Generation and functional characterization of anti-human and anti-mouse IL-36R antagonist monoclonal antibodies

Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, β and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.     

Engineering Fully Human Monoclonal Antibodies from Murine Variable Regions

Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V_H/V_L interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology.     

Engineering a high-affinity anti-IL-15 antibody: crystal structure reveals an α-helix in VH CDR3 as key component of paratope

Interleukin (IL) 15 is an inflammatory cytokine that plays an essential role in the activation, proliferation, and maintenance of specific natural killer cell and T-cell populations, and has been implicated as a mediator of inflammatory diseases. An anti-IL-15 antibody that blocked IL-15-dependent cellular responses was isolated by phage display and optimised via mutagenesis of the third complementarity-determining regions (CDRs) of variable heavy (VH) and variable light chains. Entire repertoires of improved variants were recombined with each other to explore the maximum potential sequence space. DISC0280, the most potent antibody isolated using this comprehensive strategy, exhibits a 228-fold increase in affinity and a striking 40,000-fold increase in cellular potency compared to its parent. Such a wholesale recombination strategy therefore represents a useful method for exploiting synergistic potency gains as part of future antibody engineering efforts. The crystal structure of DISC0280 Fab (fragment antigen binding), in complex with human IL-15, was determined in order to map the structural epitope and paratope. The most remarkable feature revealed lies within the paratope and is a novel six-amino-acid α-helix that sits within the VH CDR3 loop at the center of the antigen binding site. This is the first report to describe an α-helix as a principal component of a naturally derived VH CDR3 following affinity maturation.     

Antibody humanization by structure-based computational protein design

Antibodies derived from non-human sources must be modified for therapeutic use so as to mitigate undesirable immune responses. While complementarity-determining region (CDR) grafting-based humanization techniques have been successfully applied in many cases, it remains challenging to maintain the desired stability and antigen binding affinity upon grafting. We developed an alternative humanization approach called CoDAH (“Computationally-Driven Antibody Humanization”) in which computational protein design methods directly select sets of amino acids to incorporate from human germline sequences to increase humanness while maintaining structural stability. Retrospective studies show that CoDAH is able to identify variants deemed beneficial according to both humanness and structural stability criteria, even for targets lacking crystal structures. Prospective application to TZ47, a murine anti-human B7H6 antibody, demonstrates the approach. Four diverse humanized variants were designed, and all possible unique VH/VL combinations were produced as full-length IgG1 antibodies. Soluble and cell surface expressed antigen binding assays showed that 75% (6 of 8) of the computationally designed VH/VL variants were successfully expressed and competed with the murine TZ47 for binding to B7H6 antigen. Furthermore, 4 of the 6 bound with an estimated K_D within an order of magnitude of the original TZ47 antibody. In contrast, a traditional CDR-grafted variant could not be expressed. These results suggest that the computational protein design approach described here can be used to efficiently generate functional humanized antibodies and provide humanized templates for further affinity maturation.     

Antibody humanization by structure-based computational protein design

Antibodies derived from non-human sources must be modified for therapeutic use so as to mitigate undesirable immune responses. While complementarity-determining region (CDR) grafting-based humanization techniques have been successfully applied in many cases, it remains challenging to maintain the desired stability and antigen binding affinity upon grafting. We developed an alternative humanization approach called CoDAH (“Computationally-Driven Antibody Humanization”) in which computational protein design methods directly select sets of amino acids to incorporate from human germline sequences to increase humanness while maintaining structural stability. Retrospective studies show that CoDAH is able to identify variants deemed beneficial according to both humanness and structural stability criteria, even for targets lacking crystal structures. Prospective application to TZ47, a murine anti-human B7H6 antibody, demonstrates the approach. Four diverse humanized variants were designed, and all possible unique VH/VL combinations were produced as full-length IgG1 antibodies. Soluble and cell surface expressed antigen binding assays showed that 75% (6 of 8) of the computationally designed VH/VL variants were successfully expressed and competed with the murine TZ47 for binding to B7H6 antigen. Furthermore, 4 of the 6 bound with an estimated K_D within an order of magnitude of the original TZ47 antibody. In contrast, a traditional CDR-grafted variant could not be expressed. These results suggest that the computational protein design approach described here can be used to efficiently generate functional humanized antibodies and provide humanized templates for further affinity maturation.     

