Clustered H chain somatic mutations shared by anti-p-azophenylarsonate antibodies confer enhanced affinity and ablate the cross-reactive idiotype

A cluster of four consecutive CDR2 somatic mutations are shared by the VH regions of two independently isolated hybridoma antibodies with specificity for p-azophenylarsonate (Ars). The mutations appear to be derived by a series of independent events. To assess the influence of these shared somatic mutations on antibody affinity for Ars and on idiotypy, we introduced them, via site-directed mutagenesis, into the V region of an anti-Ars antibody that was otherwise unmutated and we eliminated them from the mutated context of one of the two antibodies in which they were originally found. Results of affinity measurements by fluorescence quenching and of idiotypic binding assays performed on these engineered mutants demonstrated that the shared mutations increased affinity for Ars and eliminated the predominant Id associated with strain A anti-Ars antibodies and four of five idiotypes defined by anti-idiotypic mAb. These results support the interpretation that a strong affinity-based selection pressure has favored the clonal expansion of B cells with receptors containing these mutations despite the loss of a predominant Id. Thus, in producing antibodies containing V regions conferring high affinity for Ag, the combined processes of somatic mutation and clonal selection have generated a common structural solution through parallel repeats.     

Structural characterization of idiotopes by using antibody variants generated by site-directed mutagenesis

Four anti-idiotopic mAB, 107, MB, AI, and AD8, react with mouse hybridoma protein 36-65 specific for the hapten p-azophenylarsonate. The four antiidiotypic antibodies do not react with hybridoma protein 36-71, a somatically mutated variant of 36-65 whose H and L chain V region sequence differs at 19 amino acid positions. To determine which regions of 36-65 are important for the interaction with each of the four anti-idiotypic antibodies, variants of 36-65 containing one or more of the 36-71 substitutions were generated by oligonucleotide-directed mutagenesis of the rearranged 36-65 H chain V region gene, followed by expression of mutant proteins containing either the 36-65 or the 36-71 L chain in transfected hybridoma cells. Idiotypic characterization of the mutant proteins showed that reactivity correlates with the 36-65 H chain, but some contributions from the 36-65 L chain come into play. In the 36-65 H chain V region, idiotopes were mapped to the first and third complementarity-determining regions for anti-idiotypic antibodies 107, MB, and AI, and to all three complementarity-determining regions for anti-idiotypic antibody AD8. The binding of all four anti-idiotypic antibodies to hybridoma protein 36-65 was hapten inhibitable. However, a comparison between the effect of individual 36-71 substitutions on idiotope expression and their effect on Ag-binding affinity suggests that none of the four anti-idiotypic antibodies bodies mimics the structure of Ag.     

The amino acid residues at the VH-D-JH junctions affect the affinity of anti-p-azophenylarsonate antibodies

Murine A/J anti-p-azophenylarsonate (Ars) antibodies sharing a predominant idiotype are encoded by a single combination of germ-line V region gene segments. The dominance of this idiotype among secondary immune response anti-Ars antibodies has been explained by the Ag-driven selection of favorable somatic mutants of this gene segment combination, associated with an intrinsic Ars-affinity of the germ-line V region higher than that of other possible combinations. To determine the effect of junctional diversity upon affinity for Ag, independently of somatic mutation, we determined the V region sequences and affinity for Ars of five primary response antibodies. These antibodies share identical unmutated V regions but differ only at the D gene junctions. Among the five antibodies, Ars-affinity differed up to 10-fold depending upon the identity of the amino acid residues at the VH-D and the D-JH junctions. The combination of junctional residues observed in two primary response antibodies with relatively low Ars-affinity has not been observed among secondary response antibodies. Thus the identity of junctional residues resulting from gene rearrangement prior to antigen stimulation must be taken into account in hypotheses which account for idiotype dominance by selection on the basis of affinity.     

