Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.     

Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.Key words: immunogenicity, human anti-mouse antibody, cytokine release syndrome     

基因工程抗体研究进展

临床治疗中人抗鼠抗体反应的出现使鼠源性单克隆抗体的应用受到了极大的限制。为降低其免疫原性,人们利用基因工程技术对鼠源抗体进行改造,以减少其鼠源成分。简要概述了目前研究比较多的几种基因工程抗体及其临床应用。     

The human and murine influenza-specific B cell repertoires share a common idiotope

We have dissected the human influenza-specific B cell repertoire by performing Epstein-Barr virus (EBV) limiting dilution analysis of lymphocytes obtained from donors before and after immunization with a commercially available influenza vaccine. In addition to an analysis of precursor frequency and light chain diversity, we studied sera and culture supernatants containing human anti-influenza antibodies with a panel of murine monoclonal antibodies specific for idiotopes identified on murine anti-PR8 and anti-X-31 antibodies. An idiotypic specificity present on the X-31-specific murine monoclonal PY206 has previously been shown to be shared by murine antibodies specific for PR8, X-31, and other influenza viruses. We observed little correlation among the following parameters: anti-viral titer, serum idiotope content, precursor frequency and immune status. More interestingly, there was a striking predominance of human influenza-specific antibodies that utilized lambda light chains. In addition, 12 of 26 human anti-influenza monoclonals strongly inhibited the binding of one of the murine anti-idiotopes to the labeled murine antibody, PY206. This is the same idiotope that is shared among murine antiinfluenza antibodies and all six individuals studied contained clones reactive with this anti-idiotope. Seven of these 12 idiotope-positive human antibodies gave partial cross-reactivity in a second anti-idiotypic system. These observations imply that a significant level of homology exists between the binding sites of human and murine influenza-specific antibodies and suggest that idiotypic manipulation of the human immune response to influenza virus may have important therapeutic implications.     

The interaction of murine IgG subclass proteins with human monocyte Fc receptors

The use of murine monoclonal antibodies in the immunotherapy of human disease has prompted interest in the interactions of murine IgG with Fc receptors (FcR) expressed on human effector cells. We examined the heterocytophilic interactions between monomeric murine IgG subclass proteins and the FcR expressed on human monocytic cells (peripheral blood monocytes and interferon (IFN)-gamma-induced U937 cells). All four murine IgG2a antibodies and both murine IgG3 antibodies that were tested bound to human monocyte FcR with high affinity (10(8) to 10(9) M-1). By contrast, the affinities of four murine IgG1 and four IgG2b monomers were 100-fold to 1000-fold lower than the affinity of the human IgG1-FcR interaction. A 68,000 to 72,000 dalton protein was isolated by affinity chromatography from blood monocytes and from IFN-gamma-induced U937 cells on murine IgG2a, IgG3, and human IgG immunoadsorbents. In binding assays with IFN-stimulated U937 cells, murine IgG2a and IgG3 antibodies showed complete cross-blocking with a human IgG1 myeloma protein, indicating that murine and human IgG interact with the same population of Fc-binding proteins. No evidence for heterogeneity of cross-reactive FcR was observed. The ability of murine IgG2a and IgG3 monomers to compete with human IgG1 monomers for binding to human monocyte FcR suggests the potential usefulness of antibodies of these isotypes in the immunotherapy of diseases in which monocyte- or macrophage-mediated, antibody-dependent cellular cytotoxicity may play a role in the modification or remission of disease.     

Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.     

