct variable lymphocyte receptors in lampreys: VLRD and VLRE

Jawless vertebrates possess an alternative adaptive immune system in which antigens are recognized by variable lymphocyte receptors (VLRs) generated by combinatorial assembly of leucine-rich repeat (LRR) cassettes. Three types of receptors, VLRA, VLRB, and VLRC, have been previously identified. VLRA- and VLRC-expressing cells are Tcell-like, whereas VLRB-expressing cells are B cell-like. Here, we report two types of VLRs in lampreys, VLRD and VLRE, phylogenetically related to VLRA and VLRC. The germline VLRD and VLRE genes are flanked by 39 LRR cassettes used in the assembly of mature VLRD and VLRE, with cassettes from chromosomes containing the VLRA and VLRC genes also contributing to VLRD and VLRE assemblies. VLRD and VLRE transcription is highest in the triple-negative (VLRA-/VLRB-/VLRC-) population of lymphocytes, albeit also detectable in VLRA+ and VLRC+ populations. Tissue distribution studies suggest that lamprey VLRD+ and VLRE+ lymphocytes comprise T-like sublineages of cells.

Alternative splicing derived invertebrate variable lymphocyte receptor displays diversity and specificity in immune system of crab Eriocheir sinensis.

Variable lymphocyte receptors (VLRs) play vital roles in adaptive immune system of agnathan vertebrate. In the present study, we first discover a novel VLR gene, VLR2, from an invertebrate, the Chinese mitten crab, Eriocheir sinensis. VLR2 has ten different isoforms formed via alternative splicing, which is different from that in agnathan vertebrate with the assembly of LRR modules. The longest isoform, VLR2-L, responds to Gram-positive bacteria Staphylococcus aureus challenge specifically, while shows no response to Gram-negative bacteria Vibrio parahaemolyticus challenge, confirmed by recombinant expression and bacterial binding experiments. Interestingly, VLR2s with short LRRs regions (VLR2-S8 and VLR2-S9) tend to bind to Gram-negative bacteria rather than Gram-positive bacteria. Antibacterial activity assay proves six isoforms of VLR2 have pluralistic antibacterial effects on bacteria which were never reported in invertebrate. These results suggest that the diversity and specificity of VLR2 resulted from alternative splicing and the length of the LRRs region. This pathogen-binding receptor diversity will lay the foundation for the study of immune priming. Furthermore, studying the immune function of VLR2 will provide a new insight into the disease control strategy of crustacean culture.

Review of the unique and dominant lectin pathway of complement activation in agnathans

As the most primitive vertebrates, lampreys are significant in understanding the early origin and evolution of the vertebrate innate and adaptive immune systems. The complement system is a biological response system with complex and precise regulatory mechanisms and plays an important role in innate and adaptive immunity. It consists of more than 30 distinct components, including intrinsic components, regulatory factors, and complement receptors. Complement system is the humoral backbone of the innate immune defense and complement-like factors have also been found in cyclostomes. Our knowledge as such in lamprey has dramatically increased in the recent years. The searching for complement components in the reissner lamprey Lethenteron reissneri genome database, together with published data, has unveiled the existence of all the orthologues of mammalian complement components identified thus far, including the complement regulatory proteins and complement receptors, in lamprey. This review, summarizes the key themes and recent updates on the complement system of agnathans and discusses the individual complement components of lampreys, and critically compare their functions to that of mammalian complement components. Interestingly, the adaptive immune system of agnathans differs from that of gnathostomes. Lamprey complement components also display some distinctive features, such as lampreys are characterized by the variable lymphocyte receptors (VLRs)-based alternative adaptive immunity. This review may serve as important literature for deducing the evolution of the immune system from invertebrates to vertebrates.

