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PloS One 2020Therapeutic monoclonal antibodies have the potential to work as biological therapeutics. OKT3, Herceptin, Keytruda and others have positively impacted healthcare....
Therapeutic monoclonal antibodies have the potential to work as biological therapeutics. OKT3, Herceptin, Keytruda and others have positively impacted healthcare. Antibodies evolved naturally to provide high specificity and high affinity once mature. These characteristics can make them useful as therapeutics. However, we may be missing characteristics that are not obvious. We present a means of measuring antibodies in an unbiased manner that may highlight therapeutic activity. We propose using a microarray of random peptides to assess antibody properties. We tested twenty-four different commercial antibodies to gain some perspective about how much information can be derived from binding antibodies to random peptide libraries. Some monoclonals preferred to bind shorter peptides, some longer, some preferred motifs closer to the C-term, some nearer the N-term. We tested some antibodies with clinical activity but whose function was blinded to us at the time. We were provided with twenty-one different monoclonal antibodies, thirteen mouse and eight human IgM. These antibodies produced a variety of binding patterns on the random peptide arrays. When unblinded, the antibodies with polyspecific binding were the ones with the greatest therapeutic activity. The protein target to these therapeutic monoclonals is still unknown but using common sequence motifs from the peptides we predicted several human and mouse proteins. The same five highest proteins appeared in both mouse and human lists.
Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Epitope Mapping; High-Throughput Screening Assays; Humans; Immunoglobulin M; Mice; Peptide Library; Peptides; Protein Array Analysis; Protein Binding; Proteome
PubMed: 32196507
DOI: 10.1371/journal.pone.0229080 -
Current Opinion in Biotechnology Dec 2019Despite the recent explosion in the use of monoclonal antibodies (mAbs) as drugs, it remains a significant challenge to generate antibodies with a combination of... (Review)
Review
Despite the recent explosion in the use of monoclonal antibodies (mAbs) as drugs, it remains a significant challenge to generate antibodies with a combination of physicochemical properties that are optimal for therapeutic applications. We argue that one of the most important and underappreciated drug-like antibody properties is high specificity - defined here as low levels of antibody non-specific and self-interactions - which is linked to low off-target binding and slow antibody clearance in vivo and high solubility and low viscosity in vitro. Here, we review the latest advances in characterizing antibody specificity and elucidating its molecular determinants as well as using these findings to improve the selection and engineering of antibodies with extremely high, drug-like specificity.
Topics: Antibodies, Monoclonal; Antibody Specificity; Solubility; Viscosity
PubMed: 30822699
DOI: 10.1016/j.copbio.2019.01.008 -
ACS Chemical Biology Feb 2021Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen...
Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality using yeast displayed antibodies containing noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs). To enable systematic characterization of the effects of ncAA incorporation on antibody function, we first investigated whether diversification of a single antibody loop would support the isolation of binding clones against immunoglobulins from three species. We constructed and screened a billion-member library containing canonical amino acid diversity and loop length diversity only within the third complementarity determining region of the heavy chain (CDR-H3). Isolated clones exhibited moderate affinities (double- to triple-digit nanomolar affinities) and, in several cases, single-species specificity, confirming that antibody specificity can be mediated by a single CDR. This constrained diversity enabled the utilization of additional CDRs for the installation of chemically reactive and photo-cross-linkable ncAAs. Binding studies of ncAA-substituted antibodies revealed that ncAA incorporation is reasonably well tolerated, with observed changes in affinity occurring as a function of ncAA side chain identity, substitution site, and the ncAA incorporation machinery used. Multiple azide-containing ncAAs supported copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without the abrogation of binding function. Similarly, several alkyne substitutions facilitated CuAAC without the apparent disruption of binding. Finally, antibodies substituted with a photo-cross-linkable ncAA were evaluated for ultraviolet-mediated cross-linking on the yeast surface. Competition-based assays revealed position-dependent covalent linkages, strongly suggesting successful cross-linking. Key findings regarding CuAAC reactions and photo-cross-linking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. The consistency of findings on the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with "chemically augmented" binding functions. This includes strategies for systematically introducing small molecule functionality within binding protein structures and evaluating protein-based covalent target binding. The efficient preparation and chemical diversification of antibodies on the yeast surface open up new possibilities for discovering "drug-like" protein leads in high throughput.