Human immune response to multiple injections of murine monoclonal IgG

Murine monoclonal antibody infusions in humans should induce a human anti-mouse immunoglobulin (mIgG) immune response, especially if multiple infusions over an extended period of time are necessary for therapeutic efficacy. We have administered multiple infusions of the murine monoclonal antibody T101 to patients with cutaneous T cell lymphoma (CTCL) or chronic lymphocytic leukemia (CLL). Five of 10 CTCL patients, compared with zero of six CLL patients, developed antibodies to mIgG. In those CTCL patients who did not demonstrate anti-mIgG antibodies, we were unable to correlate the lack of response to any of a large number of clinical parameters. Anti-mIgG antibodies were of both the mu and gamma isotypes and were detectable 14 days after the first infusion. Multiple infusions were associated with elevated titers. The anti-idiotypic portion of the anti-mIgG titer steadily increased with each infusion until eventually, in one patient receiving eight weekly infusions, well over one-half the serum anti-mIgG recognized only T101 and not four other murine IgG2AK antibodies tested. To increase our confidence in these findings, four separate assay systems were used to make these determinations. The identification of anti-idiotype antibodies as the dominant species of the immune response to multiple infusions of murine monoclonal antibody has major implications for future work with monoclonal antibodies. Although it has been suggested that human monoclonal antibodies would obviate an immune response, our work implies that such antibodies might still induce anti-idiotype antibodies if multiple infusions are administered.     

An active murine-human chimeric Fab antibody derived from Escherichia coli, potential therapy against over-expressing VEGFR2 solid tumors

Many recombinant murine monoclonal antibodies (mAbs) were studied under pre-clinical or clinical development and became one of the most prolific drug classes in oncology. Vascular endothelial growth factors receptor 2 (VEGFR2) has been implicated to play an important role in tumors. We have established a murine anti-VEGFR2 mAb. To reduce the shortcoming of the mAb, a murine–human chimeric Fab (cFab) named FA8H1 was constructed with gene engineering techniques and expressed as a soluble and functional protein in Escherichia coli Top10F′. Several immunological methods were used to characterize the cFab, including ELISA, affinity and kinetics assay, IP, IF, FACS, and IHC. The results illuminated that cFab maintained the specificity for the VEGFR2 antigen. The active cFab also effectively identified VEGFR2 over-expressing cells in a number of archived human cancer tissues, compared to its parental antibody. The FA8H1 provided the basis for potential therapy research against over-expressing VEGFR2 human solid tumors.     

SDR grafting--a new approach to antibody humanization

A major impediment to the clinical utility of the murine monoclonal antibodies is their potential to elicit human anti-murine antibody (HAMA) response in patients. To circumvent this problem, murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts. To that end, murine antibodies have been humanized by grafting their complementarity determining regions (CDRs) onto the variable light (V(L)) and variable heavy (V(H)) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site. However, the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-Id) response in patients. To minimize the anti-Id response, a procedure to humanize xenogeneic antibodies has been described that is based on grafting, onto the human frameworks, only the specificity determining residues (SDRs), the CDR residues that are most crucial in the antibody-ligand interaction. The SDRs are identified through the help of the database of the three-dimensional structures of the antigen-antibody complexes of known structures or by mutational analysis of the antibody-combining site. An alternative approach to humanization, which involves retention of more CDR residues, is based on grafting of the 'abbreviated' CDRs, the stretches of CDR residues that include all the SDRs. A procedure to assess the reactivity of the humanized antibody to sera from patients who had been administered the murine antibody has also been described.     

Crystal structures of human antibodies: a detailed and unfinished tapestry of immunoglobulin gene products

Sequencing of all human immunoglobulin (Ig) germline gene segments has recently been completed. However, our first glimpses of the recombined products of this combinatorial gene system were in the 1970s, in landmark publications, reporting the crystal structures of two human myeloma proteins, the Mcg lambda light chain dimer and the New IgG1(lambda) Fab. Although numerous crystal structures of murine and human antibodies have now been determined, only a relatively small proportion of the human germline genes have had their corresponding protein three-dimensional structures resolved. Therefore, further structural investigations are required before the inherent diversity of the antibody repertoire can be fully appreciated. We discuss the detailed structural information available for human antibodies with regard to their immune functions. Also discussed, is how the structural information is finding application in the 'humanization' of murine antibodies as part of their development as 'biopharmaceuticals' for the treatment of human disease.     

