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.

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.

Role of the constant region domain in the structural diversity of human antibody light chains.

Issues regarding the structural diversity (heterogeneity) of an antibody molecule have been the subject of discussion along with the development of antibody drugs. Research on heterogeneity has been extensive in recent years, but no clear solution has been reached. Heterogeneity is also observed in catalytic antibody κ light chains (CLs). In this study, we investigated how the constant region domain of CLs concerns structural diversity because it is a simple and good example for elucidating heterogeneity. By means of cation-exchange chromatography, SDS-PAGE, and 2-dimensional electrophoresis for the CL, multimolecular forms consisting of different electrical charges and molecular sizes coexisted in the solution, resulting in the similar heterogeneity of the full length of CLs. The addition of copper ion could cause the multimolecular forms to change to monomolecular forms. Copper ion contributed greatly to the enrichment of the dimer form of CL and the homogenization of the differently charged CLs. Two molecules of the CL protein bound one copper ion. The binding affinity of the ion was 48.0 µM-1 Several divalent metal ions were examined, but only zinc showed a similar effect.-Hifumi, E., Taguchi, H., Kato, R., Uda, T. Role of the constant region domain in the structural diversity of human antibody light chains.

Engineering Bifunctional Antibodies with Constant Region Fusion Architectures.

We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies have excellent in vitro activity, physiochemical properties, and stability. Among these, a Her2 × CD3 bispecific antibody (BsAb) was constructed by inserting an anti-Her2 single-chain variable fragment (ScFv) into an anti-CD3 Fab. This bispecific antibody efficiently induces targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations in vitro. Moreover, the Her2 × CD3 BsAb shows potent in vivo antitumor activity in mouse Her22+ and Her21+ xenograft models. These results demonstrate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multifunctional antibodies for therapeutic applications.

A constant threat for HIV: Fc-engineering to enhance broadly neutralizing antibody activity for immunotherapy of the acquired immunodeficiency syndrome.

Passive immunotherapy with polyclonal or hyperimmune serum immunoglobulin G (IgG) preparations provides an efficient means of protecting immunocompromised patients from microbial infections. More recently, the use of passive immunotherapy to prevent or to treat established infections with the human immunodeficiency virus (HIV) has gained much attention, due to promising preclinical data obtained in monkey and humanized mouse in vivo model systems, demonstrating that the transfer of HIV-specific antibodies can not only prevent HIV infection, but also diminish virus load during chronic infection. Furthermore, an array of broadly neutralizing HIV-specific antibodies has become available and the importance of the IgG constant region as a critical modulator of broadly neutralizing activity has been demonstrated. The aim of this review is to summarize the most recent findings with regard to the molecular and cellular mechanisms responsible for antibody-mediated clearance of HIV infection, and to discuss how this may help to improve HIV therapy via optimizing Fcγ-receptor-dependent activities of HIV-specific antibodies.

IgG subclass and heavy chain domains contribute to binding and protection by mAbs to the poly γ-D-glutamic acid capsular antigen of Bacillus anthracis.

Bacterial capsules are common targets for antibody-mediated immunity. The capsule of Bacillus anthracis is unusual among capsules because it is composed of a polymer of poly-γ-d-glutamic acid (γdPGA). We previously generated murine IgG3 monoclonal antibodies (mAbs) to γdPGA that were protective in a murine model of pulmonary anthrax. IgG3 antibodies are characteristic of the murine response to polysaccharide antigens. The goal of the present study was to produce subclass switch variants of the γdPGA mAbs (IgG3 → IgG1 → IgG2b → IgG2a) and assess the contribution of subclass to antibody affinity and protection. Subclass switch antibodies had identical variable regions but differed in their heavy chains. The results showed that a switch from the protective IgG3 to IgG1, IgG2b or IgG2a was accompanied by i) a loss of protective activity ii) a change in mAb binding to the capsular matrix, and iii) a loss of affinity. These results identify a role for the heavy chain constant region in mAb binding. Hybrid mAbs were constructed in which the CH1, CH2 or CH3 heavy chain constant domains from a non-protective, low binding IgG2b mAb were swapped into the protective IgG3 mAb. The IgG3 mAb that contained the CH1 domain from IgG2b showed no loss of affinity or protection. In contrast, swapping the CH2 or CH3 domains from IgG2b into IgG3 produced a reduction in affinity and a loss of protection. These studies identify a role for the constant region of IgG heavy chains in affinity and protection against an encapsulated bacterial pathogen.

