Human Heavy Chain Antibodies Research Articles

Human Heavy Chain Antibody Genes Elicited in Thai Dengue Patients during DENV2 Secondary Infection.

Dengue is one of the most serious mosquito-borne viral diseases occurring in humans. To combat the complexity of 4 antigenically distinct serotypes, the ideal vaccine for dengue should be able to stimulate cross-neutralizing antibodies. Recently, genetics-based immune responses have been studied to guide vaccine design against several viral pathogens. Despite a recent approval of dengue vaccine, information on genetics-based immune responses against dengue virus (DENV) is still limited. Consequently, we aimed to determine the profiles of immunoglobulin heavy chain genes from DENV2 infected patients. The immunoglobulin heavy chain variable region genes (IGHV) were amplified from peripheral blood mononuclear cells of DENV2 secondary infected patients in the acute, convalescence, and recovery phases. Antibody heavy chain genes were sequenced using next-generation sequencing, and analyzed to identify correlations with neutralizing and enhancing activities of the serum samples. IGHV1-69, 3-23, and 3-30 were frequently discovered in our Thai DENV2 infected patients. Our findings provide new data on the human B cell response during secondary DENV2 infections in Thai dengue patients that offer supportive information for dengue vaccine design and therapeutics development.

Multispecific Antibody Development Platform Based on Human Heavy Chain Antibodies.

Heavy chain-only antibodies (HCAbs) do not associate with light chains and their VH regions are functional as single domains, forming the smallest active antibody fragment. These VH regions are ideal building blocks for a variety of antibody-based biologics because they tolerate fusion to other molecules and may also be attached in series to construct multispecific antibodies without the need for protein engineering to ensure proper heavy and light chain pairing. Production of human HCAbs has been impeded by the fact that natural human VH regions require light chain association and display poor biophysical characteristics when expressed in the absence of light chains. Here, we present an innovative platform for the rapid development of diverse sets of human HCAbs that have been selected in vivo. Our unique approach combines antibody repertoire analysis with immunization of transgenic rats, called UniRats, that produce chimeric HCAbs with fully human VH domains in response to an antigen challenge. UniRats express HCAbs from large transgenic loci representing the entire productive human heavy chain V(D)J repertoire, mount robust immune responses to a wide array of antigens, exhibit diverse V gene usage and generate large panels of stable, high affinity, antigen-specific molecules.

Abstract 5620: Multispecific antibodies targeting CD38 and PD-L1 show potent tumor cytotoxicity

Abstract Multivalent antibodies targeting either CD38 alone or CD38 in conjunction with PD-L1 may yield therapeutics with superior biologic activities and provide benefit for treating malignancies expressing low levels of CD38 (DLCBL, daratumumab refractory MM, and NSCLC). Multivalent, multispecific antibodies kill CD38low cells through a variety of mechanisms including stronger and more specific engagement of CD38. Potent and directed immune checkpoint inhibition is realized by adding an anti-PD-L1 binding domain. Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Individual UniDabs targeting CD38 and PDL1 were identified using our unique sequence-based discovery platform and high-throughput lead evaluation pipeline (TeneoSeek). This robust screening workflow enables evaluation of a large diversity of natural fully human antibodies, targeting multiple epitopes on a single antigen and uncovering important sequence activity relationships. We have identified UniDabs that bind five different functional epitopes on human, cynomolgus and mouse CD38 including potent inhibitors of hydrolase and cyclase enzymatic functions. In addition, we generated a large panel of PD-L1 inhibitors that bind with high affinities human and cynomolgus PD-L1. Using different combinations and arrangements of UniDabs, a variety of multivalent antibodies were constructed and evaluated in in vitro models. Data from a range of assay types show that multivalent UniAbs targeting CD38 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action. Summary: Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Multivalent UniAbs targeting CD38 and PD-L1 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action. Citation Format: Wim van Schooten. Multispecific antibodies targeting CD38 and PD-L1 show potent tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5620.

Multispecific antibodies targeting CD38 show potent tumor-specific cytotoxicity.