Abstract 580: MM3 CAR T: A novel tumor targeting and T cell redirection platform for multiple myeloma

Abstract Novel strategies to target tumors are needed to enhance the efficacy of immune redirection therapies. The detection of tumors based on mammalian immunoglobulin scFvs has been a significant innovation in immunotherapy but is impacted by limits of immune tolerance and immune system evolution. The discovery of unique variable lymphocyte receptors (VLRs) - analogues to antibodies in evolutionarily distant jawless vertebrates - in the Cooper lab (Pancer, Nature) provides an opportunity to target antigens beyond those recognized by conventional antibodies. A particular VLR, termed MM3, was found to identify a distinct epitope specific to human plasma cells (PCs) and plasmablasts (PBs), created by CD38 dimerization with activity as an NAD+ glycohydrolase (Yu, JCI Insight). CD38 is an extensively explored target for treating multiple myeloma (MM); however, specific scFv-based targeting of CD38 on malignant plasma cells is difficult, as the protein is expressed on a variety of other immune cell lineages. Therefore, MM3 provides a unique, novel mechanism to target CD38 in a tumor-specific manner in MM and other plasma cell dyscrasias. Here, we present a CAR T cell with an MM3 binding domain replacing the standard scFv. Our CAR construct consists of the MM3 binding domain, a CD8 hinge and transmembrane domain, and 4-1BB and CD3zeta intracellular domains. Staining of MM patient bone marrow specimens revealed that the strong specificity for PCs and PBs previously observed in healthy donors was maintained in MM bone marrow, illustrating the ability of MM3 to target the malignant PC population. To assess in vitro function of the MM3 CAR T cells, flow cytometry analysis of co-cultures using healthy donor-derived MM3 CAR T cells and target cell lines revealed specific increased degranulation (CD107a expression), activation (CD69 expression), and killing (target cell Annexin V binding) over untransduced control T cells only in cell lines sensitive to MM3 binding, with no differences in negative control cell lines, demonstrating specific recognition and killing of target cells. Preliminary in vivo studies in the MISTRG6 myeloma xenograft mouse model were performed using intrafemorally injected INA-6, an IL-6-dependent MM cell line, which MM3 binds after growth in vivo. After treatment with MM3 CAR T cells via intravenous injection, we observed that the MM3 CAR T cells specifically homed to and were retained in the injected femur along with the tumor cells, suggesting tumor recognition and potential for in vivo efficacy. In summary, these data demonstrate that VLR-based targeting may allow design of novel CAR constructs. Specifically, the VLR MM3 is a promising novel, non-scFv mechanism for targeting CD38 in a malignant PC-specific manner, and the MM3 CAR T cells have shown promising preliminary activity. Therefore, the VLR-based MM3 CAR T cell platform may provide a novel strategy to improve T cell redirection to target tumors in MM. Citation Format: Samuel S. McCachren, Julia Manalo, Katherine Pendleton, Mary E. Ballestas, Kavita M. Dhodapkar, Melissa L. Kemp, Max D. Cooper, Madhav V. Dhodapkar. MM3 CAR T: A novel tumor targeting and T cell redirection platform for multiple myeloma [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 580.

Identification of lamprey variable lymphocyte receptors that target the brain vasculature

The blood–brain barrier (BBB) represents a significant bottleneck for the delivery of therapeutics to the central nervous system. In recent years, the promise of coopting BBB receptor-mediated transport systems for brain drug delivery has increased in large part due to the discovery and engineering of BBB-targeting antibodies. Here we describe an innovative screening platform for identification of new BBB targeting molecules from a class of lamprey antigen recognition proteins known as variable lymphocyte receptors (VLRs). Lamprey were immunized with murine brain microvessel plasma membranes, and the resultant repertoire cloned into the yeast surface display system. The library was screened via a unique workflow that identified 16 VLR clones that target extracellular epitopes of in vivo-relevant BBB membrane proteins. Of these, three lead VLR candidates, VLR-Fc-11, VLR-Fc-30, and VLR-Fc-46 selectively target the brain vasculature and traffic within brain microvascular endothelial cells after intravenous administration in mice, with VLR-Fc-30 being confirmed as trafficking into the brain parenchyma. Epitope characterization indicates that the VLRs, in part, recognize sialylated glycostructures. These promising new targeting molecules have the potential for brain targeting and drug delivery with improved brain vascular specificity.

New Insights Into the Lineage-Specific Expansion and Functional Diversification of Lamprey AID/APOBEC Family.

