Immunoglobulin heavy constant gamma gene evolution is modulated by both the divergent and birth-and-death evolutionary models.

Immunoglobulin G (IgG) is one of the five antibody classes produced in mammals as part of the humoral responses accountable for protecting the organisms from infection. Its antibody heavy chain constant region is encoded by the Ig heavy-chain gamma gene (IGHG). In humans, there are four IGHG genes which encode the four subclasses, each with a specialized effector function. Although four subclasses of IgG proteins have also been reported in macaques, this does not appear to be the rule for all primates. In Platyrrhini, IgG has been stated to be encoded by a single-copy gene. To date, it remains unknown how the IGHG has expanded or contracted in the primate order; consequently, we have analyzed data from 38 primate genome sequences to identify IGHG genes and describe the evolution of IGHG genes in primate order. IGHG belongs to a multigene family that evolves by the birth-death evolutionary model in primates. Whereas Strepsirrhini and Platyrrhini have a single-copy gene, in Catarrhini, it has expanded to several paralogs in their genomes; some deleted and others pseudogenized. Furthermore, episodic positive selection may have promoted a species-specific IgG effector function. We propose that IgG evolved to reach an optimal number of copies per genome to adapt their humoral immune responses to different environmental conditions. This study has implications for biomedical trials using non-human primates.

Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice.

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

Expression of immunoglobulin constant domain genes in neurons of the mouse central nervous system.

General consensus states that immunoglobulins are exclusively expressed by B lymphocytes to form the first line of defense against common pathogens. Here, we provide compelling evidence for the expression of two heavy chain immunoglobulin genes in subpopulations of neurons in the mouse brain and spinal cord. RNA isolated from excitatory and inhibitory neurons through ribosome affinity purification revealed Ighg3 and Ighm transcripts encoding for the constant (Fc), but not the variable regions of IgG3 and IgM. Because, in the absence of the variable immunoglobulin regions, these transcripts lack the canonical transcription initiation site used in lymphocytes, we screened for alternative 5' transcription start sites and identified a novel 5' exon adjacent to a proposed promoter element. Immunohistochemical, Western blot, and in silico analyses strongly support that these neuronal transcripts are translated into proteins containing four Immunoglobulin domains. Our data thus demonstrate the expression of two Fc-encoding genes Ighg3 and Ighm in spinal and supraspinal neurons of the murine CNS and suggest a hitherto unknown function of the encoded proteins.

Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes.

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt's lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sµ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

Sequence and N-glycan diversity analysis of immunoglobulin G from buffalo milk using RP-UHPLC MS/MS.

Immunoglobulin G is the abundant antibody present in the colostrum and milk of major dairy animals. In the present study, buffalo milk IgG was characterized for its amino acid sequence and glycan diversity using reverse phase liquid chromatography coupled to ESI-Q-TOF MS in tandem mode. Amino acid sequence analysis of heavy chain constant region revealed the presence of two IgG subtypes namely IgG1 and IgG3, with IgG1 being the abundant. The complete light chain constant region sequence was also determined. N-glycan sequence analysis at a highly conserved site Asn-Ser-Thr revealed the presence of mainly biantennary complex type with core fucosylation (34%), bisecting GlcNAc (19%) and sialylation with both Neu5Ac and Neu5Gc (14%). The observed glycan diversity in buffalo milk IgG is in part comparable with bovine colostrum as well as human, bovine, goat serum counterparts.

Deletions and amplifications of the IGH variable and constant regions:a novel prognostic parameter in patients with multiple myeloma.

Cytogenetic abnormalities are a recognized factor in the pathogenesis of multiple myeloma (MM). While chromosomal translocations involving the IGH gene have been investigated and reported, the implications of deletions or amplifications in the IGH gene have been less frequently examined. We conducted a retrospective analysis of 260 patients with MM from Northern Israel. Fluorescent in situ hybridization (FISH) analysis of separated CD-138 positive cells was done on bone marrow samples collected between 2016 and 2018. We used IGH break apart probes to identify IGH abnormalities and performed statistical analysis of clinical and prognostic features, comparing the different cytogenetic groups. Deletions in the variable region of the IGH (IGHv) were found in 17.3 % (n = 45) of patients and correlated with significantly worse progression free survival (PFS) after two years of follow up (p = 0.008), as well as with a worse response to 1st line treatment (p = 0.037). The median PFS was 7.1 and 17.7 months in patients with and without IGHv deletion, respectively. PFS differences remained significant (p = 0.017) in subgroup analysis of patients with high-risk cytogenetics (n = 108, 19 with IGHv deletion). Overall survival was not significantly different in the two groups. Constant region (IGHc) amplifications, were less frequently found (6.15 %, n = 16), yet significantly correlated with worse PFS after two years of follow up (p = 0.023). This difference remained valid in the high-risk subgroup (p = 0.001). In Conclusion, we identified that deletion of the IGH variable region and amplification in the IGH constant region, are both associated with poor prognosis and inferior outcome in MM.