Alteration in H chain V region affects complement activation by chimeric antibodies

Chimeric antibodies to the synthetic polypeptide (Tyr, Glu)-Ala-Lys ((T,G)-A-L) were used to examine C activation by human IgG1. Two IgG1 antibodies, which contained mouse L chains and H chains with mouse V domains and human C domains, differed only in their VH domain. Ag binding and C activation by these antibodies were analyzed by ELISA. When limiting amounts of Ag were used in the assays, the antibodies required different quantities of Ag for optimal binding, suggesting that the antibodies bind to different epitopes on the (T,G)-A-L molecule. However, when competitive inhibition assays were performed with an optimal concentration of Ag, there were no differences in relative binding affinities for (T,G)-A-L or dissociation characteristics of the antibodies. C activation was examined at optimal Ag concentration to ensure equivalent binding of two IgG1 antibodies to Ag. After combination with immobilized Ag, these two antibodies bearing different V regions exhibited marked differences in the binding of C components C1q and C3d. When present in equal amounts in the assay, antibody 10B activated C and bound more C1q and C3d than antibody B11. These results indicate that V region differences can affect C activation by IgG.     

Structural characterization of H chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies

The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.     

Structural analysis of mutants of high-affinity and low-affinity p-azophenylarsonate-specific antibodies generated by alanine scanning of heavy chain complementarity-determining region 2.

Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50-60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36-71. Each mutated H chain gene was expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain results in significant loss of binding with both mutated (36-71L) or unmutated (36-65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36-71 had minimal effect on Ab affinity when expressed with 36-71 L chain. However, in the context of unmutated L chain, of H:Gly(54) to Ala resulted in significant loss of binding, while Abs containing Asn(52) to Ala, Pro(53) to Ala, or Ile(58) to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36-65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36-65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding.     

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

T cell receptor-dependent, antigen-specific stimulation of a murine T cell clone induces a transient, VLA protein-mediated binding to extracellular matrix

Upon Ag stimulation, an arsonate-specific murine T cell clone exhibited a rapid but transient increase in cell adhesion to collagen, fibronectin, and laminin. This increase in cell adhesion was not observed when a mutant T cell clone lacking TCR expression was utilized. However, upon stimulation by phorbol esters, both parent and mutant T cell clones exhibited a similar transient increase in adhesion to the three matrix proteins. The observed cell adhesion was extensively inhibited by antibodies to the integrin beta 1 subunit, indicating the involvement of VLA proteins. Despite changes in the adhesive properties, there was essentially no difference in the expression of VLA-1, -3, -4, -5, and -6 between resting and stimulated T cells. Together these results suggest that Ag stimulation transmits signals via the TCR complex resulting in a rapid, but transient, up-regulation of matrix protein binding by VLA proteins already present at the cell surface. Because the appropriate reagents that recognize individual mouse VLA proteins were not available, we used the human T cell line Jurkat to demonstrate that T cell binding to collagen, laminin, and fibronectin is mediated largely by VLA-2, VLA-6, and a combination of VLA-5 and VLA-4, respectively.     

Increased avidity of mutant IgM antibodies caused by the absence of COOH-terminal glycosylation of the mu H chain.

We have previously described the isolation of two hybridoma variants secreting higher avidity IgM (D5 and 7F5), starting from the E11 hybridoma cell line, which produces an antibody specific for the A Ag of the ABO blood group system. In order to explain at the molecular level this increased reactivity, cDNA encoding the H and L chains of the E11, D5, and 7F5 mAb were cloned and sequenced. Comparison of the nucleotide sequences showed a single point mutation in each of the two mAb produced by the hybridoma variants. The mutations were both located in the H chain C region and caused a Ser to Phe substitution at position 565 in the D5 mAb and a Asn to Tyr substitution at position 563 in the 7F5 mAb. Both substitutions modified the consensus glycosylation sequence (Asn-X-Ser/Thr) located in the tail piece of the secretory mu-chain. The absence of glycosylation at this site was confirmed by CNBr cleavage of the [14C]mannose-labeled mAb. The two single point mutations were solely responsible for the increased avidity of the antibodies, as confirmed by site-directed mutagenesis of the E11 mu-chain and serologic analysis of the mutated E11 antibodies. We conclude that the absence of glycosylation at Asn 563 is responsible for the increased avidity of the mutant, possibly by altering the quaternary structure of the IgM polymer. To our knowledge, this is the first report that point mutations in the H chain C region can influence the reactivity of IgM mAb.     

