Native Antibody Research Articles

RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells.

Immunization-based antibody discovery platforms require robust and effective protocols for the amplification, cloning, expression, and screening of antibodies from large numbers of B-cells in order to effectively capture the diversity of an experienced Ig-repertoire. Multiplex PCR using a series of forward and reverse primers designed to recover antibodies from a range of different germline sequences is challenging because primer design requires the recovery of full length antibody sequences, low starting template concentrations, and the need for all the primers to function under the same PCR conditions. Here we demonstrate several advantages to incorporating RNase H2-dependent PCR (rh-PCR) into a high-throughput, antibody-discovery platform. Firstly, rh-PCR eliminated primer dimer synthesis to below detectable levels, thereby eliminating clones with a false positive antibody titer. Secondly, by increasing the specificity of PCR, the rh-PCR primers increased the recovery of cognate antibody variable regions from single B-cells, as well as downstream recombinant antibody titers. Finally, we demonstrate that rh-PCR primers provide a more homogeneous sample pool and greater sequence quality in a Next Generation Sequencing-based approach to obtaining DNA sequence information from large numbers of cloned antibody cognate pairs. Furthermore, the higher specificity of the rh-PCR primers allowed for a better match between native antibody germline sequences and the VL/VH fragments amplified from single B-cells.

Potent Neutralizing Native Human Monoclonal Antibody that Recognizes the Ganglioside Receptor-Binding Pocket of Tetanus Toxin

Four potent native human mAbs targeting distinct epitopes on TeNT were isolated with neutralization potency ranging from approximately 17 mg to 6 mg each equivalent to 250IU of human anti-TeNT immunoglobulin. TT0170 binds fragment B, TT0069 and TT0155 bind fragment AB. MAb TT0067 binds fragment C and blocks the binding of TeNT to gangliosides, Co-crystal structure of TT0067 with fragment C of TeNT at 2.01 A resolution demonstrated that mAb TT0067 directly occupies the W pocket of one of the receptor binding sites on TeNT, resulting in blocking the binding of TeNT to ganglioside on the surface of host cells to neutralize TeNT, and revealed at the first time at atomic level the mechanism of action by TeNT neutralizing antibody and the key neutralization epitope on the fragment C of TeNT, and provided the critical information for development of fragment C of TeNT as a better and safer tetanus vaccine.

CIRCR201-dPBD, a Novel Pyrrolobenzodiazepine Dimer-Containing Site-Specific Antibody-Drug Conjugate Targeting c-Met Overexpression Tumors.

c-Met, as a receptor expressed on the cell membrane, contributes to the growth and metastasis of tumors, as well as angiogenesis, mainly through the hepatocyte growth factor (HGF)/c-Met axis during tumor progression. Although several c-Met inhibitors, including small molecules and monoclonal antibody inhibitors, are currently being investigated, their clinical outcomes have not been promising. Development of an antibody–drug conjugate (ADC) against c-Met could be an attractive therapeutic strategy that would provide superior antitumor efficacy with broad-spectrum c-Met expression levels. In the present study, site-specific drug–conjugate technology was applied to develop an ADC using the human-mouse cross-reactive c-Met antibody and a prodrug pyrrolobenzodiazepine (PBD). The toxin payload was uniformly conjugated to the light-chain C-terminus of the native cIRCR201 antibody (drug-to-antibody ratio = 2), as confirmed using LC–MS. Using a high-throughput screening system, we found that cIRCR201-dPBD exhibited varying sensitivities depending on the expression levels of c-Met, and it induced receptor-mediated endocytosis and toxin-mediated apoptosis in 47 different cancer cell lines. cIRCR201-dPBD also showed significant antitumor activity on the MET-amplified cancer cells using in vivo xenograft models. Therefore, cIRCR201-dPBD could be a promising therapeutic strategy for tumors with c-Met expression.