The AID/APOBEC family which converts cytidine to uridine on RNA or DNA experienced dynamic expansion in primates in order to resist exogenous viruses and endogenous retrotransposons. Recently, expansion of AID/APOBEC-like homologs has also been observed in the extant jawless vertebrate lamprey. To reveal what causes such expansion and leads to the functional diversification of lamprey cytosine deaminases (CDAs), we reassessed the CDA genes in Lethenteron japonicum (Lj). We first confirmed the expansion of LjCDA1L1 (CDA1-like 1) genes and found the expression correlation of LjCDA2 and LjCDA1L2 with LjVLRs (variable lymphocyte receptors). Among up to 14 LjCDA1L1 proteins, LjCDA1L1_4a has an extremely high deamination activity on ssDNA and buDNA and, unexpectedly, on dsDNA. LjCDA1L1s can also restrict the infection of HSV-1 particles. Thus, the arms race between the host and pathogens along with the recruitment by VLR assembly may participate together to form a driving force in the expansion and diversification of the lamprey AID/APOBEC family.

Identification of Brain ECM Binding Variable Lymphocyte Receptors Using Yeast Surface Display.

Extracellular matrix (ECM) is a rich mixture of proteins and glycans secreted by cells. This includes typical ECM structures such as collagen and heparin as well as glycosylated, secreted proteins such as growth factors and peptidases. Certain components of ECM are ubiquitous among all tissue; however, each biological tissue also displays unique variations that can be identified using biopanning techniques. Here we describe using a variable lymphocyte receptor (VLR) yeast surface display library to identify selective binders to brain ECM by combining ECM biopanning with a rapid ELISA-based screen using clonal VLRs isolated directly from the yeast surface. Finally, potential ECM-binding candidates can be verified by immunostaining murine brain sections with VLRs released from the yeast surface. These methods provide a framework for the identification of tissue-selective ECM-binding VLRs using yeast surface display techniques and could easily be adapted for other binding scaffolds or ECM from other tissues.

Evolution of variable lymphocyte receptor B antibody loci in jawless vertebrates

SignificanceJawless vertebrates, lampreys and hagfish, use three types of variable leucine-rich repeat proteins as antigen receptors: VLRB on B-like cells and VLRA or VLRC on T-like lymphocytes. In their incomplete germline status, theVLRBgenes in different lamprey species uniquely possess long noncoding intervening regions, which contain different sets of transposable elements and potential donor cassettes. Phylogenetic analysis of the germlineVLRgenes reveals ongoing evolution over the last 250 million y. Comparative assessment of genomic sequences in two lamprey species yields a detailed map of theirVLRBloci. The use of this map, together with an analysis of cassette usage in a large repertoire of expressedVLRBs, supports a fragmented Lego-like model ofVLRBassembly.

Identification of Glycan-Specific Variable Lymphocyte Receptors Using Yeast Surface Display and Glycan Microarrays.

The jawless vertebrates (lamprey and hagfish) evolved a novel adaptive immune system with many similarities to that found in the jawed vertebrates, including the production of antigen-specific circulating antibodies in response to immunization. However, the jawless vertebrates use leucine-rich repeat (LRR)-based antigen receptors termed variable lymphocyte receptors (VLRs) for immune recognition, instead of immunoglobulin (Ig)-based receptors. VLR genes are assembled in developing lymphocytes through a gene conversion-like process, in which hundreds of LRR gene segments are randomly selected as template donors to generate a large repertoire of distinct antigen receptors, similar to that found within the mammalian adaptive immune system. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus) for generating libraries of anti-carbohydrate (anti-glycan) variable lymphocyte receptor B, or VLRBs. The anti-carbohydrate VLRBs are isolated using a yeast surface display (YSD) expression platform and enriched by binding to glycan microarrays through the anti-glycan VLRB. This enables both the initial identification and enrichment of individual yeast clones against hundreds of glycans simultaneously. Through this enrichment strategy a broad array of glycan-specific VLRs can be isolated from the YSD library. Subsequently, the bound yeast cells are directly removed from the microarray, the VLR antibody clone is sequenced, and the end product is expressed as a VLR-IgG-Fc fusion protein that can be used for ELISA, Western blotting, flow cytometry, and immunomicroscopy. Thus, by combining yeast surface display with glycan microarray technology, we have developed a rapid, efficient, and novel method for generating chimeric VLR-IgG-Fc proteins that recognize a broad array of unique glycan structures with exquisite specificity.

Lamprey Variable Lymphocyte Receptor Monoclonal Antibodies for Whole-Cell Surface Antigens.