Patterns of mutations and selection in antibodies to the phosphocholine-specific determinant in Proteus morganii

The contribution of somatic mutation to the generation of an antibody response was investigated by using the phosphocholine (PC) determinant in the bacterium Proteus morganii as the model Ag. The response to this determinant is restricted to a single VH/VL pair and apparently is derived from only one or two precursors per mouse. In this study we examined hybridoma antibodies from nine individual mice which produced representatives of 12 different clones. We found that all antibodies reactive with the PC Ag of P. morganii contained somatic mutations; the number ranged from 2 to 20. Two clusters of mutations were observed, one in complementarity-determining residue (CDR) 2 and the other in CDR 3 of VH. Examination of a three-dimensional model of M603, an antibody with the same V region composition as the anti-PC antibodies under study, showed that these clusters occupied an area of the binding site which presumably interacts with carrier elements of the PC epitope in P. morganii. A high incidence of recurring mutations were found in both clusters, and one of these was invariant, leading to an Asn for Asp substitution at 95. Ag binding studies with these antibodies and an additional one, which was unmutated except for the invariant substitution at 95, showed that: 1) antibodies having only the 95Asn mutation failed to bind the PC Ag of P. morganii, 2) addition of a second recurring mutation, at 52a (CDR 2), was sufficient to create strong binding to the P. morganii Ag, and 3) accumulation of mutations was directly correlated with increased binding activity for Ag. These results show that somatic mutations play a critical, if not essential, role in generating specificity for this PC Ag, and that Ag, and most likely a carrier element of the epitope, is a primary force in the continued selection and expansion of Ag-reactive B cells.     

NSm and 78-kilodalton proteins of Rift Valley fever virus are nonessential for viral replication in cell culture

Rift Valley fever viruses carrying mutations of the M gene preglycoprotein region, one lacking NSm protein expression, one lacking 78-kDa protein expression, and one lacking expression of both proteins, were compared in cell culture. All of the mutants and their parent virus produced plaques with similar sizes and morphologies in Vero E6 cells and had similar growth kinetics in Vero, C6/36, and MRC5 cells, demonstrating that the NSm and 78-kDa proteins were not needed for the virus to replicate efficiently in cell culture. A competition-propagation assay revealed that the parental virus was slightly more fit than the mutant virus lacking expression of both proteins.     

The use of peptide analogs with improved stability and MHC binding capacity to inhibit antigen presentation in vitro and in vivo

The identification of a core region for OVA 323-339, which is critical in determining binding to IAd, has enabled us to generate a series of analog peptides in which this core region was extended at both the N and C termini with different amino acid residues. When assessed for binding capacity, several peptides were shown to have increased affinity for IAd compared with the parent sequence, and in addition, some peptides had acquired binding specificities for class II MHC haplotypes not present for OVA 323-339. These peptides were next examined for their ability to inhibit T cell responses in vitro and in vivo. The correlation between binding and the ability to inhibit T cell activation in vitro was good. However, when assessed in vivo, it was clear that high Ia binding was not sufficient in itself to define the inhibitory capacity of a given peptide. That this discrepancy was due to differences in degradation of the core-extended peptides was suggested by 1) results from an inhibition of Ag presentation assay, in which the pulse period with Ag and inhibitor was extended to 20 h; and 2) direct analysis of peptide stability by using reverse phase HPLC. Finally, by protecting the peptide from degradation with N- and C-terminal substitutions of D-amino acids, the inhibitory capacity of an unstable core-extended peptide in vitro could be greatly enhanced. These data indicate that the core extension approach may be one method by which antagonists for MHC class II molecules may be generated.     

Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.     

Serine 132 is the C3 covalent attachment point on the CH1 domain of human IgG1.