CCR5 tyrosine sulfation heterogeneity generates cell surface receptor subpopulations with different ligand binding properties

BackgroundChemokine receptor tyrosine sulfation plays a key role in the binding of chemokines. It has been suggested that receptor sulfation is heterogeneous, but no experimental evidence has been provided so far. The potent anti-HIV chemokine analog 5P12-RANTES has been proposed to owe its inhibitory activity to a capacity to bind a larger pool of cell surface CCR5 receptors than native chemokines such as CCL5, but the molecular details underlying this phenomenon have not been elucidated. MethodsWe investigated the CCR5 sulfation heterogeneity and the sensitivity of CCR5 ligands to receptor sulfation by performing ELISA assays on synthetic N-terminal sulfopeptides and by performing binding assays on CCR5-expressing cells under conditions that modulate CCR5 sulfation levels. ResultsTwo commonly used anti-CCR5 monoclonal antibodies with epitopes in the sulfated N-terminal domain of CCR5 show contrasting binding profiles on CCR5 sulfopeptides, incomplete competition with each other for cell surface CCR5, and opposing sensitivities to cellular treatments that affect CCR5 sulfation levels. 5P12-RANTES is less sensitive than native CCL5 to conditions that affect cellular CCR5 sulfation. ConclusionsCCR5 sulfation is heterogeneous and this affects the binding properties of both native chemokines and antibodies. Enhanced capacity to bind to CCR5 is a component of the inhibitory mechanism of 5P12-RANTES. General significanceWe provide the first experimental evidence for sulfation heterogeneity of chemokine receptors and its impact on ligand binding, a phenomenon that is important both for the understanding of chemokine cell biology and for the development of drugs that target chemokine receptors.

Native Human Antibody to Shr Promotes Mice Survival After Intraperitoneal Challenge With Invasive Group A Streptococcus.

A vaccine against group A Streptococcus (GAS) has been actively pursued for decades. The surface receptor Shr is vital in GAS heme uptake and provides an effective target for active and passive immunization. Here, we isolated human monoclonal antibodies (mAbs) against Shr and evaluated their efficacy and mechanism. We used a single B-lymphocyte screen to discover the mAbs TRL186 and TRL96. Interactions of the mAbs with whole cells, proteins, and peptides were investigated. Growth assays and cultured phagocytes were used to study the mAbs' impact on heme uptake and bacterial killing. Efficacy was tested in prophylactic and therapeutic vaccination using intraperitoneal mAb administration and GAS challenge. Both TRL186 and TRL96 interact with whole GAS cells, recognizing the NTR and NEAT1 domains of Shr, respectively. Both mAbs promoted killing by phagocytes in vitro, but prophylactic administration of only TRL186 increased mice survival. TRL186 improved survival also in a therapeutic mode. TRL186 but not TRL96 also impeded Shr binding to hemoglobin and GAS growth on hemoglobin iron. Interference with iron acquisition is central for TRL186 efficacy against GAS. This study supports the concept of antibody-based immunotherapy targeting the heme uptake proteins to combat streptococcal infections.

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Therein, antibody fragments (e.g., single-chain Fv fragments; scFvs) with a variety of mutations are displayed on bacteriophage to generate diverse phage-antibody libraries. Rare clones with improved functions are then selected via panning against immobilized or tagged target antigens. However, this selection process often ended unsuccessful, mainly due to the biased propagation of phage-antibody clones and the competition with a large excess of undesirable clones with weaker affinities. To break radically from such panning-inherent problems, we developed a novel method, clonal array profiling of scFv-displaying phages (CAP), in which colonies of the initial bacterial libraries are examined one-by-one in microwells. Progenies of scFv-displaying phages generated are, if show sufficient affinity to target antigen, captured in the microwell via pre-coated antigen and detected using a luciferase-fused anti-phage scFv. The advantage of CAP was evidenced by its application with a small error-prone-PCR-based library (~ 105 colonies) of anti-cortisol scFvs. Only two operations, each surveying only ~ 3% of the library (9,400 colonies), provided five mutants showing 32–63-fold improved Ka values (> 1010 M−1), compared with the wild-type scFv (Ka = 3.8 × 108 M−1), none of which could be recovered via conventional panning procedures operated for the entire library.