Beyond Allotypes: The Influence of Allelic Diversity in Antibody Constant Domains.

Polymorphic diversity in antibody constant domains has long been defined by allotypic motifs that cross react with the sera of other individuals. Improvements in sequencing technologies have led to the discovery of a large number of new allelic sequences that underlie this diversity. Many of the point mutations lie outside traditional allotypic motifs suggesting they do not elicit immunogenic responses. As antibodies play an important role in immune defense and biotechnology, understanding how this newly resolved diversity influences the function of antibodies is important. This review investigates the current known diversity of antibody alleles at a protein level for each antibody isotype as well as the kappa and lambda light chains. We focus on evidence emerging for how these mutations perturb antibody interactions with antigens and Fc receptors that are critical for function, as well as the influence this might have on the use of antibodies as therapeutics and reagents.

Reprogramming the Constant Region of Immunoglobulin G Subclasses for Enhanced Therapeutic Potency against Cancer.

The constant region of immunoglobulin (Ig) G antibodies is responsible for their effector immune mechanism and prolongs serum half-life, while the fragment variable (Fv) region is responsible for cellular or tissue targeting. Therefore, antibody engineering for cancer therapeutics focuses on both functional efficacy of the constant region and tissue- or cell-specificity of the Fv region. In the functional aspect of therapeutic purposes, antibody engineers in both academia and industry have capitalized on the constant region of different IgG subclasses and engineered the constant region to enhance therapeutic efficacy against cancer, leading to a number of successes for cancer patients in clinical settings. In this article, we review IgG subclasses for cancer therapeutics, including i) IgG1, ii) IgG2, 3, and 4, iii) recent findings on Fc receptor functions, and iv) future directions of reprogramming the constant region of IgG to maximize the efficacy of antibody drug molecules in cancer patients.

Genetic removal of the CH1 exon leads to the production of hypofunctional heavy chain-only IgG2a in rats.

Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies.

Susceptibility to Plasmodium falciparum Malaria: Influence of Combined Polymorphisms of IgG3 Gm Allotypes and Fc Gamma Receptors IIA, IIIA, and IIIB.

The binding of immunoglobulin (Ig) to Fc gamma receptors (FcgR) at the immune cell surface is an important step to initiate immunological defense against malaria. However, polymorphisms in receptors and/or constant regions of the IgG heavy chains may modulate this binding. Here, we investigated whether polymorphisms located in FcgR and constant regions of the heavy chain of IgG are associated with susceptibility to P. falciparum malaria. For this purpose, a clinical and parasitological follow-up on malaria was conducted among 656 infants in southern Benin. G3m allotypes (from total IgG3) were determined by a serological method of hemagglutination inhibition. FcgRIIA 131R/H and FcgRIIIA 176F/V genotypes were determined using the TaqMan method and FcgRIIIB NA1/NA2 genotypes were assessed by polymerase chain reaction using allele-specific primers. Association analyses between the number of malaria infections during the follow-up and polymorphisms in IgG G3m allotypes and FcgR were studied independently by zero inflated binomial negative regression. The influence of combinations of G3m allotypes and FcgRIIA/FcgRIIIA/FcgRIIIB polymorphisms on the number of P. falciparum infections, and their potential interaction with environmental exposure to malaria was assessed by using the generalized multifactor dimensionality reduction (GMDR) method. Results showed that individual carriage of G3m24 single allotype and of G3m5,6,10,11,13,14,24 phenotype was independently associated with a high risk of malaria infection. A risk effect for G3m6 was observed only under high environmental exposure. FcgRIIIA 176VV single genotype and combined carriage of FcgRIIA 131RH/FcgRIIIA 176VV/FcgRIIIB NA1NA2, FcgRIIA 131HH/FcgRIIIA 176FF/FcgRIIIB NA1NA1, FcgRIIA 131HH/FcgRIIIA 176VV/FcgRIIIB NA2NA2 and FcgRIIA 131HH/FcgRIIIA 176VV/FcgRIIIB NA1NA2 genotypes were related to a high number of malaria infections. The risk was accentuated for FcgRIIIA 176VV when considering the influence of environmental exposure to malaria. Finally, the GMDR analysis including environmental exposure showed strengthened associations with a malaria risk when FcgRIIA/FcgRIIIA/FcgRIIIB genotypes were combined to G3m5,6,11,24 and G3m5,6,10,11,13,15,24 phenotypes or G3m10 and G3m13 single allotypes. Our results highlight the relevance of studying IgG heavy chain and FcgR polymorphisms, independently as well as in combination, in relation to the individual susceptibility to P. falciparum infection. The intensity of individual exposure to mosquito bites was demonstrated to impact the relationships found.