A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy

The pro-inflammatory cytokine interleukin (IL)-1β is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1β have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1β with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a >30-fold increased affinity to human IL-1β compared with its parent antibody. This anti-human IL-1β IgG also cross-reacts with mouse and monkey IL-1β, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1β pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1β monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal.     

A mechanistic PK/PD model for two anti-IL13 antibodies explains the difference in total IL-13 accumulation observed in clinical studies

IMA-638 and IMA-026 are humanized IgG1 monoclonal antibodies (mAbs) that target non-overlapping epitopes of IL-13. Separate first-in-human single ascending dose studies were conducted for each mAb. These studies had similar study designs, but mild to moderate asthmatics were recruited for the IMA-638 study and healthy subjects were recruited for the IMA-026 study. IMA-638 and IMA-026 showed similar pharmacokinetic (PK) profiles, but very different total IL-13 (free and drug bound IL-13) profiles; free IL13 was not measured. IMA-026 treatment induced a dose-dependent accumulation of total IL-13, while IMA-638 treatment led to a much smaller accumulation without any clear dose-response. To understand the differences between the two total IL-13 profiles and to predict the free IL-13 profiles for each mAb treatment, a mechanistic PK/pharmacodynamic model was developed. PK-related parameters were first fit to the mean PK profiles of each mAb separately; thereafter, the target-related parameters were fit to both total IL-13 profiles simultaneously. The IL-13 degradation rate was assumed to be the same for asthma patients and healthy subjects. The model suggests that an approximately 100× faster elimination of IL-13-IMA-638 complex than IL-13-IMA-026 complex could be responsible for the differences observed in total IL-13 profiles for the two mAbs. Furthermore, the model predicts that IMA-638 administration results in greater and more prolonged free IL-13 inhibition than equivalent dosing of IMA-026 despite similar binding K_D and PK profile. In conclusion, joint modeling of two similar molecules provided mechanistic insight that the elimination rate of mAb-target complex can regulate the degree of free target inhibition.     

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies

Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope “bins” based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.     

Binding characterization of the interleukin-13 signaling complex and development of a ternary time-resolved fluorescence resonance energy transfer assay

Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Rα1 and IL-4R. Genetic variants of IL-13 and of its receptor components have been linked to asthma. One in particular, IL-13R110Q, is associated with increased IgE levels and asthma. We characterized the interactions of the binary complexes composed of IL-13 or IL-13R110Q with IL-13Rα1 and the ternary complexes composed of IL-13 or IL-13R110Q and IL-13Rα1 with IL-4R using surface plasmon resonance and time-resolved fluorescence resonance energy transfer (TR-FRET). By both biophysical methods, we found no differences between IL-13 and IL-13R110Q binding in either the binary or the ternary complex. IL-4R bound to the IL-13/IL-13Rα1 complex with slow on and off rates, resulting in a relatively weak affinity of about 100 nM. We developed a TR-FRET assay targeting the interaction between the IL-4R and the binary complex. Two antibodies with known binding epitopes to IL-13 that block binding to either IL-13Rα1 or IL-4R inhibited the TR-FRET signal formed by the ternary complex. This assay will be useful to identify and characterize inhibitory molecules of IL-13 function.     

Important roles of CD32 in promoting suppression of IL-4 induced immune responses by a novel anti-IL-4Rα therapeutic antibody