Abstract 4054: Development of a new site-specific sulfomaleimide rebridging platform for the generation of homogeneous, stable, potent and safe antibody drug conjugates

Abstract There are currently 5 antibody drug conjugates (ADC) on the market and over 80 other ADCs evaluated in the clinic. The major drawbacks of the commonly employed bioconjugation methods based on nucleophilic coupling to lysine and cysteine are the non-selectivity and the in vivo instability. These limitations may be overcome by using alternative bioconjugation chemistries. To avoid the need for antibody engineering, disulfide-bridging linkers have recently emerged for the generation of homogeneous and stable ADCs. Here, we report a novel maleimide-based linker for site-selective bioconjugation of drugs via covalent re-bridging of reduced interchain disulfide bonds on native IgG1 antibodies. After optimization of the maleimide and linker parts, as well as of the coupling process, the sulfomaleimide re-bridging (SMR) platform was used to conjugate tubulin and DNA-targeting cytotoxic agents to model antibodies. Compared to conventional maleimide (MC)-ADCs, the SMR-ADCs were highly homogeneous by HIC-HPLC and LC-MS analysis, with DAR 4 species as the major product, and highly stable in vitro, with no deconjugation observed after incubation in different types of serum. Then, IGF-1R-targeting SMR-ADCs were synthesized with Auristatin and PNU-159682 derivatives and evaluated in in vitro and in vivo models. These ADCs exhibited potent cytotoxicity and anti-tumoral activity in 2+ and 3+ IGF-1R-expressing models at different doses (0.3 to 3 mg/kg) and schedules of administration (Q1W to Q3W, i.v.). Significant differences in in vivo efficacy were observed between SMR- and MC-ADCS constructed with Auristatin derivatives. While they were comparable in a 3+ xenograft model (MCF-7), complete regressions were indeed obtained in the 2+ CaOV3 and NCI-H2122 models after single injections of the Auristatin-based SMR-ADC, whereas the corresponding MC-ADC induced only partial responses in the same conditions. To compare their in vivo stability and tolerability, these ADCs were injected in mice at doses of 3-30 mg/kg (Q2W, i.v.). No mortality and clinical signs were observed. Body weight evolution was not affected whatever the treatment. From a histopathological perspective, no difference between both constructs could be highlighted at this point. Toxicokinetic analyses carried out from satellite animals demonstrated that there was no major difference between both ADCs and that animal exposure increased proportionally with the dose. Nevertheless, serum exposure to the payload was 2-to-7-fold higher for MC-ADC than for SMR-ADC. Our results demonstrate that the SMR platform can be applied to native IgG1 antibodies, without antibody engineering, to yield highly homogeneous and stable ADCs via cysteine cross-linking. The resulting ADCs demonstrate a therapeutic index benefit compared to analogous conventional heterogeneous ADCs. Citation Format: Jean-Francois Haeuw, Frederic Marion, Cyrille Dreyfus, Barbara Akla, Laurence Zanna, Marie-Claire Janin, Charlotte Beau-Larvor, Marion Millet, Martine Farrie, Alain Batton, Celine Thuilliez, Alain Beck, Sarah Cianferani, Thierry Clerc, Philippe Maillos, Nathalie Corvaia. Development of a new site-specific sulfomaleimide rebridging platform for the generation of homogeneous, stable, potent and safe antibody drug conjugates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4054.

Intramuscular injection of vectorized-scFvMC1 reduces pathological tau in two different tau transgenic models

With evidence supporting the prion-like spreading of extracellular tau as a mechanism for the initiation and progression of Alzheimer’s disease (AD), immunotherapy has emerged as a potential disease-modifying strategy to target tau. Many studies have proven effective to clear pathological tau species in animal models of AD, and several clinical trials using conventional immunotherapy with anti-tau native antibodies are currently active. We have previously generated a vectorized scFv derived from the conformation-dependent anti-tau antibody MC1, scFvMC1, and demonstrated that its intracranial injection was able to prevent tau pathology in adult tau mice. Here, we show that, in a prevention paradigm and in two different tau transgenic models (JNPL3 and P301S), a one-time intramuscular injection of AAV1-scFvMC1 generated a long-lasting peripheral source of anti-tau scFvMC1 and significantly reduced insoluble and soluble tau species in the brain. Moreover, our data showed that scFvMC1 was internalized by the microglia, in the absence of overt inflammation. This study demonstrates the efficacy of intramuscular delivery of vectorized scFv to target tau, and suggests a new potential application to treat AD and the other tauopathies.

Cloning antibodies from single cells in pooled sequence libraries by selective PCR.