The covalent binding of C3 (complement component C3) to antigen-antibody complexes (Ag.Ab; immune complexes (ICs)) is a key event in the uptake, transport, presentation, and elimination of Ag in the form of Ag.Ab.C3b (IC.C3b). Upon interaction of C3 with IgG.IC, C3b.C3b.IgG covalent complexes are formed that are detected on SDS-polyacrylamide gel electrophoresis by two bands corresponding to C3b.C3b (band A) and C3b.IgG (band B) covalent complexes. This allows one to evaluate the covalent binding of C3b to IgG antibodies. It has been described that C3b can attach to both the Fab (on the CH1 domain) and the Fc regions of IgG. Here the covalent interaction of C3b to the CH1 domain, a region previously described spanning residues 125-147, has been studied. This region of the CH1 domain is exposed to solvent and contains a cluster of six potential acceptor sites for ester bond formation with C3b (four Ser and two Thr). A set of 10 mutant Abs were generated with the putative acceptor residues substituted by Ala, and we studied their covalent interaction with C3b. Single (Ser-131, Ser-132, Ser-134, Thr-135, Ser-136, and Thr-139), double (positions 131-132), and multiple (positions 134-135-136, 131-132-134-135-136, and 131-132-134-135-136-139) mutants were produced. None of the mutants (single, double, or multiple) abolished completely the ability of IgG to bind C3b, indicating the presence of C3b binding regions other than in the CH1 domain. However, all mutant Abs, in which serine at position 132 was replaced by Ala, showed a significant decrease in the ability to form C3b.IgG covalent complexes, whereas the remaining mutants had normal activity. In addition we examined ICs using the F(ab')2 fragment of the mutant Abs, and only those containing Ala at position 132 (instead of Ser) failed to bind C3b. Thus Ser-132 is the binding site for C3b on the CH1 domain of the heavy chain, in the Fab region of human IgG.     

The specificity properties that distinguish members of a set of homologous anti-digoxin antibodies are controlled by H chain mutations

Five murine A/J strain anti-digoxin mAb (35-20, 40-40, 40-120, 40-140, and 40-160) have highly homologous H and L chain V regions, only differing by somatic mutation, yet differ in affinity and specificity. The availability of the VH and VL genomic clones from one hybridoma, 40-140, has now allowed studies involving in vitro mutagenesis and chain recombination among these five hybridomas. To determine the relative contributions of the mutations found in either VH or VL to the overall binding properties of these antibodies, we recombined the 40-140VH with the VL of each hybridoma. The 40-140VH gene was transfected into hybridoma variants that produce only VL. The recombinant antibodies show that the mutations present in VH, rather than in VL, affect the fine specificity properties of these antibodies, whereas, the mutations among both VH and VL chains are important in determining antigen affinity. From mutations present in VH that affect fine specificity properties, the comparison of the antibody sequences, and from the previously measured binding properties, we predicted and tested selected VH mutations for their ability to alter specificity or affinity by doing site-directed in vitro mutagenesis. The results for the somatic mutations found in this group of antibodies show: 1) VH mutations control the fine specificity properties that distinguish different members of this group; 2) in particular, VH residues 54 and 55 in CDR2 control the distinguishing characteristics of specificities between these antibodies; and 3) by mutagenesis, we had the unusual result of being able to alter Ag specificity without affecting affinity. A computer model of the 40-140 antibody binding site was generated which indicates that VH residues 54 and 55 are highly accessible.     

Binding characteristics and cross-reactivity of three different antilipid A monoclonal antibodies

A detailed characterization of binding specificity and cross-reactivity of three antilipid A murine mAb was performed. Binding characteristics of these three mAb were investigated against Ag (ReLPS, lipid A, derivatives of lipid A) in solid phase (ELISA) and in fluid phase (C consumption, inhibition studies), and upon incorporation in membranes (E: passive hemolysis assay, and liposomes: inhibition studies). Cross-reactivity with heterologous Ag was investigated in ELISA (LPS, Gram-negative bacteria) and immunoblot experiments (LPS). The binding specificity of mAb 26-5 (IgG2b), raised against synthetic lipid A, was located in the hydrophilic region of biphospholipid A and was also exposed after membrane incorporation of lipid A or after preincubation of lipid A with polymyxin B (PMX). mAb 26-20 (IgM), also raised against synthetic lipid A, showed binding specificity for the hydrophobic region of lipid A: no binding to membrane-associated lipid A could be demonstrated, and binding in ELISA could be blocked very efficiently by PMX. The reaction pattern of mAb 8-2 (IgM), raised against the heat-killed Re mutant of Salmonella typhimurium, was in part similar to that of mAb 26-20. However, inhibition of binding with PMX was less efficient and a high specificity for ReLPS, also after membrane incorporation of this Ag, was demonstrated. In contrast to mAb 26-5 and 26-20, mAb 8-2 showed extensive cross-reactivity with heterologous LPS preparations and heat-killed as well as live Gram-negative bacteria. It is concluded that each of the three mAb binds to a different antigenic epitope in lipid A and that exposure of those epitopes for antibody binding is restricted in a differential manner, depending on mode of Ag presentation. The here defined reaction patterns provide a basis for the interpretation of potential inhibitory effects on in vitro and in vivo biologic (and toxic) activities of endotoxins and Gram-negative bacteria.     