Lamprey antibodies, the variable lymphocyte receptor B proteins (VLRB), have unique properties that make them promising alternatives to jawed vertebrate immunoglobulin domain antibodies. These leucine-rich repeat proteins exhibit a diversity on par with that of jawed vertebrate antibodies but are structurally completely distinct. VLRB antibodies have been successfully raised to a variety of antigens. A procedure for high-throughput screening of full-length lamprey VLRB libraries using whole cells is described here. Lamprey antibodies against cell surface antigens can be generated and screened quickly using this method.

Characterization of the variable lymphocyte receptor B genes of a freshwater nonparasitic lamprey species, Lampetra hubbsi

Agnathans possess a convergent adaptive immune system in comparison to that of jawed vertebrates. In lieu of immunoglobulins, agnathans deploy variable lymphocyte receptors (VLRs), single chain protein effector molecules consisting of leucine rich repeat modules. Foundational work for this discovery utilized the parasitic sea lamprey, Petromyzon marinus. However, for several reasons, it is desirable to employ a local species for further studies of lamprey immunity. A disjunct freshwater species from the Kings River of California, Lampetra hubbsi, was evaluated for this purpose. Validation that its adaptive immune system was analogous to that of P. marinus entailed detailed examination of its immune tissue organization and of its VLRB cDNA transcripts. The VLRB molecules showed high degrees of homology with P. marinus VLRB. Furthermore, hemato-lymphopoietic tissue expression of VLRB protein was confirmed. We conclude that L. hubbsi should be a viable alternative for studying the lamprey adaptive immune system and for generation of monoclonal antibodies.

Targeting T-Cell Malignancies with Chimeric Antigen Receptors Using Two Structurally Different Antigen Binding Domains Against CD5

Patients with relapsed T-cell acute lymphoblastic leukemia (T-ALL) have extremely poor survival when treated with chemotherapy alone. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a chance of cure for these patients, but attaining clinical remission in order to undergo HSCT remains the biggest therapeutic challenge. Chimeric antigen receptor (CAR) T-cell therapy has been used with great success in relapsed/refractory B-ALL patients. The same approach, however, is difficult in targeting T-cell disease given the lack of a T-lymphoblast specific surface antigen. The use of natural killer (NK) cells as the effector cell provides a potential solution. We are testing two different CAR structures directed against CD5, a pan T-cell marker; one consisting of a more traditional immunoglobulin (Ig) based single chain variable fragment (scFv), while the other comprising of a variable lymphocyte receptor (VLR) as the antigen recognition domain. VLRs are single chain crescent shaped proteins that represent the functional unit of the adaptive immune system in jawless vertebrates. Our previous work has shown that a VLR can be used as an alternative for CAR-based antigen recognition. Here, we directly compare a VLR to its scFv counterpart in a CAR setting. The single chain structure of a VLR allows for the rapid creation of novel CARs, compared to the corresponding use of a scFv-CAR, in which the variable heavy and light sequences need further engineering for adapting to CAR technologies. Thus, our hypothesis was that natural killer cells engineered to express an anti-CD5 CAR can specifically be used to target T-cell malignancies, with the VLR-CAR being equal or superior to scFv-based cassettes. We constructed two second generation CARs using the VLR or scFv sequence targeting CD5 as our antigen recognition domain. Both CD5-CAR constructs showed similar activity when tested in CD5-positive Jurkat cells. For our cytotoxicity studies, we used the IL-2 dependent natural killer cell line NK-92. To generate uniform populations of CAR-expressing NK cells we transduced NK-92 cells with a dual lentiviral construct expressing the CD5-CAR and green fluorescent protein (GFP), and then selected for the positively transduced cells using flow sorting for GFP. In vitro cytotoxicity studies showed significantly increased killing using the CD5-CAR expressing NK-92 cells compared to unmodified NK-92 cells (p<0.05), for both VLR and scFv modified cells. We then tested the two CD5-CARs in a T-cell leukemia mouse model. NOD scid gamma (NSG) mice were intravenously injected with luciferase expressing Jurkat leukemia cells to establish the T-cell leukemia xenograft model. Four doses of NK-92 cells were injected over a two week period starting on day 7 post-tumor injection. Bioluminescence imaging was used to monitor tumor burden. The CD5-scFv-CAR NK-92 treated mice group had a significant survival advantage over the other groups (p<0.01). Interestingly, the CD5-VLR-CAR NK-92 treated mice did not have a significant advantage over naïve NK-92 or saline treated groups. Given the superior in vivo cytotoxicity demonstrated in our studies, we plan to use the CD5-scFV-CAR going forward as we transition to primary cells. Overall, these studies provide the foundation to move these studies into preclinical testing as a treatment for relapsed T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Engineering CD5-Targeted Chimeric Antigen Receptors and Edited T Cells for the Treatment of T-Cell Leukemia

