Human-derived Antibody Research Articles

295. Humanized Mice and Convalescent Humans Yield an Antibody Panel against the Highly Immunogenic Decorin Binding Protein A (DbpA) of the Lyme Disease Spirochete, Borrelia Burgdorferi

Abstract Background Lyme disease is the most commonly diagnosed tick-borne infection, affecting more than 300000 people in the United States annually. The disease is principally caused by the transmission of the spirochete bacteria, by the black-legged (deer) ticks. With a renewed call for interest in a Lyme disease vaccine, antibodies relevance continues due to their efficacy, safety and versatile nature. Humanized mice and humans recovering from the disease have successfully been used to generate therapeutic antibodies against Ebola and SARS-CoV-2 diseases. Methods Our study used humanized mice and convalescent patients to generate recombinant human antibodies that recognize decorin-binding protein A (DbpA), a lipoprotein that is proposed to function as spirochete adhesin. The mAbs expressed as human IgG-Fc fragments were characterized for reactivity with recombinant DbpA, native DbpA on the surface of live Borrelia burgdorferi, borreliacidal activity in the presence of human complement and, finally subject to epitope mapping using an array of methodologies. Results The predominant lineage of humanized mice antibodies utilized VH5-51, VH3-20, VH3-23 and VH3-53 all paired with VK1-33 families. Other common families included VH4-31 paired with VK1-39, VH4-4 and VH4-8 paired with VK-9. The human derived antibody utilized VH3-30 paired with VK1-33. We observed an overlap in the repertoire of kappa chains between humanized mice and human-derived mAbs. Conclusion Combining the humanized mice and human platforms enabled the identification of antibodies with a high binding and specificity to DbpA, which have potential prophylactic and therapeutic properties. Disclosures All Authors: No reported disclosures

Antitumor Immune Mechanisms of the Anti-Complement Factor H Antibody GT103.

Development of novel therapeutic antibodies that not only kill tumor cells but modulate the adaptive immune response has the potential to produce long term anti-cancer immunity and a durable clinical response. We previously reported the discovery of anti-complement factor H (CFH) autoantibodies in lung cancer patients that were associated with early stage disease and exceptional outcomes. The human monoclonal antibody GT103, produced from a single CFH autoantibody-expressing B cell of a lung cancer patient, recognizes a conformationally distinct epitope on tumor cells, kills tumor cells, and inhibits tumor growth in animal studies. Recent experiments have shown that GT103 restructures the tumor microenvironment and initiates a robust antitumoral adaptive immune response. The current study further elucidates several mechanisms by which GT103 kills tumor cells and drives the immune program. Here we show GT103 has specificity for tumor cells without binding to native soluble CFH or normal tissues. GT103 causes complement C3 split product deposition on tumor cells in vitro and in vivo, triggers antibody-dependent cellular phagocytosis, and increases translocation of the danger associated molecular pattern molecule calreticulin to the plasma membrane. We also demonstrate that GT103 causes B cell activation in vitro and in vivo, and that GT103 antitumor activity in vivo is B cell dependent. The complex mechanism of GT103, a tumor specific antibody that kills tumor cells and stimulates an immune response, supports further development of this human-derived antibody as a novel therapeutic option for patients with lung cancer.

N501Y and K417N Mutations in the Spike Protein of SARS-CoV-2 Alter the Interactions with Both hACE2 and Human-Derived Antibody: A Free Energy of Perturbation Retrospective Study

The N501Y and K417N mutations in the spike protein of SARS-CoV-2 and their combination gave rise to questions, but the data on their mechanism of action at the molecular level were limited. In this study, we present free energy perturbation (FEP) calculations, performed at the end of December 2020, for the interactions of the spike S1 receptor-binding domain (RBD) with both the ACE2 receptor and an antibody derived from COVID-19 patients. Our results showed that the S1 RBD-ACE2 interactions were significantly increased whereas those with the STE90-C11 antibody dramatically decreased. The K417N mutation in a combination with N501Y fully abolished the antibody effect. However, Lys417Asn seems to have a compensatory mechanism of action increasing the S1 RBD-ACE2 free energy of binding. This may explain the increased spread of the virus observed in the U.K. and South Africa and also gives rise to an important question regarding the possible human immune response and the success of the already available vaccines. Notably, when the experimental data became available confirming our calculations, it was demonstrated that protein-protein FEP can be a useful tool for providing urgent data to the scientific community.

Blood group systems

Blood group systems

A human-derived antibody targets misfolded SOD1 and ameliorates motor symptoms in mouse models of amyotrophic lateral sclerosis.