Altered hapten recognition by two anti-digoxin hybridoma variants due to variable region point mutations

Two spontaneous variants of the murine anti-digoxin antibody-producing hybridoma cell line 26-10 were isolated by two-color fluorescence-activated cell sorting on the basis of altered hapten binding. The variable region sequences of the antibodies produced by the mutant lines revealed that each contains a single amino acid change in the heavy chain second complementarity determining region. A Tyr to His change at position 50 leads to a 40-fold reduction in affinity for digoxin. A Ser to Phe mutation at position 52 results in a 300-fold reduction in affinity for digoxin. A competition assay involving 33 digoxin analogues was used to examine the specificity of hapten binding of 26-10 and the two mutant antibodies. The position 50 mutant has a distinct specificity change; it exhibits a preference for digoxin congeners containing a hydroxyl group at the steroid 12 position, whereas the 26-10 parent does not. The affinities of all three antibodies for hapten are progressively lowered by substitutions of increasing size at the digoxin steroid D ring 16 position. Although 26-10 binds digoxin and its genin form equally, 12 and 16 steroid position substitutions which lower affinity also confer a preference for a sugar at the steroid 3 position. These results suggest that position 50 contributes to specificity of the antibody and that alterations of the hapten can lead to differences in recognition, possibly through a shift in hapten orientation within the binding site.     

Mutants of Chlamydomonas with Aberrant Responses to Sulfur Deprivation

In the absence of sulfur, Chlamydomonas reinhardtii, a unicellular green alga, increases its rate of sulfate import and synthesizes several periplasmic proteins, including an arylsulfatase (Ars). These changes appear to help cells acclimate to a sulfur-deficient environment. The elevated rate of sulfate import results from an increase in the capacity and affinity of the transport system for sulfate. The synthesis of Ars, a periplasmic enzyme that cleaves sulfate from aromatic compounds, enables cells to use these molecules as a source of sulfur when free sulfate is not available. To characterize the ways in which C. reinhardtii perceives changes in the sulfur status of the environment and regulates its responses to these changes, we mutagenized cells and isolated strains exhibiting aberrant accumulation of Ars activity. These mutants were characterized for Ars activity, ars mRNA accumulation, periplasmic protein accumulation, and sulfate transport activity when grown in both sulfur-sufficient and sulfur-deficient conditions. All of the mutants exhibited pleiotropic effects with respect to several of these responses. Strains harboring double mutant combinations were constructed and characterized for Ars activity and ars mRNA accumulation. From the mutant phenotypes, we inferred that both positive and negative regulatory elements were involved in the acclimation process. Both the epistatic relationships among the mutations and the effects of the lesions on the responses of C. reinhardtii to sulfur limitation distinguished these mutants from similar mutants in Neurospora crassa.     

Potentiation of anti-carcinoembryonic antigen immunotoxin cytotoxicity by monoclonal antibodies reacting with co-expressed carcinoembryonic antigen epitopes

The initial step in ricin A-chain (RTA)-immunotoxin-mediated cell cytotoxicity involves binding to the target cell Ag through the antibody moiety. One of the factors influencing this is the affinity of the antibody component for the target cell Ag. Multiple epitopes on carcinoembryonic Ag have been mapped providing a range of mAb of known specificity. These have been used to show that the cytotoxicity of an immunotoxin containing RTA conjugated to an anti-carcinoembryonic Ag mAb (228-RTA) is potentiated by mAb recognizing different epitopes. The potentiating antibodies also increased the level of target cell binding of antibody 228. Cross-linking of cell bound antibody was not involved because monovalent fragments of a potentiating antibody were effective. The potentiating antibodies modified the binding affinity of 228 antibody increasing the t1/2 of antibody at the tumor cell surface. This increased the dwell time of cell bound antibody and using conjugates of 228 linked to albumin-tetramethylrhodamine it was shown to enhance conjugate endocytosis. These investigations indicate that enhanced antibody affinity leads to increased endocytosis of bound immunoconjugate and potentiates cytotoxicity.