Carbohydrate chains on IgG2b: a requirement for efficient feedback immunosuppression

The in vitro immunosuppressive ability of a monoclonal IgG2b-anti-trinitrophenyl antibody lacking carbohydrate chains (GORK-Tm) has been compared with the immunosuppressive ability of the intact antibody (GORK). The carbohydrate depletion of GORK-Tm was achieved by culturing the GORK hybridoma cells in media containing tunicamycin, an inhibitor of glycosylation. Both antibodies have been shown to have the same antigen and protein A-binding capacity, although GORK-Tm is deficient in complement fixation, antibody-dependent cellular cytotoxicity, binding to Fc receptors on macrophages, and rapid elimination of antigen-antibody complexes from the circulation. It is shown in these studies that although GORK antibodies are efficient immunosuppressors, suppressing up to 95% of the plaque-forming cell response, GORK-Tm antibody preparations with identical hemagglutinating and antigen-binding capacity are highly deficient in suppressing the immune response. These findings are of interest in two ways. First, it shows that the carbohydrate chains are of great importance for the immunosuppressive effects of IgG. Secondly, it also shows that the isotype IgG2b, in addition to all other murine isotypes, can efficiently suppress the humoral immune response.     

In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

The Fc effector functions of immunoglobulin G (IgG) antibodies are in part determined by structural features of carbohydrates linked to each of the paired gamma heavy chains in the antibody constant domain (C(H)2). One glycoform that has been shown to be advantageous is G2, where both arms of complex bi-antennary N-glycans terminate in galactose. In vitro treatment with glycosyltransferases can remodel heterogeneous IgG glycoforms, enabling preparation of IgG molecules with homogeneous glycan chains. Here we describe optimization of conditions for use of a soluble recombinant galactosyltransferase in vitro to remodel glycans of human serum IgG, and we demonstrate a scaled-up reaction in which >98% of neutral glycans attached to 1 kg IgG are converted to the G2 glycoform. Removal of glycosylation reagents from the product is achieved in one step by affinity chromatography on immobilized Protein A.     

Characterization of a mutated IgA2 antibody of the m(1) allotype against the epidermal growth factor receptor for the recruitment of monocytes and macrophages

IgA antibodies constitute an important part of the mucosal immune system, but their immunotherapeutic potential remains rather unexplored, in part due to biotechnological issues. For example, the IgA2m(1) allotype carries an unusual heavy and light chain pairing, which may confer production and stability concerns. Here, we report the generation and the biochemical and functional characterization of a P221R-mutated IgA2m(1) antibody against the epidermal growth factor receptor (EGFR). Compared with wild type, the mutated antibody demonstrated heavy chains covalently linked to light chains in monomeric as well as in joining (J)-chain containing dimeric IgA. Functional studies with wild type and mutated IgA2m(1) revealed similar binding to EGFR and direct effector functions such as EGFR down-modulation and growth inhibition. Furthermore, both IgA molecules triggered similar levels of indirect tumor cell killing such as antibody-dependent cell-mediated cytotoxicity (ADCC) by isolated monocytes, activated polymorphonuclear cells, and human whole blood. Interestingly, the dimeric IgA antibodies demonstrated higher efficiency in direct as well as in indirect effector mechanisms compared with their respective monomeric forms. Both wild type and mutated antibody triggered effective FcαRI-mediated tumor cell killing by macrophages already at low effector to target cell ratios. Interestingly, also polarized macrophages mediated significant IgA2-mediated ADCC. M2 macrophages, which have been described as promoting tumor growth and progression, may convert to ADCC-mediating effector cells in the presence of EGFR-directed antibodies. In conclusion, these results provide further insight into the immunotherapeutic potential of recombinant IgA antibodies for tumor immunotherapy and suggest macrophages as an additional effector cell population.     

Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies

Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan^®) and anti-Her2/neu IgG1 trastuzumab (Herceptin^®). ADCC is triggered upon the binding of lymphocyte receptors (FcγRs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.     