Mutations in the gene encoding superoxide dismutase 1 (SOD1) lead to misfolding and aggregation of SOD1 and cause familial amyotrophic lateral sclerosis (FALS). However, the implications of wild-type SOD1 misfolding in sporadic forms of ALS (SALS) remain unclear. By screening human memory B cells from a large cohort of healthy elderly subjects, we generated a recombinant human monoclonal antibody (α-miSOD1) that selectively bound to misfolded SOD1, but not to physiological SOD1 dimers. On postmortem spinal cord sections from 121 patients with ALS, α-miSOD1 antibody identified misfolded SOD1 in a majority of cases, regardless of their SOD1 genotype. In contrast, the α-miSOD1 antibody did not bind to its epitope in most of the 41 postmortem spinal cord sections from non-neurological control (NNC) patients. In transgenic mice overexpressing disease-causing human SOD1G37R or SOD1G93A mutations, treatment with the α-miSOD1 antibody delayed the onset of motor symptoms, extended survival by up to 2 months, and reduced aggregation of misfolded SOD1 and motor neuron degeneration. These effects were obtained whether α-miSOD1 antibody treatment was administered by direct brain infusion or peripheral administration. These results support the further development of α-miSOD1 antibody as a candidate treatment for ALS involving misfolding of SOD1.

Structural and kinetic basis for the selectivity of aducanumab for aggregated forms of amyloid-\u03b2

Aducanumab, a human-derived antibody targeting amyloid-β (Aβ), is in Phase 3 clinical trials for the treatment of Alzheimer’s disease. Biochemical and structural analyses show that aducanumab binds a linear epitope formed by amino acids 3–7 of the Aβ peptide. Aducanumab discriminates between monomers and oligomeric or fibrillar aggregates based on weak monovalent affinity, fast binding kinetics and strong avidity for epitope-rich aggregates. Direct comparative studies with analogs of gantenerumab, bapineuzumab and solanezumab demonstrate clear differentiation in the binding properties of these antibodies. The crystal structure of the Fab fragment of aducanumab bound to its epitope peptide reveals that aducanumab binds to the N terminus of Aβ in an extended conformation, distinct from those seen in structures with other antibodies that target this immunodominant epitope. Aducanumab recognizes a compact epitope that sits in a shallow pocket on the antibody surface. In silico analyses suggest that aducanumab interacts weakly with the Aβ monomer and may accommodate a variety of peptide conformations, further supporting its selectivity for Aβ aggregates. Our studies provide a structural rationale for the low affinity of aducanumab for non-pathogenic monomers and its greater selectivity for aggregated forms than is seen for other Aβ-targeting antibodies.

In Animal and Cellular Models, Human-Derived Alpha-Synuclein Antibody is Found to Attenuate Spread of Toxic Protein

In Animal and Cellular Models, Human-Derived Alpha-Synuclein Antibody is Found to Attenuate Spread of Toxic Protein

Mechanism of Action and In Vivo Efficacy of a Human-Derived Antibody against Staphylococcus aureus α-Hemolysin

The emergence and spread of multi-drug-resistant strains of Staphylococcus aureus in hospitals and in the community emphasize the urgency for the development of novel therapeutic interventions. Our approach was to evaluate the potential of harnessing the human immune system to guide the development of novel therapeutics. We explored the role of preexisting antibodies against S. aureus α-hemolysin in the serum of human individuals by isolating and characterizing one antibody with a remarkably high affinity to α-hemolysin. The antibody provided protection in S. aureus pneumonia, skin, and bacteremia mouse models of infection and also showed therapeutic efficacy when dosed up to 18h post-infection in the pneumonia model. Additionally, in pneumonia and bacteremia animal models, the therapeutic efficacy of the α-hemolysin antibody appeared additive to the antibiotic linezolid. To better understand the mechanism of action of this isolated antibody, we solved the crystal structure of the α-hemolysin:antibody complex. To our knowledge, this is the first report of the crystal structure of the α-hemolysin monomer. The structure of the complex shows that the antibody binds α-hemolysin between the cap and the rim domains. In combination with biochemical data, the structure suggests that the antibody neutralizes the activity of the toxin by preventing binding to the plasma membrane of susceptible host cells. The data presented here suggest that protective antibodies directed against S. aureus molecules exist in some individuals and that such antibodies have a therapeutic potential either alone or in combination with antibiotics.

A novel human-derived antibody against NY-ESO-1 improves the efficacy of chemotherapy.

