Final analysis of the prospective WSG-AGO EC-Doc versus FEC phase III trial in intermediate-risk (pN1) early breast cancer: efficacy and predictive value of Ki67 expression.

BACKGROUND: Taxane-based adjuvant chemotherapy is standard in node-positive (N+) early breast cancer (BC). The magnitude of benefit in intermediate-risk N+ early BC is still unclear. WSG-AGO epiribicine and cyclophosphamide (EC)-Doc is a large trial evaluating modern taxane-based chemotherapy in patients with 1-3 positive lymph nodes (LNs) only. PATIENTS AND METHODS: A total of 2011 BC patients (18-65 years, pN1) were entered into a randomized phase III trial comparing 4 × E90C600 q3w followed by 4 × docetaxel 100 q3w (n = 1008) with the current standard: 6 × F500E100C500 q3w (n = 828) or C600M40F600 d1, 8× q4w (n = 175). Primary end point was event-free survival (EFS); secondary end points were overall survival (OS), toxicity, translational research, and quality of life. Central tumor bank samples were evaluable in a representative collective (n = 772; 40%). Ki-67 was assessed centrally in hormone receptor-positive disease as a surrogate marker for the distinction of luminal A/B-like tumors. RESULTS: Baseline characteristics were well balanced between study arms in both main study and central tumor bank subset. At 59-month median follow-up, superior efficacy of EC-Doc [versus FEC (a combination of 5-fluorouracil, epirubicin, and cyclophosphamide)] was seen in EFS and OS: 5-year EFS: 89.8% versus 87.3% (P = 0.038); 5-year OS: 94.5% versus 92.8% (P = 0.034); both tests one-tailed. EC-Doc caused more toxicity. In hormone receptor-positive (HR)+ disease, only high-Ki-67 tumors (≥ 20%) derived significant benefit from taxane-based therapy: hazard ratio = 0.39 (95% CI 0.18-0.82) for EC-Doc versus FEC (test for interaction; P = 0.01). CONCLUSION: EC-Doc significantly improved EFS and OS versus FEC in intermediate-risk BC (1-3 LNs) within all subgroups as defined by local pathology. In HR+ disease, patients with luminal A-like tumors may be potentially over-treated by taxane-based chemotherapy. CLINICAL TRIAL NUMBER: ClinicalTrials.gov, NCT02115204.

Nucleotide sequence of chimpanzee Fc and hinge regions.

The nucleotide sequence of the Fc and hinge regions of a chimpanzee monoclonal antibody has been determined. Most of the sequence is similar to the human IgG1 sequence. However, the chimpanzee hinge regions differs from the human hinge region in six of 48 nucleotides, which leads to three amino acid substitutions. Two of the amino acid changes are not conservative and may lead to differences in flexibility of the hinge. The chimp hinge sequence seems to be a combination of the human IgG1 hinge and the hinge sequence of a human IgG pseudogene. The implications of this difference for the evolution of human IgG subgroup is discussed. Despite differences in the hinge regions, the chimpanzee monoclonal antibody differs from the most closely related human IgG1 allotype only slightly more than the two most distantly related human allotypes differ from each other.

Characterization and relative orientation of epitopes for monoclonal antibodies and antisera to human chorionic gonadotropin.

We analyzed ten monoclonal antibodies and three polyclonal antisera directed against human chorionic gonadotropin by immunoassay in order to determine which pairs of antibodies are capable of binding simultaneously to hCG. A relative orientation of epitopes on the surface of hCG could be inferred from a matrix of data describing the abilities of particular antibody pairs to inhibit competitively or enhance the binding of each other. These results indicated that the binding site of each of the antibodies could be assigned to one of three different regions of the hCG molecule, and at least one antibody binding within each region exhibited a substantial preference for hCG relative to human luteinizing hormone. One of these regions is the COOH-terminal portion of the hCG beta subunit. A second region contains the binding site for the SB6 rabbit antiserum (which has been widely employed in clinical studies) as well as the binding sites of several monoclonal antibodies. Antibodies to this second region characteristically bind both the whole hormone and the free beta subunit. A third region contains the binding sites of a previously reported human antiserum and also several monoclonal antibodies. These antibodies characteristically react preferentially with the intact hormone relative to the free subunits. The information contained in the type of epitope map described here can be used as the rational basis for the design of two-site immunoradiometric assays for hCG and/or its subunits.

The reversal of multidrug resistance in multicellular tumor spheroids by SDZ PSC 833.