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

Human IgG2 antibodies may exist in at least three distinct structural isomers due to disulfide shuffling within the upper hinge region. Antibody interactions with Fc gamma receptors and the complement component C1q contribute to immune effector functions. These interactions could be impacted by the accessibility and structure of the hinge region. To examine the role structural isomers may have on effector functions, a series of cysteine to serine mutations were made on a human IgG2 backbone. We observed structural homogeneity with these mutants and mapped the locations of their disulfide bonds. Importantly, there was no observed difference in binding to any of the Fc gamma receptors or C1q between the mutants and the wild‐type IgG2. However, differences were seen in the apparent binding affinity of these antibodies that were dependent on the selection of the secondary detection antibody used.     

Effect of a tail piece cysteine deletion on biochemical and functional properties of an epidermal growth factor receptor-directed IgA2 m(1) antibody

Antibodies of human IgA isotype are critical components of the mucosal immune system, but little is known about their immunotherapeutic potential. Compared with IgG antibodies, IgA molecules carry a C-terminal tail piece extension of 18 amino acids with a free cysteine at position 471. This cysteine is required for the formation of dimeric IgA antibodies, but may impair molecular characteristics of monomeric IgA antibodies as therapeutic reagents. Thus, we generated and characterized a d471-mutated antibody against the epidermal growth factor receptor (EGFR) and compared it to its respective IgA2 m(1) wild type antibody. Both wild type and mutated IgA antibodies demonstrated similar EGFR binding and were similarly efficient in inhibiting EGF binding and in blocking EGF-mediated cell proliferation. Recruitment of Fc-mediated effector functions like antibody-dependent cell-mediated cytotoxicity by monocytes, macrophages or PMN was similar, but the d471-mutated IgA exhibited different biochemical properties compared with wild type antibody. As expected, mutated IgA did not form stable dimers in the presence of human joining (J)-chain, but we also observed reduced levels of dimeric aggregates in the absence of J-chain. Furthermore, glycoprofiling revealed different glycosylation patterns for both antibodies, including considerably less mannosylation of d471-mutated antibodies. Overall, our results demonstrate that the deletion of the C-terminal cysteine of IgA2 did not affect the investigated effector functions compared with wild type antibody, but it improved biochemical properties of an IgA2 m(1) antibody against EGFR, and may thereby assist in exploring the immunotherapeutic potential of recombinant IgA antibodies.     

Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous beta1, 4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II

The effector functions elicited by IgG antibodies strongly depend on the carbohydrate moiety linked to the Fc region of the protein. Therefore several approaches have been developed to rationally manipulate these glycans and improve the biological functions of the antibody. Overexpression of recombinant beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in production cell lines leads to antibodies enriched in bisected oligosaccharides. Moreover, GnT-III overexpression leads to increases in non-fucosylated and hybrid oligosaccharides. Such antibody glycovariants have increased antibody-dependent cellular cytotoxicity (ADCC). To explore a further variable besides overexpression of GnT-III, we exchanged the localization domain of GnT-III with that of other Golgi-resident enzymes. Our results indicate that chimeric GnT-III can compete even more efficiently against the endogenous core alpha1,6-fucosyltransferase (alpha1,6-FucT) and Golgi alpha-mannosidase II (ManII) leading to higher proportions of bisected non-fucosylated hybrid glycans ("Glyco-1" antibody). The co-expression of GnT-III and ManII led to a similar degree of non-fucosylation as that obtained for Glyco-1, but the majority of the oligosaccharides linked to this antibody ("Glyco-2") are of the complex type. These glycovariants feature strongly increased ADCC activity compared to the unmodified antibody, while Glyco-1 (hybrid-rich) features reduced complement-dependent cytotoxicity (CDC) compared to Glyco-2 or unmodified antibody. We show that apart from GnT-III overexpression, engineering of GnT-III localization is a versatile tool to modulate the biological activities of antibodies relevant for their therapeutic application.     

Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region

Chimeric mouse-human IgG was used to study the structural and functional roles of the carbohydrate present in the CH2 domain of human IgG molecules. To remove this N-linked carbohydrate, Asn-297, the oligosaccharide attachment residue, was changed to either Gln (a conservative replacement) or His for IgG1 or Lys for IgG3 (nonconservative replacements) by site-directed mutagenesis. Carbohydrate-deficient antibodies are properly assembled and secreted and bind Ag and protein A. However, aglycosylated IgG are more sensitive to most proteases than their corresponding wild-type IgG, indicating some conformational changes have occurred. Aglycosylated IgG do not bind to the human Fc gamma RI and do not activate C; depending on the isotype, C1q binding ability is either completely lost (IgG1) or dramatically decreased (IgG3). The serum half-life in mice of aglycosylated IgG1-Gln remains the same as wild-type IgG1, 6.5 +/- 0.5 days, whereas aglycosylated IgG3-Gln has a shorter half-life, 3.5 +/- 0.2 days, compared to that of wild-type IgG3, 5.1 +/- 0.4 days. These results indicate the carbohydrate interposed between CH2 domain of human IgG is necessary to maintain the appropriate structure for the maintenance of many of the effector functions dependent on the CH2 domain.     

Importance of antibody isotype in monoclonal anti-idiotype therapy of a murine B cell lymphoma. A study of hybridoma class switch variants

An initial panel of four syngeneic monoclonal antibodies directed against the idiotype of a murine B cell lymphoma was used to treat this tumor in vivo. The antibody in the panel of the IgG2a isotype was more effective in treatment than the other antibodies, which were of the IgG1 and IgG2b isotypes. To independently assess the role of antibody isotype in mediating antitumor effects, switch variant hybridoma families were isolated from the hybridomas secreting the less effective IgG1 and IgG2b antibodies. A family isolated from an IgG1-secreting parent consisted of IgG1-, IgG2b-, and IgG2a-secreting members, and an IgG2a variant was isolated from an IgG2b-secreting parent for another family. Antibody members of each family differed only in heavy chain composition and were the same with respect to their light chains and their affinity and specificity for idiotype. The IgG2a members of both families were superior to the other members in inhibiting tumor growth with an order of effectiveness of IgG2a greater than IgG1 greater than IgG2b. These in vivo results paralleled the abilities of these different isotype antibodies to mediate antibody-dependent cellular cytolysis in vitro. For the IgG2b----IgG2a family, in vivo treatment with the IgG2a member given i.p. after i.p. tumor challenge at one-tenth the dose of the IgG2b member was still superior to the latter. At one-hundredth the dose of the IgG2b, the IgG2a was still superior to the latter when the antibodies were given i.p. and tumors subcutaneously. These data and those showing that the clearance of these antibodies from the serum differed in only a relatively minor way indicate that the IgG2a antibodies in this system had greater antitumor effects primarily by virtue of their greater capacity for host effector interaction.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type B polysaccharide. Demonstration of three types of V regions and their association with H and L chain isotypes

The antibody response to Haemophilus influenzae type b polysaccharide (Hib-PS) is pauciclonal but can vary between different individuals. To estimate the size of this antibody repertoire we examined the constant and V regions of human IgG anti-Hib antibodies from 14 individuals at the clonal level using various serologic and IEF methods. Examination of H chains showed that 11 of 14 individuals produced both IgG1 and IgG2 antibodies, two individuals produced only IgG2 and one individual produced only IgG1 antibody. All 14 individuals produced kappa-containing antibody clones and three persons also produced significant lambda-containing antibody clones. V region heterogeneity was examined by comparing cross-reactivity of anti-Hib-PS antibodies to the capsular polysaccharide of Escherichia coli K100 carbohydrate (K100 CHO). These studies showed that clones of IgG anti-Hib-PS antibodies cross-reactive with K100 CHO were present in 5 of 14 (36%) individuals and also revealed at least three types of V regions among these antibodies. The first type has no cross-reaction with K100 CHO and was found in 13 of the 14 individuals. The second type, found in three of 14 individuals, cross-reacts with K100 CHO and uses a lambda L chain V region. The third type, found in 2 of 14 individuals, cross-reacts with K100 CHO and uses a kappa L chain V region. Although the lambda type V region was found only in association with IgG2, the other two V region types associate with both IgG1 and IgG2. Thus, five IgG antibody clones are serologically discernable. An individual generally responds to Hib-PS by expressing several clones selected from these discernable antibody clones. Indeed, we can observe six individual response patterns among these 14 individuals and conclude that considerable variability in individual responses to Hib-PS can be achieved with very few V regions.     