Biosensor surface functionalization by a simple photochemical immobilization of antibodies: experimental characterization by mass spectrometry and surface enhanced Raman spectroscopy.

Surface functionalization is a key step in biosensing since it is the basis of an effective analyte recognition. Among all the bioreceptors, antibodies (Abs) play a key role thanks to their superior specificity, although the available immobilization strategies suffer from several drawbacks. When gold is the interacting surface, the recently introduced Photochemical Immobilization Technique (PIT) has been shown to be a quick, easy-to-use and very effective method to tether Abs oriented upright by means of thiols produced via tryptophan mediated disulphide bridge reduction. Although the molecular mechanism of this process is quite well identified, the detailed morphology of the immobilized antibodies is still elusive due to inherent difficulties related to the microscopy imaging of Abs. The combination of Mass Spectrometry, Surface-Enhanced Raman Spectroscopy and Ellman's assay demonstrates that Abs irradiated under the conditions in which PIT is realized show only two effective disulphide bridges available for binding. They are located in the constant region of the immunoglobulin light chain so that the most likely position Ab assumes is side-on, i.e. with one Fab (i.e. the antigen binding portion of the antibody) exposed to the solution. This is not a limitation of the recognition efficiency in view of the intrinsic flexibility of the Ab structure, which makes the free Fab able to sway in the solution, a feature of great importance in many biosensing applications.

Plasma Pharmacokinetics of High-Affinity Transferrin Receptor Antibody-Erythropoietin Fusion Protein is a Function of Effector Attenuation in Mice.

Erythropoietin (EPO) is a potential therapeutic for Alzheimer's disease (AD); however, limited blood-brain barrier (BBB) penetration reduces its applicability as a CNS therapeutic. Antibodies against the BBB transferrin receptor (TfRMAbs) act as molecular Trojan horses for brain drug delivery, and a fusion protein of EPO and TfRMAb, designated TfRMAb-EPO, is protective in a mouse model of AD. TfRMAbs have Fc effector function side effects, and removal of the Fc N-linked glycosylation site by substituting Asn with Gly reduces the Fc effector function. However, the effect of such Fc mutations on the pharmacokinetics (PK) of plasma clearance of TfRMAb-based fusion proteins, such as TfRMAb-EPO, is unknown. To examine this, the plasma PK of TfRMAb-EPO (wild-type), which expresses the mouse IgG1 constant heavy chain region and includes the Asn residue at position 292, was compared to the mutant TfRMAb-N292G-EPO, in which the Asn residue at position 292 is mutated to Gly. Plasma PK was compared following IV, IP, and SQ administration for doses between 0.3 and 3 mg/kg in adult male C57 mice. The results show a profound increase in clearance (6- to 8-fold) of the TfRMAb-N292G-EPO compared with the wild-type TfRMAb-EPO following IV administration. The clearance of both the wild-type and mutant TfRMAb-EPO fusion proteins followed nonlinear PK, and a 10-fold increase in dose resulted in a 7- to 11-fold decrease in plasma clearance. Following IP and SQ administration, the Cmax values of the TfRMAb-N292G-EPO mutant were profoundly (37- to 114-fold) reduced compared with the wild-type TfRMAb-EPO, owing to comparable increases in plasma clearance of the mutant fusion protein. The wild-type TfRMAb fusion protein was associated with reticulocyte suppression, and the N292G mutation mitigated this suppression of reticulocytes. Overall, the beneficial suppression of effector function via the N292G mutation may be offset by the deleterious effect this mutation has on the plasma levels of the TfRMAb-EPO fusion protein, especially following SQ administration, which is the preferred route of administration in humans for chronic neurodegenerative diseases including AD.

Applications of the immunoglobulin Cw fragment (IgCw) composed of the constant regions of heavy and light (CH and CL) chains.