[Correlation between macro- and micro-stability ch2 domains of human IGG2 and their biological activity. 1. Ansalysis the calorimetric and optical melting curves].

Human myeloma immunoglobulin second subclass LOM and SIN, their Fc fragment and firstly obtained hFc fragment in which there is not only low portion of the hinge region, but also its core portion (Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys), have been studied by number of physical methods (scanning calorimetry, fluorescence spectroscopy, analytical centrifugation). Joint analysis of calorimetric and optical melting curves revealed that only first (low-temperature) heat absorption peak at all the melting curves corresponds to the melting of the two CH2 domains. It was shown that CH2 domains of intact IgG2 are destabilized relative to those domains in hFc and Fc fragments.

Constant domain-regulated antibody catalysis.

Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.

Cloning of a hamster anti-mouse CD79B antibody sequences and identification of a new hamster immunoglobulin lambda constant IGLC gene region.

Anti-CD79 antibodies have been effective at targeting B cell lymphoma cells and depleting B cells in animal models. In order to engineer recombinant antibodies with additional effector functions in mice, we cloned and sequenced the full-length cDNAs of the heavy and light chain of a hamster anti-mouse CD79B antibody. Although hamster antibodies represent a unique source of monoclonal antibodies against mouse, rat, and human antigens, sequence information of hamster immunoglobulins (IG) is sparse. Here, we report a new hamster (Cricetulus migratorius) IG lambda constant (IGLC) gene region that is most homologous to mouse IGLC2 and IGLC3.

Destabilization of CH2 domains in intact IgG2 is accompanied by reduced ability to inhibit complement system factor C1.

Fc fragments (hFc) of human myeloma IgG2 proteins LOM and SIN having core hinge (Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys) were first obtained by a modified proteolytic procedure. The thermostability of CH2 domains inside of standard Fc, hFc fragments, and intact IgG2 LOM and SIN was studied by fluorescence spectroscopy. It was found that CH2 domains of intact IgG2 are destabilized. The destabilization is accompanied by reduced ability of IgG2 to inhibit the activation of complement system by classical pathway. This could be due to the decrease in the affinity of CH2 domains to factor C1q.

The constant region contributes to the antigenic specificity and renal pathogenicity of murine anti-DNA antibodies.

Affinity for DNA and cross-reactivity with renal antigens are associated with enhanced renal pathogenicity of lupus autoantibodies. In addition, certain IgG subclasses are enriched in nephritic kidneys, suggesting that isotype may determine the outcome of antibody binding to renal antigens. To investigate if the isotype of DNA antibodies affects renal pathogenicity by influencing antigen binding, we derived IgM, IgG1, IgG2b and IgG2a forms of the PL9-11 antibody (IgG3 anti-DNA) by in vitro class switching or PCR cloning. The affinity and specificity of PL9-11 antibodies for nuclear and renal antigens were analyzed using ELISA, Western blotting, surface plasmon resonance (SPR), binding to mesangial cells, and glomerular proteome arrays. Renal deposition and pathogenicity were assayed in mice injected with PL9-11 hybridomas. We found that PL9-11 and its isotype-switched variants had differential binding to DNA and chromatin (IgG3>IgG2a>IgG1>IgG2b>IgM) by direct and competition ELISA, and SPR. In contrast, in binding to laminin and collagen IV the IgG2a isotype actually had the highest affinity. Differences in affinity of PL9-11 antibodies for renal antigens were mirrored in analysis of specificity for glomeruli, and were associated with significant differences in renal pathogenicity in vivo and survival. Our novel findings indicate that the constant region plays an important role in the nephritogenicity of antibodies to DNA by affecting immunoglobulin affinity and specificity. Increased binding to multiple glomerular and/or nuclear antigens may contribute to the renal pathogenicity of anti-DNA antibodies of the IgG2a and IgG3 isotype. Finally, class switch recombination may be another mechanism by which B cell autoreactivity is generated.

The human G1m1 allotype associates with CD4+ T-cell responsiveness to a highly conserved IgG1 constant region peptide and confers an asparaginyl endopeptidase cleavage site.