We investigated whether antibodies against intracellular tumor-associated antigens support tumor-specific immunity when administered together with a treatment that destroys the tumor. We propose that released antigens form immune complexes with the antibodies, which are then efficiently taken up by dendritic cells. We cloned the first human monoclonal antibodies against the Cancer/Testis (CT) antigen, NY-ESO-1. We tested whether the monoclonal anti-NY-ESO-1 antibody (12D7) facilitates cross-presentation of a NY-ESO-1-derived epitope by dendritic cells to human CD8+ T cells, and whether this results in the maturation of dendritic cells in vitro. We investigated the efficacy of 12D7 in combination with chemotherapy using BALB/c mice bearing syngeneic CT26 tumors that express intracellular NY-ESO-1. Human dendritic cells that were incubated with NY-ESO-1:12D7 immune complexes efficiently stimulated NY-ESO-1(157-165)/HLA-A2-specific human CD8+ T cells to produce interferon-γ, whereas NY-ESO-1 alone did not. Furthermore, the incubation of dendritic cells with NY-ESO-1:12D7 immune complexes resulted in the maturation of dendritic cells. Treatment of BALB/c mice that bear CT26/NY-ESO-1 tumors with 5-fluorouracil (5-FU) plus 12D7 was significantly more effective than chemotherapy alone. We propose systemic injection of monoclonal antibodies (mAbs) against tumor-associated antigens plus a treatment that promotes the local release of those antigens resulting in immune complex formation as a novel therapeutic modality for cancer.

Abstract 4741: Therapeutic efficacy of a human-derived antibody against cancer-testis antigen NY-ESO-1

Abstract Rationale: It is accepted that the immune system can control cancer and a favorable clinical course correlates with high infiltration by CD8+ T cells in different cancer entities. Thus, boosting cancer-specific immunity is an interesting therapeutic modality. Cancer-testis antigens (CTA) are promising target antigens due to their tumor-restricted expression pattern and high immunogenicity. NY-ESO-1 is one of the best characterized CTA, and spontaneous anti-NY-ESO-1 humoral and cellular responses were described in patients with various cancers. We propose that antibodies (Abs) against intracellular CTA have therapeutic potential because they may facilitate the uptake and presentation of antigens by dendritic cells (DC), which supports cancer-specific T cell responses. This effect may be more pronounced when Abs are used in combination with therapies that result in death, and thus antigen release, of cancer cells. We report here the characterization of the first human anti-NY-ESO-1 monoclonal Ab. Methods: Human-derived monoclonal Ab 12D7 was obtained by molecular cloning from memory B cells of a melanoma patient, ZH-311. To characterize the binding of 12D7 we performed ELISA, Biacore analysis and tissue staining. In addition, we used 12D7 to immunoprecipitate native NY-ESO-1 from human melanoma cell line SK-MEL-37. As a proof of concept, we compared the activation of NY-ESO-1-specific CD8+ T cell clones by DC fed with NY-ESO-1 vs. NY-ESO-1/12D7 immune complexes (IC) in vitro. To determine the therapeutic effect in vivo, we treated BALB/c mice bearing syngeneic CT26/NY-ESO-1 colorectal tumors with 5-FU +/- 12D7. To investigate Ab accumulation in established tumors 12D7 was labeled with FITC and injected into mice 2 days after 5-FU treatment. Results: Human monoclonal 12D7 specifically bound to NY-ESO-1 with high affinity (pM range) and precipitated endogenous NY-ESO-1 from SK-MEL-37. The uptake and cross-presentation of NY-ESO-1 by DC was greatly enhanced when NY-ESO-1 was complexed with 12D7 before feeding. Importantly, uptake of NY-ESO-1/12D7 IC, but not NY-ESO-1, resulted in maturation of DCs as determined by upregulation of co-stimulatory molecules. Finally, 12D7 significantly improved the therapeutic efficacy of 5-FU treatment in established CT26/NY-ESO-1 tumors. Ab accumulation in CT26/NY-ESO-1 tumors was enhanced when mice were treated with 5-FU as compared with untreated mice. Conclusion: We propose that the release of intracellular tumor antigens by chemotherapy and the accumulation of Ab to such antigens results in the local formation of IC. These IC are efficiently cross-presented to tumor-specific CD8+ T cells by DCs and may induce in situ maturation of the latter, both of which support tumor-specific effector function and thus contributes to tumor control. Our results suggest that targeting intracellular antigens, more specifically CTA, with Abs is a useful strategy for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4741. doi:10.1158/1538-7445.AM2011-4741