We report the production and application of a recombinant IgCw molecule, which is composed of only the constant domains of the heavy (CH) and light (CL) chains, lacking a variable (V) domain. We produced IgCw, especially human IgCw-γκ (98 kDa), composed of two human Cγ chains (37 kDa each) and two Cκ chains (12 kDa each), using HEK293F cell culture. We found that the yield of IgCw-γκ protein was ∼20 mg/L, which was comparable to that of full-size IgG; it bound to Fcγ receptor-positive cells with a low background noise on Fcγ receptor-negative cells; and IgCw-γκ can be used as a reference for measurement of Ig concentration. Moreover, Cγ and Cκ chains were easily isolated from IgCw-γκ by a single step of affinity chromatography in the presence of a reducing agent. These results demonstrate that the IgCw molecule has the potential to be used for certain in vitro and in vivo applications as an alternative to an irrelevant isotype control IgG, and to be used a favorable antigen for acquiring isotype-specific antibodies by immunizing animals.

Developmental regulation of DNA cytosine methylation at the immunoglobulin heavy chain constant locus.

DNA cytosine methylation is involved in the regulation of gene expression during development and its deregulation is often associated with disease. Mammalian genomes are predominantly methylated at CpG dinucleotides. Unmethylated CpGs are often associated with active regulatory sequences while methylated CpGs are often linked to transcriptional silencing. Previous studies on CpG methylation led to the notion that transcription initiation is more sensitive to CpG methylation than transcriptional elongation. The immunoglobulin heavy chain (IgH) constant locus comprises multiple inducible constant genes and is expressed exclusively in B lymphocytes. The developmental B cell stage at which methylation patterns of the IgH constant genes are established, and the role of CpG methylation in their expression, are unknown. Here, we find that methylation patterns at most cis-acting elements of the IgH constant genes are established and maintained independently of B cell activation or promoter activity. Moreover, one of the promoters, but not the enhancers, is hypomethylated in sperm and early embryonic cells, and is targeted by different demethylation pathways, including AID, UNG, and ATM pathways. Combined, the data suggest that, rather than being prominently involved in the regulation of the IgH constant locus expression, DNA methylation may primarily contribute to its epigenetic pre-marking.

Human cytomegalovirus-specific T-cell receptor engineered for high affinity and soluble expression using mammalian cell display.

T-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV). However, TCRs are more difficult to express and engineer than antibodies, and advanced methods are needed to enable their widespread use. Here, we engineered the human CMV-specific TCR RA14 for high-affinity and robust soluble expression. To achieve this, we adapted our previously reported mammalian display system to present TCR extracellular domains and used this to screen CDR3 libraries for clones with increased pMHC affinity. After three rounds of selection, characterized clones retained peptide specificity and activation when expressed on the surface of human Jurkat T cells. We obtained high yields of soluble, monomeric protein by fusing the TCR extracellular domains to antibody hinge and Fc constant regions, adding a stabilizing disulfide bond between the constant domains and disrupting predicted glycosylation sites. One variant exhibited 50 nm affinity for its cognate pMHC, as measured by surface plasmon resonance, and specifically stained cells presenting this pMHC. Our work has identified a human TCR with high affinity for the immunodominant CMV peptide and offers a new strategy to rapidly engineer soluble TCRs for biomedical applications.

Safety, pharmacokinetics, and biomarkers of F-652, a recombinant human interleukin-22 dimer, in healthy subjects.

F-652 is a recombinant fusion protein consisting of two human interleukin-22 (IL-22) molecules linked to an immunoglobulin constant region (IgG2-Fc). IL-22 plays critical roles in promoting tissue repair and suppressing bacterial infection. The safety, pharmacokinetics (PK), tolerability, and biomarkers of F-652 were evaluated following a single dose in healthy male volunteers in a randomized, double-blind, placebo-controlled study. Following single-dose subcutaneous (SC) injection of F-652 at 2.0 µg/kg into healthy subjects, six out of six subjects experienced delayed injection site reactions, which presented as erythematous and/or discoid eczematous lesions 10 to 17 days post-dosing. F-652 was then administered to the healthy subjects via an intravenous (IV) infusion at 2.0, 10, 30, and 45 µg/kg. No severe adverse event (SAE) was observed during the study. Among the IV-dosed cohorts, eye and skin treatment emergent adverse events (TEAEs) were observed in the 30 and 45 µg/kg cohorts. F-652 IV dosing resulted in linear increases in Cmax and AUC(0-t), and the T1/2 ranged from 39.4 to 206 h in the cohorts. An IV injection of F-652 induced dose-dependent increases in serum marker serum amyloid A, C-reactive protein, and FIB, and decreased serum triglycerides. The serum levels of 36 common pro-inflammatory cytokines/chemokines were not altered by the treatment of F-652 at 45 µg/kg. In conclusion, IV administration of F-652 to healthy male volunteers is safe and well-tolerated and demonstrates favorable PK and pharmacodynamic properties. These results warrant further clinical development of F-652 to treat inflammatory diseases.