The human G1m1 allotype comprises two amino acids, D12 and L14, in the CH3 domain of IGHG1. Although the G1m1 allotype is prevalent in human populations, ~40% of Caucasiods are homozygous for the nG1m1 allotype corresponding to E12 and M14. Peptides derived from the G1m1 region were tested for their ability to induce CD4+ T-cell proliferative responses in vitro. A peptide immediately downstream from the G1m1 sequence was recognized by CD4+ T cells in a large percentage of donors (peptide CH315-29). CD4+ T-cell proliferative responses to CH315-29 were found at an increased frequency in nG1m1 homozygous donors. Homozygous nG1m1 donors possessing the HLA-DRB1*07 allele displayed the highest magnitudes of proliferation. CD4+ T cells from donors homozygous for nG1m1 proliferated to G1m1-carrying Fc-fragment proteins, whereas CD4+ T cells from G1m1 homozygous donors did not. The G1m1 sequence creates an enzymatic cleavage site for asparaginyl endopeptidase in vitro. Proteolytic activity at D12 may allow the presentation of the CH315-29 peptide, which in turn may result in the establishment of tolerance to this peptide in G1m1-positive donors. Homozygous nG1m1 patients may be more likely to develop CD4+ T-cell-mediated immune responses to therapeutic antibodies carrying the G1m1 allotype.

Inducible DNA breaks in Ig S regions are dependent on AID and UNG.

Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cmu) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination. Uracil DNA glycosylase (UNG) is required for most CSR activity although its role is disputed. Here we use ligation-mediated PCR to detect DSBs in S regions in splenic B cells undergoing CSR. We find that the kinetics of DSB induction corresponds with AID expression, and that DSBs are AID- and UNG-dependent and occur preferentially at G:C basepairs in WRC/GYW AID hotspots. Our results indicate that AID attacks cytosines on both DNA strands, and staggered breaks are processed to blunt DSBs at the initiating ss break sites. We propose a model to explain the types of end-processing events observed.

Characterization of immunoglobulin heavy chain knockout rats.

The rat is a species frequently used in immunological studies but, until now, there were no models with introduced gene-specific mutations. In a recent study, we described for the first time the generation of novel rat lines with targeted mutations using zinc-finger nucleases. In this study, we compare immune development in two Ig heavy-chain KO lines; one with truncated Cµ and a new line with removed JH segments. Rats homozygous for IgM mutation generate truncated Cµ mRNA with a de novo stop codon and no Cγ mRNA. JH-deletion rats showed undetectable mRNA for all H-chain transcripts. No serum IgM, IgG, IgA and IgE were detected in these rat lines. In both lines, lymphoid B-cell numbers were reduced >95% versus WT animals. In rats homozygous for IgM mutation, no Ab-mediated hyperacute allograft rejection was encountered. Similarities in B-cell differentiation seen in Ig KO rats and ES cell-derived Ig KO mice are discussed. These Ig and B-cell-deficient rats obtained using zinc-finger nucleases-technology should be useful as biomedical research models and a powerful platform for transgenic animals expressing a human Ab repertoire.

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.

Monoclonal alloantibodies specific for the constant region of T cell antigen receptors.

We established three distinct monoclonal antibodies (7C5, 7D1; IgM and 6A4; IgG1) by the fusion of P3U1 and BALB/c (Igh-1a) spleen cells hyperimmunized with T cell blasts from the immunoglobulin heavy chain (Igh) allotype congenic CB-20 (Igh-1b) mice. The 7C5 or 6A4 antibody reacts with the constant region determinants on the antigen-binding molecule (Ct) of the antigen-specific suppressor T cell factor (TsF) or augmenting T cell factor (TaF), respectively. The 7D1 antibody, however, recognizes the shared determinants on the Ct molecules of TsF and TaF. Genetic studies of determinants recognized by these monoclonal antibodies have also suggested that the distinct Ct molecules of TsF and TaF are encoded by two discrete genes linked to the Igh-1b genes, which are located on the right side of the variable genes of Igh on the 12th chromosome. By using the immunoadsorbent columns of 6A4 antibody and anti-I-Ab, TaF, in a manner similar to TsF, was demonstrated to be composed of two chains, i.e., the Ct molecules and the I-A-encoded products. Furthermore, the Ct-bearing molecules were shown to possess the antigen-binding moiety.

Engineered humanized dimeric forms of IgG are more effective antibodies.