Topics: Alkynes; Amino Acid Sequence; Amino Acids; Animals; Antibody Specificity; Azides; Cattle; Click Chemistry; Complementarity Determining Regions; Cross-Linking Reagents; Cycloaddition Reaction; Equidae; Immunoglobulin G; Peptide Library; Protein Engineering; Rabbits; Ultraviolet Rays
PubMed: 33482061
DOI: 10.1021/acschembio.0c00865 -
Cold Spring Harbor Protocols Dec 2020Verifying that a new antibody recognizes its target can be difficult. In this protocol, expression of a target protein in embryos is either knocked down using...
Verifying that a new antibody recognizes its target can be difficult. In this protocol, expression of a target protein in embryos is either knocked down using CRISPR-Cas9 technology (for zygotic proteins) or enhanced by microinjection of a synthetic mRNA (for maternal proteins). Western blotting analysis is then performed. If the antibody recognizes the target protein, the western blot will show a relatively weak band for CRISPR-injected embryos and a relatively strong band for RNA-injected embryos. This represents a straightforward, powerful strategy for confirming antibody specificity in .
Topics: Animals; Antibody Specificity; Blotting, Western; CRISPR-Cas Systems; Electrophoresis, Polyacrylamide Gel; Embryo, Nonmammalian; Female; Microinjections; Oocytes; RNA, Messenger; Recombinant Proteins; Xenopus laevis
PubMed: 33037077
DOI: 10.1101/pdb.prot105601 -
Methods in Molecular Biology (Clifton,... 2023Monoclonal antibodies (MAbs) can be used to detect and quantify protein biomarker antigens (Ag). Systematic screening with an enzyme-linked immunosorbent assay (Butler,...
Monoclonal antibodies (MAbs) can be used to detect and quantify protein biomarker antigens (Ag). Systematic screening with an enzyme-linked immunosorbent assay (Butler, J Immunoass, 21(2-3):165-209, 2000) [1] can be used to identify matched Ab-Ag pairs. A method is described for identifying MAbs that recognize cardiac biomarker creatine kinase isoform MB. Cross-reactivity with skeletal muscle biomarker creatine kinase isoform MM and brain biomarker creatine kinase isoform BB is also examined.
Topics: Antibody Specificity; Enzyme-Linked Immunosorbent Assay; Creatine Kinase; Antibodies, Monoclonal; Muscle, Skeletal; Sensitivity and Specificity
PubMed: 36795358
DOI: 10.1007/978-1-0716-2903-1_4 -
MAbs 2021The human IgG3 subclass is conspicuously absent among the formats for approved monoclonal antibody therapies and Fc fusion protein biologics. Concern about the potential... (Review)
Review
The human IgG3 subclass is conspicuously absent among the formats for approved monoclonal antibody therapies and Fc fusion protein biologics. Concern about the potential for rapid degradation, reduced plasma half-life, and increased immunogenicity due to marked variation in allotypes has apparently outweighed the potential advantages of IgG3, which include high affinity for activating Fc receptors, effective complement fixation, and a long hinge that appears better suited for low abundance targets. This review aims to highlight distinguishing features of IgG3 and to explore its functional role in the immune response. We present studies of natural immunity and recombinant antibody therapies that elucidate key contributions of IgG3 and discuss historical roadblocks that no longer remain clearly relevant. Collectively, this body of evidence motivates thoughtful reconsideration of the clinical advancement of this distinctive antibody subclass for treatment of human diseases. ADCC - Antibody-Dependent Cell-mediated CytotoxicityADE - Antibody-dependent enhancementAID - Activation-Induced Cytidine DeaminaseCH - Constant HeavyCHF - Complement factor HCSR - Class Switch RecombinationEM - Electron MicroscopyFab - Fragment, antigen bindingFc - Fragment, crystallizableFcRn - Neonatal Fc ReceptorFcγR - Fc gamma ReceptorHIV - Human Immunodeficiency VirusIg - ImmunoglobulinIgH - Immunoglobulin Heavy chain geneNHP - Non-Human Primate.