Remission reinduction poses a great challenge in treating relapsed T-cell malignancies. These patients have poor outcomes when treated with chemotherapy alone. Treatment of relapsed B-cell malignancies using chimeric antigen receptor (CAR) T-cell therapy has had great success. However, the same approach will be difficult to apply to the treatment of T-cell malignancies due to the lack of T-lymphoblast specific antigens. Therefore, T cells modified with anti-T cell CARs target themselves, potentially resulting in fratricide of CAR T cells and killing of normal T cells. We have been investigating mechanistic approaches to evade fratricide-based limitations of CAR therapy for T-cell leukemias. Since the majority of T-cell leukemias express CD5, we hypothesized that if CD5 expression was knocked down/out in effector T cells, the issue of fratricide would be eliminated. We used CRISPR/Cas9 genome editing to knock out CD5 expression in CD5-positive T cells, resulting in CD5-negative T cells that were used as CAR-expressing effector cells. In order to target CD5, CARs were generated using two structurally different antigen recognition sequences - a traditional immunoglobulin-based single chain variable fragment (scFv) and a lamprey-derived variable lymphocyte receptor (VLR). We have previously shown this VLR can be used as an alternative for CAR-based antigen recognition. Here, we compare non-CRISPR T cells to CD5-edited T cells when modified with either a CD5-VLR-CAR or CD5-scFv-CAR. Our in vitro studies show both CARs yield comparable T-cell activation in non-CRISPR T cells and that CD5-edited CAR-modified T cells have a substantial reduction in self-activation. Furthermore, CD5-edited CAR-modified cells become activated in the presence of CD5-positive target cells to a greater degree than do non-CRISPR CAR-modified T cells (p<0.05 at all effector:target ratios of 2:1, 1:1 and 1:5). We, and others, have shown a decrease in cell surface CD5 protein expression following lentiviral gene transfer of a CD5-CAR transgene cassette in non-CD5 edited T cells using both VLR and scFv-based vectors. This downregulation has been speculated to be due to the interaction of the cell surface CAR with the target antigen. We also show that CD5-CAR expression in CD5-edited CAR-modified T cells is increased compared to CD5-CAR expression in non-CRISPR CAR-modified T cells (see Figure). Therefore, the interaction of the CAR with the cell surface antigen not only decreases antigen levels, but CAR levels as well. The decreased fratricide, increased CAR expression and activation of CD5-edited CAR-modified T cells in the presence of target cells should lead to more potent and durable anti-leukemia effects. Overall, our preclinical data show an advantage to using CD5-edited effector T cells when utilizing CAR-T cell therapy to target difficult to treat relapsed T-cell malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Detection and Neutralization of SARS-CoV-2 Using Non-conventional Variable Lymphocyte Receptor Antibodies of the Evolutionarily Distant Sea Lamprey.

SARS-CoV-2 is a newly emerged betacoronavirus and the causative agent for the COVID-19 pandemic. Antibodies recognizing the viral spike protein are instrumental in natural and vaccine-induced immune responses to the pathogen and in clinical diagnostic and therapeutic applications. Unlike conventional immunoglobulins, the variable lymphocyte receptor antibodies of jawless vertebrates are structurally distinct, indicating that they may recognize different epitopes. Here we report the isolation of monoclonal variable lymphocyte receptor antibodies from immunized sea lamprey larvae that recognize the spike protein of SARS-CoV-2 but not of other coronaviruses. We further demonstrate that these monoclonal variable lymphocyte receptor antibodies can efficiently neutralize the virus and form the basis of a rapid, single step SARS-CoV-2 detection system. This study provides evidence for monoclonal variable lymphocyte receptor antibodies as unique biomedical research and potential clinical diagnostic reagents targeting SARS-CoV-2.