人源化抗体研究历程及发展趋势

单克隆抗体从问世到目前广泛应用于临床,经历了一段曲折的发展历程。其中人源化抗体是一个重要的里程碑,并伴随着一系列重大的技术革新,如PCR技术、抗体库技术、转基因动物等。人源化抗体的形式也从最初的嵌合抗体、改型抗体等逐步发展为今天的人抗体。抗体人源化已经成为治疗性抗体的发展趋势,同时各种抗体衍生物也不断涌现,它们从不同角度克服抗体本身的应用局限,也为治疗人类疾病提供了更多利器。对单克隆抗体进行改造使之应用于临床治疗,不仅需要对抗体效应机制进行更细致深入的研究,同时还有赖于对人类免疫系统调控机制的全面精确认识。     

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.     

Antitumour activity of a chimeric antibody against the leucocyte antigen CD48

Preclinical studies with the murine anti-CD48 antibody, mHuLym3 (IgG2a) have shown it to be a potentially useful therapeutic reagent in the treatment of human leukaemia and lymphoma. For clinical use, humanised antibodies can have a number of advantages over their original murine version, including mediation of higher effector cell function with human cells, longer serum half-life and lower immunogenicity. In this study, we have produced a mouse/human chimeric HuLym3 antibody (cHuLym3) where the murine antibody constant regions have been replaced with human constant regions. We report the production and preclinical characterisation of the antibody, cHuLym3, with potent in vitro and in vivo antitumour activity. The genes encoding the variable heavy and light chains were amplified by the polymerase chain reaction, sequenced and cloned into eukaryotic expression vectors containing the human light- and heavy-chain constant regions (κ and IgG1). The chimeric and murine HuLym3 antibodies had similar cell-binding specificity and affinity. In the human Raji cell severe combined immunodeficient mouse model the i.v. injection of cHuLym3 and mHuLym3 produced similar antitumour responses. Doses of cHuLym3 and mHuLym3 (100 μg) on days 1, 2 and 4 after i.v. Raji cell injection produced a 40% longer time to hind-leg paralysis than when a control antibody was used. cHuLym3 had more potent activity than mHuLym3 in antibody-dependent cellular cytotoxicity (ADCC) assays in vitro, with human peripheral blood mononuclear cells as effectors. Up to 60% specific cell lysis was observed with cHuLym3 in ADCC assays. These properties suggest that anti-CD48 antibodies may be useful in the treatment of a number of diseases including lymphoid leukaemias and lymphoma. Received: 5 May 1999 / Accepted: 12 August 1999     

The ability of cytotoxic T cells to recognize HLA-A2.1 or HLA-B7 antigens expressed on murine cells correlates with their epitope specificity

Two groups of human and murine cytotoxic T lymphocyte (CTL) clones specific for human leukocyte antigen (HLA)-A2 or -B7 can be distinguished based on their ability to kill murine transfectants expressing these molecules. The clones which do not recognize murine transfectants exhibited greatly reduced conjugate formation with these cells, indicating that the inability to lyse these cells occurs in recognition and binding. No systematic differences in inhibitory titer between the two types of CTL clones were seen with anti-CD8 (Lyt-2), anti-LFA-1, or monoclonal antibodies against HLA class I molecules. However, blocking with anti-HLA class I monoclonal antibodies suggested that different CTL clones recognized spatially separate epitopes on HLA-A2 and -B7. In addition, a correlation between the inability to recognize murine transfectants and fine specificity was seen. Eight of nine clones which did not lyse murine transfectants also failed to recognize human cells expressing HLA-A2.2 or -A2.3. In contrast only 5 of 12 clones which lysed transfectants failed to recognize the variant molecules. Analogous data were obtained with human CTL clones raised against HLA-A2.1. These findings suggest that CTL clones that do not lyse murine cells expressing appropriate antigens recognize epitopes that have been altered or lost as a consequence of expression on the murine cell surface. It is suggested that the loss of HLA-associated epitopes on the murine cell surface may be due to differences between mouse and human cells in the processing or presentation of class I-associated peptides.     