Topics: AIDS Vaccines; Animals; Antibody Diversity; Antibody Specificity; Cancer Vaccines; Glycosylation; Humans; Immunity, Humoral; Immunoglobulin Class Switching; Immunoglobulin G; Pneumococcal Vaccines; Protein Engineering; Protein Processing, Post-Translational; Structure-Activity Relationship; Vaccines
PubMed: 33602056
DOI: 10.1080/19420862.2021.1882028 -
Clinical and Experimental Immunology Apr 2023Anti-centromere (ACA) and antimitochondrial antibodies (AMA) are specific for limited-cutaneous systemic sclerosis (lcSSc) and primary biliary cholangitis (PBC),...
Anti-centromere (ACA) and antimitochondrial antibodies (AMA) are specific for limited-cutaneous systemic sclerosis (lcSSc) and primary biliary cholangitis (PBC), respectively, and can coexist in up to 25 and 30% of SSc and PBC patients. Here, we evaluated whether anti-centromeric protein A (CENP-A) antibodies cross-react with mitochondrial antigens. To this end, sera from two lcSSc patients (pt1 and pt4), one of them (pt4) also affected by PBC, were used as the source of ACA, previously shown to recognize different groups of amino acids (motifs) in the CENP-A region spanning amino acids 1-17 (Ap1-17). Pt1 and pt4 Ap1-17-specific IgG were purified by affinity-chromatography on insolubilized Ap1-17-peptide column and tested by western blotting with nuclear and cytoplasmic protein extract from HeLa cells. Immunoreactive proteins were identified by mass spectrometry and validated by immunodot. The results showed that affinity-purified SSc/PBC pt4 anti-Ap1-17 and not SSc pt1 anti-Ap1-17 Ab, specifically cross-reacted with the E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC-E2), the major mitochondrial autoantigen in PBC. Sequence homology analysis indicated that the motif A-x-x-P-x-A-P recognized by pt4 anti-Ap1-17 IgG and shared by CENP-A and PDC-E2, is also expressed by some members of the Human Herpesvirus family, suggesting that they may trigger the production of these cross-reacting antibodies.
Topics: Humans; Autoantibodies; Centromere Protein A; Pyruvate Dehydrogenase Complex; Liver Cirrhosis, Biliary; HeLa Cells; Scleroderma, Systemic; Autoantigens; Immunoglobulin G; Amino Acids; Antibody Specificity
PubMed: 36715304
DOI: 10.1093/cei/uxad012 -
Transplantation Feb 2021Advances in genetic engineering, particularly CRISPR/Cas9, have resulted in the development of a triple glycan-knockout (TKO) pig. There is minimal human antipig... (Review)
Review
Advances in genetic engineering, particularly CRISPR/Cas9, have resulted in the development of a triple glycan-knockout (TKO) pig. There is minimal human antipig antibody binding to TKO pig cells. The TKO background has decreased antibody binding to a sufficiently low level that any additional xenoantigens expressed on the cells can now be more easily detected. One of these xenoantigens is the swine major histocompatibility complex, termed swine leukocyte antigens (SLA). SLA are the homolog to HLAs, a protein complex expressed on human tissue capable of stimulating the development of new antibodies in allotransplantation. These antibodies can result in graft failure through hyperacute, acute, or chronic rejection. Our knowledge of SLA, particularly in the last 5 years, has grown considerably. The presence, cause, and methods to detect anti-SLA antibodies will need to be carefully considered for the first clinical trial of xenotransplantation. The focus of this review is to summarize the role of SLA in xenotransplantation and consider whether it will prove to be a major barrier. Techniques are now available to mutate target SLA amino acids to ensure that cross-reactive anti-HLA antibodies no longer bind to SLA on the cells of the organ-source pigs. While deletion of SLA expression is possible, it would render the pig at risk for infectious complications. The ideal organ-source pig for HLA highly sensitized recipients may therefore be 1 with site-specific mutations to eliminate cross-reactive binding.