Comparative Analysis of B-Cell Receptor Repertoires Induced by Live Yellow Fever Vaccine in Young and Middle-Age Donors.

Age-related changes can significantly alter the state of adaptive immune system and often lead to attenuated response to novel pathogens and vaccination. In present study we employed 5'RACE UMI-based full length and nearly error-free immunoglobulin profiling to compare plasma cell antibody repertoires in young (19-26 years) and middle-age (45-58 years) individuals vaccinated with a live yellow fever vaccine, modeling a newly encountered pathogen. Our analysis has revealed age-related differences in the responding antibody repertoire ranging from distinct IGH CDR3 repertoire properties to differences in somatic hypermutation intensity and efficiency and antibody lineage tree structure. Overall, our findings suggest that younger individuals respond with a more diverse antibody repertoire and employ a more efficient somatic hypermutation process than elder individuals in response to a newly encountered pathogen.

Genetic Removal of the CH1 Exon Enables the Production of Heavy Chain-Only IgG in Mice.

Nano-antibodies possess great potential in many applications. However, they are naturally derived from heavy chain-only antibodies (HcAbs), which lack light chains and the CH1 domain, and are only found in camelids and sharks. In this study, we investigated whether the precise genetic removal of the CH1 exon of the γ1 gene enabled the production of a functional heavy chain-only IgG1 in mice. IgG1 heavy chain dimers lacking associated light chains were detected in the sera of the genetically modified mice. However, the genetic modification led to decreased expression of IgG1 but increased expression of other IgG subclasses. The genetically modified mice showed a weaker immune response to specific antigens compared with wild type mice. Using a phage-display approach, antigen-specific, single domain VH antibodies could be screened from the mice but exhibited much weaker antigen binding affinity than the conventional monoclonal antibodies. Although the strategy was only partially successful, this study confirms the feasibility of producing desirable nano-bodies with appropriate genetic modifications in mice.

Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions.

The advantages of site-specific over stochastic bioconjugation technologies include homogeneity of product, minimal perturbation of protein structure/function, and - increasingly - the ability to perform structure activity relationship studies at the conjugate level. When selecting the optimal location for site-specific payload placement, many researchers turn to in silico modeling of protein structure to identify regions predicted to offer solvent-exposed conjugatable sites while conserving protein function. Here, using the aldehyde tag as our site-specific technology platform and human IgG1 antibody as our target protein, we demonstrate the power of taking an unbiased scanning approach instead. Scanning insertion of the human formylglycine generating enzyme (FGE) recognition sequence, LCTPSR, at each of the 436 positions in the light and heavy chain antibody constant regions followed by co-expression with FGE yielded a library of antibodies bearing an aldehyde functional group ready for conjugation. Each of the variants was expressed, purified, and conjugated to a cytotoxic payload using the Hydrazinyl Iso-Pictet-Spengler ligation to generate an antibody-drug conjugate (ADC), which was analyzed in terms of conjugatability (assessed by drug-to-antibody ratio, DAR) and percent aggregate. We searched for insertion sites that could generate manufacturable ADCs, defined as those variants yielding reasonable antibody titers, DARs of ≥ 1.3, and ≥ 95% monomeric species. Through this process, we discovered 58 tag insertion sites that met these metrics, including 14 sites in the light chain, a location that had proved refractory to the placement of manufacturable tag sites using in silico modeling/rational approaches.

Genomic organization and adaptive evolution of IGHC genes in marine mammals.

Immunoglobulins are important elements of the adaptive immune system that bind to an immense variety of microbial antigens to neutralize infectivity and specify effector functions. In the present study, the immunoglobulin heavy chain constant region (IGHC) genes from marine mammals were identified and compared with those of their terrestrial relatives to explore their genomic organization and evolutionary characteristics. The genomic organization of marine mammal IGHC genes was shown to be conservative with other eutherian mammals. Stronger signals of positive selection on IGHC were revealed in terrestrial mammals than that in marine mammals with the branch-site model, displaying different selective pressure, which might suggest their divergent adaptations to contrasted environments.