Engineering the variable region of therapeutic IgG antibodies

Since the first generation of humanized IgG1 antibodies reached the market in the late 1990s, IgG antibody molecules have been extensively engineered. The success of antibody therapeutics has introduced severe competition in developing novel therapeutic monoclonal antibodies, especially for promising or clinically validated targets. Such competition has led researchers to generate so-called second or third generation antibodies with clinical differentiation utilizing various engineering and optimization technologies. Parent IgG antibodies can be engineered to have improved antigen binding properties, effector functions, pharmacokinetics, pharmaceutical properties and safety issues. Although the primary role of the antibody variable region is to bind to the antigen, it is also the main source of antibody diversity and its sequence affects various properties important for developing antibody therapeutics. Here we review recent research activity in variable region engineering to generate superior antibody therapeutics.     

The impact of glycosylation on monoclonal antibody conformation and stability

Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy. However, the modified molecule could have unusual behavior during development due to the alteration of its intrinsic properties and stability. In this study, we focused on the differences between glycosylated and deglycosylated antibodies, as aglycosyl antibodies are often chosen when effector function is not desired or unimportant. We selected three human IgG1 antibodies and used PNGase F to remove their oligosaccharide chains. Although there were no detected secondary or tertiary structural changes after deglycosylation, other intrinsic properties of the antibody were altered with the removal of oligosaccharide chains in the Fc region. The apparent molecular hydrodynamic radius increased after deglycosylation based on size-exclusion chromatography analysis. Deglycosylated antibodies exhibited less thermal stability for the CH2 domain and less resistance to GdnHCl induced unfolding. Susceptibility to proteolytic cleavage demonstrated that the deglycosylated version was more susceptible to papain. An accelerated stability study revealed that deglycosylated antibodies had higher aggregation rates. These changes may impact the development of aglycosyl antibody biotherapeutics.     

Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems. Multiple effector mechanisms result from cellular activation mediated through a family of IgG-Fc receptors differentially expressed on leucocytes. It is established that glycosylation of IgG-Fc is essential for recognition and activation of these ligands. IgG antibodies predominate in human serum and most therapeutic antibodies are of the IgG class.The IgG-Fc is a homodimer of N-linked glycopeptide chains comprised of two immunoglobulin domains (Cgamma2, Cgamma3) that dimerise via inter-heavy chain disulphide bridges at the N-terminal region and non-covalent interactions between the C-terminal Cgamma3 domains. The overall shape of the IgG-Fc is similar to that of a "horseshoe" with a majority of the internal space filled by the oligosaccharide chains, only attached through asparagine residues 297.To investigate the influence of individual sugar (monosaccharide) residues of the oligosaccharide on the structure and function of IgG-Fc we have compared the structure of "wild-type" glycosylated IgG1-Fc with that of four glycoforms bearing consecutively truncated oligosaccharides. Removal of terminal N-acetylglucosamine as well as mannose sugar residues resulted in the largest conformational changes in both the oligosaccharide and in the polypeptide loop containing the N-glycosylation site. The observed conformational changes in the Cgamma2 domain affect the interface between IgG-Fc fragments and FcgammaRs. Furthermore, we observed that the removal of sugar residues permits the mutual approach of Cgamma2 domains resulting in the generation of a "closed" conformation; in contrast to the "open" conformation which was observed for the fully galactosylated IgG-Fc, which may be optimal for FcgammaR binding. These data provide a structural rationale for the previously observed modulation of effector activities reported for this series of proteins.     