Topics: Animals; Animals, Genetically Modified; Antibodies, Heterophile; Antibody Specificity; Antigens, Heterophile; Graft Rejection; Graft Survival; Histocompatibility Antigens Class I; Humans; Species Specificity; Sus scrofa; Transplantation Tolerance; Transplantation, Heterologous
PubMed: 32433239
DOI: 10.1097/TP.0000000000003303 -
PLoS Pathogens May 2023The modestly efficacious HIV-1 vaccine regimen (RV144) conferred 31% vaccine efficacy at 3 years following the four-shot immunization series, coupled with rapid waning...
The modestly efficacious HIV-1 vaccine regimen (RV144) conferred 31% vaccine efficacy at 3 years following the four-shot immunization series, coupled with rapid waning of putative immune correlates of decreased infection risk. New strategies to increase magnitude and durability of protective immunity are critically needed. The RV305 HIV-1 clinical trial evaluated the immunological impact of a follow-up boost of HIV-1-uninfected RV144 recipients after 6-8 years with RV144 immunogens (ALVAC-HIV alone, AIDSVAX B/E gp120 alone, or ALVAC-HIV + AIDSVAX B/E gp120). Previous reports demonstrated that this regimen elicited higher binding, antibody Fc function, and cellular responses than the primary RV144 regimen. However, the impact of the canarypox viral vector in driving antibody specificity, breadth, durability and function is unknown. We performed a follow-up analysis of humoral responses elicited in RV305 to determine the impact of the different booster immunogens on HIV-1 epitope specificity, antibody subclass, isotype, and Fc effector functions. Importantly, we observed that the ALVAC vaccine component directly contributed to improved breadth, function, and durability of vaccine-elicited antibody responses. Extended boosts in RV305 increased circulating antibody concentration and coverage of heterologous HIV-1 strains by V1V2-specific antibodies above estimated protective levels observed in RV144. Antibody Fc effector functions, specifically antibody-dependent cellular cytotoxicity and phagocytosis, were boosted to higher levels than was achieved in RV144. V1V2 Env IgG3, a correlate of lower HIV-1 risk, was not increased; plasma Env IgA (specifically IgA1), a correlate of increased HIV-1 risk, was elevated. The quality of the circulating polyclonal antibody response changed with each booster immunization. Remarkably, the ALVAC-HIV booster immunogen induced antibody responses post-second boost, indicating that the viral vector immunogen can be utilized to selectively enhance immune correlates of decreased HIV-1 risk. These results reveal a complex dynamic of HIV-1 immunity post-vaccination that may require careful balancing to achieve protective immunity in the vaccinated population. Trial registration: RV305 clinical trial (ClinicalTrials.gov number, NCT01435135). ClinicalTrials.gov Identifier: NCT00223080.
Topics: Humans; Antibody Formation; HIV-1; HIV Infections; Immunization, Secondary; Antibody Specificity; HIV Antibodies; AIDS Vaccines; HIV Seropositivity; HIV Envelope Protein gp120
PubMed: 37256916
DOI: 10.1371/journal.ppat.1011359 -
Cells Aug 2023Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-rich plaques within arterial walls. T cells play a pivotal role in the... (Review)
Review
Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-rich plaques within arterial walls. T cells play a pivotal role in the pathogenesis of atherosclerosis in which they help orchestrate immune responses and contribute to plaque development and instability. Here, we discuss the recognition of atherosclerosis-related antigens that may trigger T cell activation together with additional signaling from co-stimulatory molecules and lesional cytokines. Although few studies have indicated candidates for the antigen specificity of T cells in atherosclerosis, further research is needed. Furthermore, we describe the pro-atherogenic and atheroprotective roles of diverse subsets of T cells such as CD4 helper, CD8 cytotoxic, invariant natural killer, and γδ T cells. To classify and quantify T cell subsets in atherosclerosis, we summarize current methods to analyze cellular heterogeneity including single cell RNA sequencing and T cell receptor (TCR) sequencing. Further insights into T cell biology will help shed light on the immunopathology of atherosclerosis, inform potential therapeutic interventions, and pave the way for precision medicine approaches in combating cardiovascular disease.
Topics: Humans; T-Lymphocytes; Atherosclerosis; Cardiovascular Diseases; Antibody Specificity; Arteries
PubMed: 37681883
DOI: 10.3390/cells12172152