Antibodies function by binding to antigens. Antibodies must be cloned and expressed to determine their binding characteristics, but current methods for high-throughput antibody sequencing yield antibody DNA pooled from many cells and do not readily permit cloning of antibodies from single B cells. We present a strategy for retrieving and cloning antibody DNA from single cells within a pooled library of cells. Our strategy, called selective PCR for antibody retrieval (SPAR), takes advantage of the unique sequence barcodes attached to individual cDNA molecules during sample preparation to enable specific amplification by PCR of antibody heavy- and light-chain cDNA originating from a single cell. We show through computational analysis that most human antibodies sequenced using typical high-throughput methods can be retrieved using SPAR, and experimentally demonstrate retrieval of full-length antibody variable region cDNA from three cells within pools of ~5,000 cells. SPAR enables rapid low-cost cloning and expression of native human antibodies from pooled single-cell sequence libraries for functional characterization.

Complementary epitopes and favorable developability of monoclonal anti-LAMP1 antibodies generated using two transgenic animal platforms.

Monoclonal antibodies (mAbs) for therapeutic applications should be as similar to native human antibodies as possible to minimize their immunogenicity in patients. Several transgenic animal platforms are available for the generation of fully human mAbs. Attributes such as specificity, efficacy and Chemistry, Manufacturing and Controls (CMC) developability of antibodies against a specific target are typically established for antibodies obtained from one platform only. In this study, monoclonal antibodies (mAbs) cross-reactive against human and cynomolgus LAMP1 were derived from the human immunoglobulin transgenic TRIANNI mouse and OmniChicken® platforms and assessed for their specificity, sequence diversity, ability to bind to and internalize into tumor cells, expected immunogenicity and CMC developability. Our results show that the two platforms were complementary at providing a large diversity of mAbs with respect to epitope coverage and antibody sequence diversity. Furthermore, most antibodies originating from either platform exhibited good manufacturability characteristics.

FLUORESCENT MODIFICATION OF THE MONOCLONAL ANTIBODIES OLIGOSACCHARIDES BY FLUORESCEIN-5-THIOSEMICARBAZIDE

Introduction Monoclonal antibodies (Mabs) are a good tool for diagnosing human pathologies. They are used as conjugates with fluorescent and other dyes. The classical approach of creating such conjugates is reduced to chemical reactions using the protein base of the Mab. At the same time, for a number of Mabs, conjugate production is accompanied by embedding the label into the antigen binding site, which leads to a decrease or complete loss of the specific activity of the conjugate. To get out of this situation, the synthesis of fluorescent conjugates by methods of carbohydrate chemistry through spatially distant from the active center oligosaccharides of antibodies is proposed. Objective To obtain high activity ICO series Mab conjugates based on the reaction of covalent inclusion of the fluorescent label in the oligosaccharide sequence of the Mab. Materials and methods We used Mab series ICO of high purity. Oligosaccharides Mabs oxidized to aldehyde groups, were subjected to interaction with fluoriscine-5-thiosemicarboside, followed by the reduction of hydrazone derivative borgidrides. The resulting covalent conjugate was investigated in a flow cytometry. Results The synthesis of a fluorescent conjugate using monoclonal antibodies oligosaccharides was worked out. Modified monoclonal antibodies retain specific binding to target cells inherent in the native antibody. The resulting conjugates remained active for a long time during storage. Conclusion An alternative method for conjugation of immunofluorescent, allowing to obtain conjugates of high activity, has been developed.

Pickering emulsions with ethiodized oil and nanoparticles for slow release of intratumoral anti-CTLA4 immune checkpoint antibodies

BackgroundIntratumorous immunotherapy for cancer is currently thriving. The aim of such local strategy is to improve the therapeutic index of these treatments, for higher on-target/on-tumor activity and less on-target/off-tumor adverse...

Evaluation of hydrophobic-interaction chromatography resins for purification of antibody-drug conjugates using a mimetic model with adjustable hydrophobicity.