Humanized IgG1 M195 (HuG1-M195), a complementarity determining region-grafted recombinant monoclonal antibody, is reactive with CD33, an antigen expressed on myelogenous leukemia cells. M195 is in use in trials for the therapy of acute myelogenous leukemia. Since biological activity of IgG may depend, in part, on multimeric Fab and Fc clustering, homodimeric forms of HuG1-M195 were constructed by introducing a mutation in the gamma 1 chain CH3 region gene to change a serine to a cysteine, allowing interchain disulfide bond formation at the COOH terminal of the IgG. Despite similar avidity, the homodimeric IgG showed a dramatic improvement in the ability to internalize and retain radioisotope in target leukemia cells. Moreover, homodimers were 100-fold more potent at complement-mediated leukemia cell killing and antibody-dependent cellular cytotoxicity using human effectors. Therefore, genetically engineered multimeric constructs of IgG may have advantages relative to those forms that are found naturally.

Influence of the macromolecular form of a B cell epitope on the expression of antibody variable and constant region structure.

We investigated the influence of the macromolecular form of an epitope on the structure of antibody variable and constant regions expressed by the B cell population participating in an immune response to that epitope. Hybridomas were constructed from strain A/J mice undergoing either primary or secondary immune responses to p-azophenylarsonate conjugated to Brucella abortus (Ars-Bruc). We determined the sequences of the V genes expressed by hybridomas selected on the basis of expression of a single VH gene segment known to encode a large family of anti-Ars antibodies. These sequences were compared with the sequences of V genes expressed by a previously characterized panel of hybridomas isolated in the same way during the primary and secondary responses of A/J mice to Ars-KLH. The repertoire of Ars-specific V domains expressed among primary and secondary hybridomas elicited with these two forms of Ars were similar, as were the differences between primary and secondary V region somatic mutational alteration and affinity for Ars. In contrast, predominant expression of IgG2 anti-Ars antibodies was elicited in the secondary Ars-Bruc response, whereas secondary anti-Ars antibodies elicited with Ars-KLH are predominantly IgG1. Thus, differences in the macromolecular form of Ars clearly influence the isotypic profile of the anti-Ars response, but the expression, diversification, and selection of V domains elicited with this hapten are not greatly affected by such differences. Our results suggest that while isotype regulation is highly perceptive of the macromolecular form of a B cell epitope, V region regulation is primarily influenced by the molecular structure of that epitope.

Role of repetitive antigen patterns for induction of antibodies against antibodies.

Antibody responses against antibodies, such as rheumatoid factors, are found in several immunopathological diseases and may play a role in disease pathogenesis. Experience shows that they are usually difficult to induce experimentally. Antibodies specific for immunoglobulin constant regions (anti-allotypic) or for variable regions (anti-idiotypic) have been investigated in animal models; the latter have even been postulated to regulate antibody and T cell responses via network-like interactions. Why and how such anti-antibodies are induced during autoimmune diseases, has remained largely unclear. Because repetitively arranged epitopes in a paracrystalline structure of a viral envelope cross-link B cell receptors efficiently to induce a prompt T-independent IgM response, this study used immune complexes containing viruses or bacteria to evaluate the role of antigen pattern for induction of anti-antibody responses. We present evidence that antibodies bound to strictly ordered, but not to irregularly arranged, antigens dramatically enhance induction of anti-antibodies, already after a single immunization and without using adjuvants. The results indicate a novel link between anti-antibody responses and infectious agents, and suggest a similar role for repetitive self-antigens such as DNA or collagen involved in chronic immunopathological diseases.

T helper subset differentiation in the absence of invariant chain.

The outcome of murine infection with Leishmania major is regulated by major histocompatibility complex class II-restricted T helper cells. Invariant chain-deficient (Ii -/-) mice have impaired ability to present major histocompatibility complex class II-restricted antigens, and reduced numbers of CD4+ T cells. Despite these deficits, C57BL/6 Ii -/- mice controlled L. major infection comparably to wild-type mice. As assessed by mRNA analysis and in vitro antigen restimulation for IFN-gamma, Ii -/- mice had normal induction of Th1 subset differentiation even though antigen-dependent proliferation of their lymph node cells was substantially compromised. In addition, BALB/c Ii -/- mice exhibited a progressive course of infection and Th2 effector cell development that were comparable to that seen in wild-type BALB/c mice. We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro. In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-gamma-or IL-4-secreting T helper cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic. These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation signals may facilitate the full differentiation of T helper subsets during limiting presentation of antigenic peptides.