57 Background: Multivalent antibodies targeting either CD38 alone or CD38 in conjunction with PD-L1 may yield therapeutics with superior biological activities and provide benefit for treating malignancies expressing low levels of CD38 (MCL, NHL, T cell lymphomas and Daratumumab refractory MM). Multivalent, multispecific antibodies kill CD38low cells through a variety of mechanisms including stronger and more specific engagement of CD38. Potent and directed immune checkpoint inhibition is realized by adding an anti-PD-L1 binding domain. Teneobio’s discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Methods: Individual UniDabs targeting CD38 and PDL1 were identified using our unique sequence-based discovery platform and high-throughput lead evaluation pipeline (TeneoSeek). This robust screening workflow enables evaluation of a large diversity of natural fully human antibodies, targeting multiple epitopes on a single antigen and uncovering important sequence activity relationships. UniDabs from transgenic rats are ideal building blocks for the generation of potent and highly manufacturable multivalent antibody therapeutics. Results: We have identified UniDabs that efficiently block PD-1/PD-L1 interaction as well as additional UniDabs that bind to five different functional epitopes on human CD38. Using different combinations and arrangements of UniDabs, a variety of multivalent antibodies were constructed and evaluated in in vitro models. Specific combinations of UniDabs show more potent cytotoxic effects than Daratumumab for multiple mechanisms including CDC and direct apoptosis. Conclusions: Data from a range of assay types show that multivalent UniAbs targeting CD38 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action.

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics.

Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics.

Glypican-3 targeted human heavy chain antibody as a drug carrier for hepatocellular carcinoma therapy.

Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with (125)I- and (111)In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with (111)In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.

High Frequency of Monoclonal Immunoglobulins Exhibiting Natural Autoantibody-Like Activity in Patients with Multiple Myeloma and Waldenström Macroglobulinemia

Abstract 2893Serum monoclonal immunoglobulins (M-Igs) of patients with monoclonal gammopathies are known to possess antibody activity against autoantigens, bacterial antigens and haptens, but their incidence is controversial. So far, high incidence of autoantibodies (autoAb) have been demonstrated against actin, Ro/SS-A and La/SS-B for M-Igs of all three classes (IgG, IgA, IgM) and against Fc fragment of IgG and the Ii group exclusively for IgM M-Ig. Other less frequent antiboby specificities of M-Igs include anti-myosin, anti-tubulin, anti-thyroglobulin and anti-DNA. M-Igs with anti-actin activity were found to express polyreactivity, which is a property of natural autoAbs (NAbs). NAbs are germ-line encoded, occur in all species, circulate in low concentrations and belong to all Ig classes. NAbs play important immunoregulatory role and anti-F(ab')2 reactivity is crucial for the establishment of the idiotypic network.The aim of this study was to investigate the frequency of human M-Igs that exhibit NAb-like activity in patients with monoclonal gammopathies and to explore possible correlations with their clinical features. Thus, 151 M-Igs (124 of IgG class: 71IgG-kappa/53 IgG-lambda and 27 of IgA class: 16 IgA-kappa/11 IgA-lambda) from patients with symptomatic multiple myeloma (MM; n=136), asymptomatic MM (n=8) or MGUS (n=7) and 50 M-Igs from patients with Waldenström Macroglobulinemia (WM) or IgM-MGUS (43 IgM-kappa and 7 IgM-lambda), without any autoimmune diseases, were studied by an in-house ELISA for polyreactivity against six autoantigens (native DNA, actin, tubulin, myosin, carbonic anhydrase, thyroglobulin) and the non-self hapten TriNitroPhenyl (TNP). Sera were also tested for antibody activity against F(ab’)2 fragment of human polyclonal IgGs. Most of these antigens are known targets of NAbs. Sera were first screened with enzyme-coupled human heavy chain antibodies (anti-G, anti-M and anti-A), and reactivity–with the exception of anti-F(ab’)2–was further attributed to the M-Ig using enzyme-coupled light chain antibodies (anti-kappa and anti-lambda).A significant proportion (18.2%: 35/192 evaluated patients) of pathological sera exhibited anti-F(ab')2 activity (27 from IgG-MM, 3 from IgA-MM and 5 from WM patients). Among the total M-Igs tested, 9.5% (18/189) recognised actin, 8.7% (17/194) carbonic anhydrase, 4.6% (9/194) thyroglobulin, 3.7% (7/186) TNP, 3.5% (7/196) tubulin, 2.5% (5/193) myosin and 1.5% (3/188) DNA. M-Igs reacting with actin, carbonic anhydrase, tubulin, myosin and TNP were found to belong to all three Ig classes (IgG, IgA, and IgM). Among the IgG M-Igs tested, the most frequent reactivity was that for thyroglobulin (7.6%), while among the IgA and IgM M-Igs the highest incidence observed was for actin and carbonic anhydrase (23%, 23% and 16.6%, 15.2%, respectively). Positive IgM and IgG M-Ig had either kappa or lambda light chain in a similar proportion, while positive IgA were mostly IgA-lambda (16/18). A high percentage of positive M-Igs from MM and WM patients were polyreactive (46%), i.e., reacted with at least two panel antigens. Polyreactivity was a prominent characteristic of IgA (6/6) and IgM (9/13) but not of IgG (6/36) M-Ig. Most frequent polyreactivity profile was that for actin, carbonic anhydrase and TNP.There was no significant correlation of immune-related features of symptomatic WM (immune cytopenias, cold agglutinin disease, cryoglobulinemia) or of MM characteristics (ISS, CRAB, LDH, plasma cell infiltration) with any of the detected M-Ig specificities or their NAb-like profile.In conclusion, we report for the first time anti-carbonic anhydrase and anti-TNP activity of the M-Igs from patients with monoclonal gammopathies. We also report high incidence of anti- F(ab')2 activity in the sera of these patients as well as of polyreactive M-Igs. Our findings suggest that the M-Igs with NAb activity might reflect the expansion of a clone normally producing a NAb. To-date, it has been assumed that a naive B-cell is first driven to clonal expansion by antigenic stimulation and that subsequently, an oncogenic stimulus results in malignant transformation. Assuming that certain malignant monoclonal gammopathies arise from the proliferation of a clone normally producing a NAb which recognise F(ab')2 and hence is probably involved in the regulation of the NAbs network, it may also be postulated that this may result in deregulation of this network. Disclosures:No relevant conflicts of interest to declare.