Antibody drug conjugates are cytotoxic pharmaceuticals, designed to destroy malignant cells. A cytotoxic molecule is attached to an antibody that binds specific to a cancer‐cell surface. Given the high toxicity of the drugs, strict safety standards have to be kept. For this reason, an antibody drug conjugates model was developed with fluorescein 5‐isothiocyanate as the nontoxic payload surrogate. Due to the similar hydrophobicity, this model is used to establish a suitable purification process and characterization method for antibody drug conjugates. Because of the pH dependent solubility of fluorescein, the hydrophobicity of conjugates can be modulated by the pH value. Based on the complex heterogeneity and hydrophobicity of the conjugates a chromatographic purification is challenging. Hydrophobic interaction chromatography is used for analytical as well as for preparative separation. Because of the increased hydrophobicity of the conjugates compared to native antibody, hydrophobic interaction chromatography often suffer from resolution and recovery problems. Conjugates were separated differing on the number of payloads attached to the antibody. For this matter, the drug–antibody ratio is determined and used as a quantitative term. The conjugates are purified at high recoveries and resolution by step gradients using suitable resins, allowing the separation of the target drug–antibody ratio.

Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity

“Antibody-breeding” has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the VH and VL domains of parent antibodies to generate diverse libraries of single-chain Fv fragments (scFvs), from which evolved mutants are selected. We produced an scFv against estradiol-17β with 11 amino acid substitutions and a >100-fold improved affinity constant (Ka = 1.19 × 1010 M−1) over the parent scFv, enabling immunoassays with >30-fold higher sensitivity. We systematically analyzed contributions of these substitutions to the affinity enhancement. Comparing various partial scFv revertants based on their Kas indicated that a revertant with four substitutions (VH-L100gQ, VL-I29V, -L36M, -S77G) exhibited somewhat higher affinity (Ka = 1.46 × 1010 M−1). Finally, the VH-L100gQ substitution, occurring in VH complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and VL-I29V and/or VL-L36M cooperated significantly. These findings encouraged us to reconsider the potential of VH-CDR3-targeting mutagenesis, which has been frequently attempted. The substitution(s) wherein might enable a “high rate of return” in terms of selecting mutants with dramatically enhanced affinities. The “high risk” of generating a tremendous excess of “junk mutants” can be overcome with the efficient selection systems that we developed.

Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminases.

Site-specific bioconjugation technologies are frequently employed to generate homogeneous antibody-drug conjugates (ADCs) and are generally considered superior to stochastic approaches like lysine coupling. However, most of the technologies developed so far require undesired manipulation of the antibody sequence or its glycan structures. Herein, we report the successful engineering of microbial transglutaminase enabling efficient, site-specific conjugation of drug-linker constructs to position HC-Q295 of native, fully glycosylated IgG-type antibodies. ADCs generated via this approach demonstrate excellent stability in vitro as well as strong efficacy in vitro and in vivo. As it employs different drug-linker structures and several native antibodies, our study additionally proves the broad applicability of this approach.

A Plug-and-Play Approach for the De Novo Generation of Dually Functionalized Bispecifics

Diseases are multifactorial, with redundancies and synergies between various pathways. However, most of the antibody-based therapeutics on the market interact with only one target, thus limiting their efficacy. The targeting of multiple epitopes could improve the therapeutic index of treatment and counteract mechanisms of resistance. To this effect, a new class of therapeutics has emerged: bispecific antibodies. Bispecific formation using chemical methods is rare and low-yielding and/or requires a large excess of one of the two proteins to avoid homodimerization and heterogeneity. In order for chemically prepared bispecifics to deliver their full potential, high-yielding, modular, and reliable cross-linking technologies are required. Herein, we describe a novel approach not only for the rapid and high-yielding chemical generation of bispecific antibodies from native antibody fragments, but also for the site-specific dual functionalization of the resulting bioconjugates. Based on orthogonal clickable functional groups, this strategy enables the assembly of functionalized bispecifics with controlled loading in a modular and convergent manner.

Proof of site-specificity of antibody-drug conjugates produced by chemical conjugation technology: AJICAP first generation

Antibody-drug conjugates (ADCs) have become major biopharmaceutical drugs in the field of oncology. Traditional ADCs possess a stochastic distribution of cytotoxic payloads linked to several different amino acid residues of the antibody. This heterogeneous nature of stochastic ADCs results in a complex conjugation-site characterization. To improve upon traditional ADC technology, we have developed a chemical conjugation platform, termed AJICAP™, for site-specific modification of native antibodies using a class of IgG Fc-affinity reagents (Yamada et al., 2019). Here, we report further investigation focusing on peptide mapping of the AJICAP™-ADC to confirm the exact conjugation position of the first generation AJICAP™-ADC. Neutral pH pretreatment for peptide mapping prevented undesired PTMs such as succinimide ring hydrolysis. Mirroring comparison using the purified ADC visibly indicated that Lys248 in the Fc region was conjugated to the drug-linker. MS/MS analysis also provided evidence to support Lys248 conjugation. Finally, extracted ion-chromatogram methodology suggested the site-specificity of AJICAP™ conjugation. Purified ADCs by preparative HIC-HPLC showed clear visual results and more than 93% sequence coverage by a single enzymatic digestion. The analytical strategy described herein demonstrated a robust analytical methodology for revealing the conjugation site of ADCs.