Multidrug resistance (MDR) is a major impediment to the effective treatment of cancer. We have used multicellular tumor spheroids (MTS) as a model to investigate whether MDR can be reversed in a three dimensional structure. MTS are tightly associated aggregates of tumor cells that exhibit many of the properties of solid tumors. A human MDR breast carcinoma cell line was selected by exposure to taxol under monolayer conditions. The sensitive (parental) and drug-resistant phenotypes were retained when the cells were grown as MTS. Thus, the three dimensional conditions in this novel model system did not affect the MDR phenotype. SDZ PSC 833 is an efficient MDR reversing agent as determined under monolayer conditions and is currently being evaluated in clinical trials. Resistance to taxol and doxorubicin of the MDR cells grown as MTS was almost completely reversed by SDZ PSC 833. Our results suggest that SDZ PSC 833 has the potential to reverse the MDR phenotype in solid tumors.

Further characterization of the fate of human monoclonal antibodies in rhesus monkeys.

We have shown previously that human monoclonal antibodies are not very immunogenic in rhesus monkeys, with only one monkey out of five mounting an anti-monoclonal antibody response. Two additional monkeys have been injected multiple times with much larger amounts of one human monoclonal antibody. No anti-antibody response has been detected in these monkeys. Structural changes that occur in the monoclonal antibodies over time in vivo have been investigated by Western Blots using anti-idiotypic antisera. Sodium dodecyl sulfate gel electrophoresis reveals that very little antibody has altered molecular weight. Isoelectric focusing patterns change more dramatically. Forms of the antibodies with lower isoelectric points appear in the serum. These forms have a similar in vivo half-life as the freshly prepared antibody. Very low pI forms of the monoclonal antibodies are not detected in the serum. Isoelectric focusing can be used to determine the in vivo or in vitro condition of a monoclonal antibody preparation. Finally, the monkey anti-human IgG that arose in the single monkey studied previously has been further characterized.

Rhesus monkey responses to multiple injections of human monoclonal antibodies.

Five rhesus monkeys were injected multiple times over several months with two different human IgG1 monoclonal antibodies, both directed against human cytomegalovirus. Three monkeys each injected four times with monoclonal antibody EV2-7 for over 200 days showed no response other than a normal decay in antibody level. The in vivo half life of this antibody was substantially longer when measured with an idiotype-specific two site immunoassay than with radiolabeled antibody, indicating that the iodination procedure greatly affected the stability of the antibody. Although there was considerable individual variation in the half-life of EV2-7, from 8.9 to 30.5 days, the half-life was fairly long, especially considering the size of the monkeys. Two monkeys were injected with monoclonal antibody EV1-15. One monkey has responded in a similar manner to the EV2-7-injected monkeys. However, the other monkey has produced an anti-idiotypic antibody (or antibodies) of high affinity. It is possible that this response was triggered by the unusual physical nature of antibody EV1-15 or the effect of the species difference between human and rhesus monkey. In any case, the results from these five monkeys indicate that human monoclonal antibodies should have a significant advantage over mouse monoclonal antibodies for in vivo therapeutic applications.

Further characterization of cooperative interactions of monoclonal antibodies.

We reported previously that mixtures of some monoclonal antibodies directed against the glycoprotein hormone human chorionic gonadotropin (hCG) had a higher affinity for the antigen than either monoclonal antibody separately. The synergistic interaction could no longer be detected when one of the antibodies was replaced with its F(ab) fragment. This cooperative interaction has now been further characterized. One-half of 10 possible pairs prepared from five IgG1 monoclonal antibodies against hCG result in a synergistic interaction. The addition of an IgG2b monoclonal antibody to one of the IgG1 monoclonal antibodies also induces a cooperative interaction, which shows that the effect is not subclass restricted. Cooperative interactions between antibodies are also not restricted to solution conditions; adsorption of one antibody to a solid support appears to increase the cooperative effect. Indeed, one pair of antibodies that failed to bind hCG synergistically in solution did so when one antibody was bound to a solid surface. The liquid phase antibody also has an effect on the specificity of the solid phase antibody. The sensitivity of the solid phase assay system has enabled us to develop a rapid method of determining if two monoclonal antibodies can bind to an antigen simultaneously. A quantitative theoretical model has been devised that successfully predicts the cooperative behavior observed between antibodies and should be useful in devising conditions that result in sensitive solid phase radioimmunoassays.

Monoclonal antibodies to alpha-chain regions of human fibrinogen that participate in polymer formation.