ABSTRACT

Asthma is characterized by airway hyperresponsiveness and inflammation, as well as underlying structural changes to the airways. Interleukin-4 (IL-4) is a key T-helper type 2 (Th2) cytokine that plays important roles in the pathogenesis of atopic and eosinophilic asthma. We developed a novel humanized anti-IL-4Rα antibody that can potently inhibit IL-4/IL-13-mediated TF-1 cell proliferation. Using monocytes isolated from human peripheral blood mononuclear cells (PBMCs), we revealed a critical role of CD32 in modulating the immune responses of monocytes in response to blockade of IL-4Rα signaling pathway. We, therefore, devised a new strategy to increase the efficacy of the anti-IL-4Rα monoclonal antibody for the treatment of asthma and other atopic diseases by co-engaging CD32 and IL-4Rα on monocytic cells by choosing IgG classes or Fc mutations with higher affinities for CD32. The antibody with selectively enhanced affinity for CD32A displayed superior suppression of IL-4-induced monocytes’ activities, including the down-regulation of CD23 expression. Intriguingly, further analysis demonstrated that both CD32A and CD32B contributed to the enhancement of antibody-mediated suppression of CD23 expression from monocytes in response to blockade of IL-4Rα signaling. Furthermore, inhibition of IgE secretion from human PBMC by the antibody variants further suggests that the complex allergic inflammation mediated by IL-4/IL-4Rα signaling might result from a global network where multiple cell types that express multiple FcγRs are all involved, of which CD32, especially CD32A, is a key mediator. In this respect, our study provides new insights into designing therapeutic antibodies for targeting Th2 cytokine-mediated allergic pathogenesis.     

A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy

The pro-inflammatory cytokine interleukin (IL)-1β is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1β have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1β with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a >30-fold increased affinity to human IL-1β compared with its parent antibody. This anti-human IL-1β IgG also cross-reacts with mouse and monkey IL-1β, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1β pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1β monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal.     

Synthetic antibody libraries focused towards peptide ligands

Synthetic antibody libraries have proven immensely useful for the de novo isolation of antibodies without the need for animal immunization. Recently, focused libraries designed to recognize particular classes of ligands, such as haptens or proteins, have been employed to facilitate the selection of high-affinity antibodies. Focused libraries are built using V regions encoding combinations of canonical structures that resemble the structural features of antibodies that bind the desired class of ligands and sequence diversity is introduced at residues typically involved in recognition. Here we describe the generation and experimental validation of two different single-chain antibody variable fragment libraries that efficiently generate binders to peptides, a class of molecules that has proven to be a difficult target for antibody generation. First, a human anti-peptide library was constructed by diversifying a scaffold: the human variable heavy chain (V_H) germ line gene 3-23, which was fused to a variant of the human variable light chain (V_L) germ line gene A27, in which L1 was modified to encode the canonical structure found in anti-peptide antibodies. The sequence diversity was introduced into 3-23 (V_H) only, targeting for diversification residues commonly found in contact with protein and peptide antigens. Second, a murine library was generated using the antibody 26-10, which was initially isolated based on its affinity to the hapten digoxin, but also binds peptides and exhibits a canonical structure pattern typical of anti-peptide antibodies. Diversity was introduced in the V_H only using the profile of amino acids found at positions that frequently contact peptide antigens. Both libraries yielded binders to two model peptides, angiotensin and neuropeptide Y, following screening by solution phage panning. The mouse library yielded antibodies with affinities below 20 nM to both targets, although only the V_H had been subjected to diversification.     

Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments

Multispecific antibody formats provide a promising platform for the development of novel therapeutic concepts that could facilitate the generation of safer, more effective pharmaceuticals. However, the production and use of such antibody-based multispecifics is often made complicated by: 1) the instability of the antibody fragments of which they consist, 2) undesired inter-subunit associations, and 3) the need to include recombinant heterodimerization domains that confer distribution-impairing bulk or enhance immunogenicity. In this paper, we describe a broadly-applicable method for the stabilization of human or humanized antibody Fv fragments that entails replacing framework region IV of a V_κ1/V_H3-consensus Fv framework with the corresponding germ-line sequence of a λ-type V_L chain. We then used this stable Fv framework to generate a novel heterodimeric multispecific antibody format that assembles by cognate V_L/V_H associations between 2 split variable domains in the core of the complex. This format, termed multispecific antibody-based therapeutics by cognate heterodimerization (MATCH), can be applied to produce homogeneous and highly stable antibody-derived molecules that simultaneously bind 4 distinct antigens. The heterodimeric design of the MATCH format allows efficient in-format screening of binding domain combinations that result in maximal cooperative activity.     

Structure-based Development of Human Interleukin-1β-Specific Antibody That Simultaneously Inhibits Binding to Both IL-1RI and IL-1RAcP

Interleukin-1β (IL-1β) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1β regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1β to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1β initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1β neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1β. We validated IgG26AW-neutralizing antibodies specific for IL-1β in vivo to prevent human IL-1β-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1β also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1β complex structure. Meanwhile, SPR experiments showed that IL-1β bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1β/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1β, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1β is critical to pathogenesis.     

Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain

Currently, almost all U.S. Food and Drug Administration-approved therapeutic antibodies and the vast majority of those in clinical trials are full-size antibodies mostly in an immunoglobulin G1 format of about 150 kDa in size. Two fundamental problems for such large molecules are their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules [e.g., on the human immunodeficiency virus envelope glycoprotein (Env)] that are accessible by molecules of smaller size. We have identified a phage-displayed heavy chain-only antibody by panning of a large (size, ∼ 1.5 × 10¹⁰) human naive Fab (antigen-binding fragment) library against an Env and found that the heavy chain variable domain (V_H) of this antibody, designated as m0, was independently folded, stable, highly soluble, monomeric, and expressed at high levels in bacteria. m0 was used as a scaffold to construct a large (size, ∼ 2.5 × 10¹⁰), highly diversified phage-displayed human V_H library by grafting naturally occurring complementarity-determining regions (CDRs) 2 and 3 of heavy chains from five human antibody Fab libraries and by randomly mutating four putative solvent-accessible residues in CDR1 to A, D, S, or Y. The sequence diversity of all CDRs was determined from 143 randomly selected clones. Most of these V_Hs were with different CDR2 origins (six of seven groups of V_H germlines) or CDR3 lengths (ranging from 7 to 24 residues) and could be purified directly from the soluble fraction of the Escherichia coli periplasm. The quality of the library was also validated by successful selection of high-affinity V_Hs against viral and cancer-related antigens; all selected V_Hs were monomeric, easily expressed, and purified with high solubility and yield. This library could be a valuable source of antibodies targeting size-restricted epitopes and antigens in obstructed locations where efficient penetration could be critical for successful treatment.     

Germline humanization of a non-human primate antibody that neutralizes the anthrax toxin, by in vitro and in silico engineering

Fab 35PA₈₃ is an antibody fragment of non-human primate origin that neutralizes the anthrax lethal toxin. Human antibodies are usually preferred when clinical use is envisioned, even though their framework regions (FR) may carry mutations introduced during affinity maturation. These hypermutations can be immunogenic and therefore FR that are encoded by human germline genes, encountered in IgMs and thus part of the “self” proteins, are preferable. Accordingly, the proportion of FR residues in 35PA₈₃ that were encoded by human V and J germline genes, i.e. the germinality index (GI) of 35PA₈₃, was increased in a multistep cumulative approach. In a first step, the FR1 and FR4 residues of 35PA₈₃ were changed simultaneously into their counterparts coded by 35PA₈₃'s closest human germline genes, without prior modelling. The resulting derivative of 35PA₈₃ had the same affinity as its parental Fab. In a second step, the 3D structures of this first 35PA₈₃ derivative, carrying the same type of residue changes but in the FR2 and FR3 regions, were modelled in silico from sequences. Some of the changes in FR2 or FR3 modified the predicted peptide backbone. The changes that did not seem to alter the structure were introduced simultaneously in the Fab by an in vitro method and resulted in a loss of reactivity, which could however be fully restored by a single point mutation. The final 35PA₈₃ derivative had a GI higher than that of a fully human Fab, which had neutralization properties similar to 35PA₈₃ and which was used as a benchmark in this study.     

126Gln is the residue of human IL-2 binding to IL-2R γ subunit

The 126Gln of human interleukin-2 (IL-2) is a conserved amino acid residue. After substitution of 126Gln with Asp, the binding abilities of this mutant to different composites of IL-2 receptor (R) subunits have been determined. Results show that 126AspIL-2 has higher affinity to IL-2R α β γ complex and normal affinity to IL-2R α β complex, but loses its binding ability to IL-2R β γ complex, demonstrating that the 126Gln is the residue of human IL-2 which binds to IL-2R γ subunit. Project supported by the “863” Project of China.     

Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody

The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with (125)I or (131)I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.     

Structure-based design of a protein immunogen that displays an HIV-1 gp41 neutralizing epitope

Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K_d of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.     

Structural insights into humanization of anti-tissue factor antibody 10H10

Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.