Heterogeneity of the human phosphocholine-specific B cell repertoire

We have utilized a highly efficient method of culturing small numbers of Epstein Barr virus (EBV)-infected cells to analyze the heterogeneity of human antibodies specific for phosphocholine (PC). Lymphocytes from peripheral blood or tonsils of individuals who had no evidence of recent pneumococcal infection were infected with EBV and cultured at limiting dilution. After correction for the cloning efficiency, between 1/1500 and 1/10,000 B cells produced specific anti-PC antibodies by our criteria. Examination of the heterogeneity of these antibodies revealed that most individuals had an overwhelming predominance of anti-PC antibodies with kappa-light chain. Fine specificity analysis of 39 monoclonal anti-PC antibodies demonstrated that the IgM antibodies examined displayed significant binding site diversity, whereas the IgA PC-specific clones were much less heterogeneous. In general, the human anti-PC antibodies had a much higher relative affinity (Krel) for choline and glycerophosphocholine than the murine antibody families. Through examination of the human PC-specific B cell repertoire we have drawn some interesting parallels with the well-defined murine clonotype families and have begun to dissect the human response to this naturally occurring antigenic determinant.     

Microencapsulation technology: A novel method for monoclonal antibody production

Monoclonal antibodies have been envisioned as useful agents for human therapeutic and diagnostic applications in vivo. Recent results from human clinical trials suggest that this potential is becoming a reality. Attention is now shifting to the development of methods to produce monoclonal antibodies of a quality acceptable for widespread human use and in sufficient quantity to be a commercially viable product. Microencapsulation technology has been demonstrated to be suited to the large-scale production of both human and murine monoclonal antibodies of high purity and activity, for use in applications both in vitro and in vivo.     

Generation of human antibody fragments against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>using a phage display chain shuffling approach

Background  

Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials.     

Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application.     

Epitope mapping reveals the binding mechanism of a functional antibody cross-reactive to both human and murine programmed death 1

Of the inhibitory checkpoints in the immune system, programmed death 1 (PD-1) is one of the most promising targets for cancer immunotherapy. The anti-PD-1 antibodies currently approved for clinical use or under development bind to human PD-1 (hPD-1), but not murine PD-1. To facilitate studies in murine models, we developed a functional antibody against both human and murine PD-1, and compared the epitopes of such antibody to a counterpart that only bound to hPD-1. To quickly identify the epitopes of the 2 antibodies, we used alanine scanning and mammalian cell expression cassette. The epitope identification was based on PD-1-binding ELISA and supported by affinity ranking of surface plasmon resonance results. The hPD-1 epitopes of the 2 functional antibodies were also compared with the binding region on hPD-1 that is responsible for PD-L1 interaction. In silico modeling were conducted to explain the different binding modes of the 2 antibodies, suggesting a potential mechanism of the antibody cross-species binding.     

Function blocking antibodies to neuropilin-1 generated from a designed human synthetic antibody phage library

Non-immune (na?ve) antibody phage libraries have become an important source of human antibodies. The synthetic phage antibody library described here utilizes a single human framework with a template containing human consensus complementarity-determining regions (CDRs). Diversity of the libraries was introduced at select CDR positions using tailored degenerate and trinucleotide codons that mimic natural human antibodies. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 semaphorins, is expressed on endothelial cells and neurons. NRP1 is required for vascular development and is expressed widely in the developing vasculature. To investigate the possibility of function blocking antibodies to NRP1 as potential therapeutics, and study the consequence of targeting NRP1 in murine tumor models, panels of antibodies that cross-react with human and murine NRP1 were generated from a designed antibody phage library. Antibody (YW64.3) binds to the CUB domains (a1a2) of NRP1 and completely blocks Sema3A induced neuron collapse; antibody (YW107.4.87) binds to the coagulation factor V/VIII domains (b1b2) of NRP1 and blocks VEGF binding and VEGF induced cell migration. YW107.4.87 inhibits tumor growth in animal xenograft models. These antibodies have provided valuable tools to study the roles of NRP1 in vascular and tumor biology.