A novel domain antibody rationally designed against TNF-α using variable region of human heavy chain antibody as scaffolds to display antagonistic peptides

Neutralizing of TNF-α has been proved effective in treatment of some autoimmune diseases, e.g. rheumatoid arthritis and Crohn's disease. Low molecular weight synthetic peptides can mimic the binding sites of TNF-α receptors and block the activity of TNF-α. In order to stabilize the conformation, increase the affinity and bioactivity, in this study, heavy chain variable region of human antibody was used as a scaffold to simultaneously display three peptides, which were designed on the interaction between TNF-α and it's neutralizing monoclonal antibody. On the basis of the structural character and physical–chemical property of the families of seven kinds of heavy chain variable regions (VH) in human antibodies, the fifth type of VH was screened as scaffold to display the antagonist peptide. Based on the computer-guided molecular design method, a novel domain antibody against TNF-α (named as ATD5) was designed as TNF-α antagonist. The theoretical study showed that ATD5 was more stable than displayed antagonist peptide. The binding activity with TNF-α was higher than free peptides. After expression and purification in Escherichia coli, ATD5 could bind directly with TNF-α and inhibit the binding of TNF-α to its two receptors, TNFR1 and TNFR2. ATD5 could also reduce the TNF-α-mediated cytotoxicity and inhibit TNF-α-meditated caspase activation on L929 cells in a dose dependent manner. The activity of ATD5 was significantly stronger than three peptides displayed by ATD5. This study provides a novel strategy for the development of new TNF-α inhibitors. This study demonstrates that it is possible to screen potential antagonists of TNF-α using in vitro analysis systems in combination with the computer-aided modeling method.

Identification of a human heavy chain antibody fragment directed against human platelet alloantigen 1a by phage display library.

The human platelet alloantigen HPA-1a (PlA1) is responsible for most cases of post-transfusion purpura and neonatal alloimmune thrombocytopenia in the Caucasian population. HPA-1a and HPA-1b are two allelic forms of the platelet membrane glycoprotein IIIa (GPIIIa) gene that differ by a single amino acid. In this report, we describe the development of a recombinant heavy chain antibody fragment capable of distinguishing between the homozygous forms of HPA-1a and HPA-1b. This antibody fragment was isolated from the lymphocytes of an immunized individual through the use of a phage display library system. The recombinant antibody fragment reacted with human platelet lysates from HPA-1a homozygous donors, the HPA-1a form of recombinant N-terminal GPIIIa and intact HPA-1a platelets, but did not react with platelet lysate from HPA-1b homozygous donors, reduced HPA-1a form of platelet GPIIIa or other platelet glycoproteins. This HPA-1a specific human antibody fragment works well in common laboratory assays such as ELISA and flow cytometry, which can assist in identifying HPA-1b homozygous individuals who are known to have a higher risk for developing neonatal alloimmmune thrombocytopenia and post-transfusion purpura. Thus, selection of recombinant antibody fragment using phage display offers a promising alternative to hybridoma technology for the production of human antibodies against human alloantigens and holds potential as a technique in therapeutic applications.

Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome.

We have developed a method for the introduction of yeast artificial chromosomes (YACs) into transgenic mice. An 85 kilobase (kb) fragment of the human heavy chain immunoglobulin gene was cloned as a YAC, and embryonic stem cell lines carrying intact, integrated YACs were derived by co-lipofection of the YAC with an unlinked selectable marker. Chimaeric founder animals were produced by blastocyst injection, and offspring transgenic for the YAC were obtained. Analysis of serum from these offspring for human heavy chain antibody subunits demonstrated expression of the YAC-borne immunoglobulin gene fragment. Co-lipofection may prove to be a highly-successful means of producing transgenic mice containing large gene fragments in YACs.