Macrophagic Activation Syndrome (MAS) Revealing Systemic Lupus Erythematosus

Introduction: Macrophagic activation syndrome (SAM) or hemophagocytic lymphohistiocytosis is an anatomoclinical entity resulting from an inappropriate proliferation and activation of macrophagic cells. This rare but serious syndrome can be primary or secondary to certain pathologies dominated by infections and neoplasias. More rarely, it is secondary to connectivitis, in particular to systemic lupus erythematosus (LES). The aim of this work is to report the case of SAM revealing a systemic lupus erythematosus and to discuss the triggering factors. Observation: A 27-year-old patient who presents to the emergency room for arthralgia evolving for a week in a context of fever and deterioration of the general state hospitalized in the nephrology department for bicytopenia and functional renal failure. The hemogram showed anemia at 10.7 g/dL, normocytic, normochromic, aregenerative (reticulocytes at 18 G/L), hyperleukocytosis at 10.5 G/L and platelets at the lower limit of normal at 158 G/L (338 G/L in September 2006). In view of argenerative anemia, a myelogram was performed showing the presence of hemophagocytosis figures. The remainder of the biological examination revealed hyperferritinaemia at 1017 ng/mL with an increase in LDH 930 U/Let a CRP at 18.15 mg/L. The patient was transferred to the internal medicine department for etiological assessment, clinical and paraclinical examinations showed arthritis with deep ADP and pleural mass, leuconeutropenia with native DNA and positive antinuclear antibodies (ANA) as well as the coombs test which was positive, The diagnosis of SLE was retained in her on the criteria of the ACR.

Streamlined Expressed Protein Ligation: Site-Specific Antibody-Drug Conjugate.

Protein semisynthesis is a powerful tool for studying proteins and has contributed to a better understanding of protein structure and function and also driven innovations in protein science. Expressed protein ligation (EPL) is a widely used method to generate chemically modified proteins. However, EPL has some limitations, particularly relevant to modify challenging proteins such as antibodies. The method termed streamlined expressed protein ligation (SEPL) overcomes some of the problems of EPL, and other methods of protein semisynthesis, to generate challenging modified proteins such as antibody-drug conjugates (ADCs). ADCs targeting highly cytotoxic molecules to cancer cells, offer an attractive strategy to selectively eliminate tumor cells with improved therapeutic index than the antibodies or cytotoxic molecules themselves. Despite the potential of ADCs, the development of such complex molecules is challenging. We provide here protocols to prepare site-specifically modified ADCs by streamlined expressed protein ligation (SEPL), which does not require the incorporation of unnatural modifications into the antibody. Therefore, fully native antibodies, with only the desired cytotoxic molecules attached, can be generated.

Human IgA binds a diverse array of commensal bacteria.

In humans, several grams of IgA are secreted every day in the intestinal lumen. While only one IgA isotype exists in mice, humans secrete IgA1 and IgA2, whose respective relations with the microbiota remain elusive. We compared the binding patterns of both polyclonal IgA subclasses to commensals and glycan arrays and determined the reactivity profile of native human monoclonal IgA antibodies. While most commensals are dually targeted by IgA1 and IgA2 in the small intestine, IgA1+IgA2+ and IgA1-IgA2+ bacteria coexist in the colon lumen, where Bacteroidetes is preferentially targeted by IgA2. We also observed that galactose-α terminated glycans are almost exclusively recognized by IgA2. Although bearing signs of affinity maturation, gut-derived IgA monoclonal antibodies are cross-reactive in the sense that they bind to multiple bacterial targets. Private anticarbohydrate-binding patterns, observed at clonal level as well, could explain these apparently opposing features of IgA, being at the same time cross-reactive and selective in its interactions with the microbiota.