Adalimumab (HumiraTM) in ophthalmology: A review of the literature

Tumor Necrosis Factor alpha (TNF-α) is a pleiotropic cytokine which plays a primary role in the induction of inflammation in autoimmune diseases. The newest anti-TNF-α agent is adalimumab (Humira, Abbott Pharmaceutical Inc.), a human-derived antibody. This review summarizes the characteristics of adalimumab, highlighting its clinical use in systemic and ocular inflammatory disorders, and the possible therapeutic strategies. Adalimumab has been successfully used for the treatment of rheumatoid arthritis, ankylosing spondylitis, and psoriasis arthritis. More recently, adalimumab has shown promising qualities in controlling intraocular inflammations, even though this has been used prevalently as a rescue therapy for unresponsive cases. This biologic agent was also used in pediatric cases, showing a good safety and efficacy profile. Albeit no direct comparison with other biologics has been done, and adalimumab seems to be equivalent to the other anti-TNF-α, the switching to adalimumab can offer a better uveitic control. Adalimumab is a promising drug for the treatment of uveitis, even though further studies are needed on its application as a primary therapy in uveitis.

Generation and characterization of recombinant human antibodies specific for native laminin epitopes: potential application in cancer therapy.

Laminins are specific cellular regulators that directly and indirectly control activities such as cell attachment and migration, differentiation and polarity, proliferation and apoptosis, and protease expression. Considering the centrality of these issues to tumor progression, the generation of human-derived antibody fragments able to modulate laminin-regulated biological functions would allow the development of new strategies to improve treatment of cancer patients. In this report, we explore the use of phage display technology to isolate human anti-laminin antibody fragments. A library of single chain antibodies was selected using intact mouse laminin, and five different clones were identified. All the antibodies were specific for their cognate antigen, as revealed by lack of cross-reactivity with other components of the basement membranes. A more extensive characterization of the panel indicated that these antibodies recognize the native protein through conformational epitopes. All of them reduced tumor cell attachment to laminin, suggesting that domains of the laminin molecule that are recognized by these antibodies likely bind to cell-surface receptors. The antibody fragments bind to mouse, rat and human laminin. and show strong immunohistochemical reactivity with basement membranes in human and murine tissue sections. Their properties make them ideal candidates for in vivo applications.

Immunological properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127).

We examined the immunological properties of the recombinant hepatitis B surface antigen (r-HBsAg) which was expressed in mammalian cell (C127). The cross-immunity of r-HBsAg and plasma-derived hepatitis B surface antigen (p-HBsAg) were tested using Western blotting and ELISA with guinea pig polyclonal antibody and naturally infected human-derived antibody and the both antigens show the same results in their response pattern and intensity, which indicate they have a good cross-immunity. From the measurement of ED50 after formalin- or heat-inactivation, both r-HBsAg and p-HBsAg showed ED50 of 0.2-0.3 in formalin-inactivation, while r-HBsAg was 0.05-0.09 and p-HBsAg was 0.03-0.07 in heat-inactivation, which means heat-inactivation method is 3-4 times superior in immunogenicity. In the immunopersistency test performed in guinea pig for the period of 3 months with two different adjuvants, antibody titer was 34.2 with muramyl dipeptide adjuvant, which was 1.8 times greater than the antibody titer of 18.9 with AlPO4 adjuvant. The mutagenicity of r-HBsAg, examined by Ames test and micronucleus test, were all negative. In conclusion, r-HBsAg has the same cross-immunity with p-HBsAg, and heat-inactivation method and muramyl dipeptide adjuvant allow development of r-HBsAg vaccine with excellent immunogenicity.

Cloning and biological characterization of human single-chain Fv fragments that mediate neutralization of HIV-1.

To develop recombinant single-chain Fv fragments against HIV-1 gp120. A panel of human monoclonal antibody Fv fragments were generated against the HIV-1 gp120 by affinity selection from an antibody library expressed on the surface of filamentous phage. The library was prepared from peripheral blood lymphocytes of an asymptomatic HIV-1-infected mother with a high neutralization titer. This mother did not transmit HIV-1 to her offspring (non-transmitter). Heavy and light chains were initially amplified separately and combined by splicing by overlap extension to generate Fv fragments. Several clones expressing single-chain Fv fragments bind strongly to HIV-1 gp120 and several were found to neutralize cell-free HIV-1IIIB. Gross epitope mapping suggest that different clones bound to different functional regions on the envelope. The clones also exhibited sequence diversity. This strategy of cloning resulted in the development of functional human-derived antibody reagents with different anti-HIV-1 biological properties in vitro. These recombinant Fv fragments have potential utility as immune reagents, as well as in the design of potential immunotherapeutics. In addition, these antibody reagents may provide information on the relationship between humoral immunity and maternal-fetal (vertical) HIV-1 transmission.