Monoclonal antibodies to streptococcal group A carbohydrate. I. A dominant idiotypic determinant is located on Vk

In an effort to better define the antibody repertoire to streptococcal group A carbohydrate (GAC), somatic cell hybrids were prepared from A/J mice immunized with streptococcal vaccine. Most antibodies were IgG3K and IgMK, while 2 of 26 antibodies were lambda type. Each of the IgG3 antibodies had a distinct isoelectric point consistent with previous estimates of clonal repertoires of approximately 200. IEF analysis of the L chains, however, showed that about half of the antibodies produce a common L chain, called VK1GAC, previously identified in A/J anti-GAC serum antibodies. Additional support for the structural similarity of these L chains was gained by developing an idiotype antiserum to VK1GAC. All proteins with the common L chain spectrotype react strongly with anti-VK1GAC. Thus, it appears that anti-GAC antibodies are composed of H chains bearing a few VH regions pairing with a few L chains.     

Engineering the variable region of therapeutic IgG antibodies

Since the first generation of humanized IgG1 antibodies reached the market in the late 1990s, IgG antibody molecules have been extensively engineered. The success of antibody therapeutics has introduced severe competition in developing novel therapeutic monoclonal antibodies, especially for promising or clinically validated targets. Such competition has led researchers to generate so-called second or third generation antibodies with clinical differentiation utilizing various engineering and optimization technologies. Parent IgG antibodies can be engineered to have improved antigen binding properties, effector functions, pharmacokinetics, pharmaceutical properties and safety issues. Although the primary role of the antibody variable region is to bind to the antigen, it is also the main source of antibody diversity and its sequence affects various properties important for developing antibody therapeutics. Here we review recent research activity in variable region engineering to generate superior antibody therapeutics.Key words: antibody therapeutics, variable region, engineering, affinity, pharmacokinetics, stability, immunogenicity     

Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control

Elite controllers (ECs) represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC) that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune–recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.     

Engineering a human IgG2 antibody stable at low pH

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid‐induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti‐2,4‐dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti‐DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain—namely, F300Y, V309L, and T339A (IgG2_YLA)—reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen‐binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. II. IgG antibodies contain VH genes from a single VH family and VL genes from at least four VL families

To define the V gene family repertoire of human IgG anti-Haemophilus influenzae type b polysaccharide antibodies, we purified six IgG1 and nine IgG2 anti-Hib-PS antibodies to monoclonality from immune serum of six individuals and performed N-terminal amino acid sequence analysis. Of the 15 clonal antibodies we examined, all H chain V regions were of the VHIII family. In contrast, the L chains of these antibodies were clearly from at least four different VL families; VKI, VKII, VKIII, and V lambda. Interestingly. VL family expression correlated with the cross-reactivity of these antibodies to the capsular carbohydrate of Escherichia coli K100. VKII antibodies did not cross-react, whereas antibodies expressing V lambda, VKI, or VKIII generally cross-reacted. We conclude that L chain V regions are very important contributors to the limited heterogeneity in this antibody repertoire.     

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.     

Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens

Human antibodies were isolated by phage display from a naturally expressed human antibody repertoire. Antibody selection was carried out against the epithelial cell adhesion molecule (EpCAM) or 17-1A antigen, that in a clinical trial had been successfully used as a target for antibody therapy of minimal residual colorectal cancer. VH chains were selected from the human IgD repertoire expressed on naive B2 and autoreactive B1 lymphocytes. By guiding the selection through a murine template antibody, two EpCAM-specific human antibodies, HD69 and HD70, were obtained that closely resembled the murine therapeutic 17-1A antibody in their binding properties when expressed as complete huIgG1 molecules in CHO cells. However, both human antibodies recruited human cytotoxic effector cells far more efficiently than the murine 17-1A antibody used for clinical trials. Therefore, and in view of the long in vivo half-life of human IgG1 antibodies, HD69 and HD70 are regarded as highly promising third generation versions of the murine therapeutic antibody. Because of their origin from an evolutionary conserved germline VH repertoire, they are expected to exhibit minimal immunogenicity in patients. Received: 16 November 2000 / Accepted: 11 January 2001