Monoclonal antibodies have been generated against a cross-link-containing derivative of alpha polymer (alpha XLCNBr), isolated following CNBr digestion of fibrin [Sobel, J. H., Ehrlich, P. H., Birken, S., Saffran, A. J., & Canfield, R. E. (1983) Biochemistry (preceding paper in this issue)]. One cloned cell line (F-102) was chosen for characterization based on its apparent specificity for the A alpha-chain region A alpha 518-584 (CNBr X). A second line (F-103) was selected because of its anti-A alpha 241-476 (CNBr VIII) properties. These two regions of the A alpha chain have previously been implicated as major contributors to the cross-linking process that leads to alpha-polymer formation. Radioimmunoassays have been developed, employing the immunoglobulins produced by clones F-102 and F-103. These assays have been applied, in conjunction with high-performance liquid chromatography purified tryptic and chymotryptic derivatives of CNBr VIII and CNBr X, to localize the respective determinants involved in antibody binding. In each case, virtually full immunoreactivity was exhibited by both the CNBr fragment and a single tryptic or chymotryptic peptide originating from it. These findings indicate that sequence-specific, rather than conformational, determinants were operative in the generation of antibodies F-102 and F-103. The epitope recognized by F-102 was localized to the region of A alpha 540-554, while the F-103 binding site resided within A alpha 259-276. When these radioimmunoassays were applied to study the relative immunoreactivity exhibited by a variety of fibrinogen derivatives, the results obtained support earlier suggestions that the COOH-terminal portion of the A alpha chain contains regions of random conformation.

Mixing two monoclonal antibodies yields enhanced affinity for antigen.

We observed that mixing monoclonal antibodies directed against various epitopes of human chorionic gonadotropin (hCG) can increase the sensitivity of antigen-binding assays. Depending on the antibody pair chosen, the affinity of the mixture was as much as 10-fold higher than either of the monoclonal antibodies assayed separately. Because hCG does not have any repeating sequences, these results do not reflect a change in avidity of an individual antibody. The increased avidity of the mixture can be detected in both solid-phase assays and liquid-phase double antibody radioimmunoassays. This was not a general property of all monoclonal antibodies raised against hCG. Certain antibodies, apparently reacting with the same region of the antigen molecule, do not bind simultaneously and do not result in enhanced affinity when mixed. In addition, certain other pairs of antibodies do not bind cooperatively even though they can bind simultaneously. The mechanism for the increase in affinity depends on the formation of a multicomponent complex. If one of the antibodies of a pair that results in enhanced affinity upon mixing is replaced by its F(ab) fragment, the enhancement is no longer detectable, indicating it is unlikely the enhancement is due to an allosteric effect. Although the F(ab')2 fragment shows some enhancement when mixed with another antibody, it is not as effective as the intact antibody.

Potential of primate monoclonal antibodies to substitute for human antibodies: nucleotide sequence of chimpanzee Fab fragments.

The nucleotide sequence of the mRNA that codes for Fab fragments from two chimpanzee monoclonal antibodies has been determined. Both antibodies have high affinity and good specificity for digoxin. Four domains from the two antibodies have been sequenced: the constant domains of the kappa and lambda light chains, the variable domain of the lambda light chain, and the CH1 domain of the IgG1 heavy chain. There are very few differences between the chimpanzee and human sequences; the nucleotide sequences differ by less than 2%.

Characterization of human monoclonal antibodies directed against hepatitis B surface antigen.

Four hybridoma cell lines stably secreting human monoclonal antibodies directed against hepatitis B surface antigen have been isolated. The monoclonal antibodies have been characterized by determining several allotypes, measuring affinity for HBsAg, binding to two HBsAg subtypes, and kinetics of antibody binding to solid adsorbed antigen. In addition, the relative position of the epitopes have been determined by competition in binding to HBsAg. The results indicate that human anti-HBsAg monoclonal antibodies of quite different properties can be isolated. Pharmacokinetics of one antibody have been measured in two rhesus monkeys. This monoclonal antibody, OST 577, has been compared to hyperimmune gamma globulins in a Bureau of Biologics approved radioimmunoassay. It is expected that these antibodies will be useful in preventing and treating hepatitis B.

Genetic alterations in the gene encoding the major HBsAg: DNA and immunological analysis of recurrent HBsAg derived from monoclonal antibody-treated liver transplant patients.

A gene region encoding a segment of the major surface protein, HBsAg, of hepatitis B virus was analyzed from serum samples after orthotopic liver transplantation of three hepatitis B virus chronic carrier patients treated with a human anti-hepatitis B virus monoclonal antibody (SDZ OST 577). Each of these three patients became HBsAg negative after transplantation and therapy with the human anti-hepatitis B virus monoclonal antibody but returned to HBsAg positivity (first detected 143,251 and 252 days after the transplantation). Polymerase chain reaction DNA amplification was performed on DNA from serum samples showing low levels of recurrent HBsAg and reduced antigen reactivity with SDZ OST 577 antibody. Polymerase chain reaction DNA included a 230-bp highly conserved, major S gene region that was cloned into M13 bacteriophage; analysis of this DNA segment provided a consensus of DNA sequences for the serum samples exhibiting altered reactivity with the therapeutic monoclonal. Analysis of independent DNA clones from serum samples of patients exhibiting low but detectable recurrent serum levels of posttherapy HBsAg revealed the presence of S protein variant sequences when compared with polymerase chain reaction DNA derived from the original infected liver or pretherapy serum HBsAg. Genetic variation was predominant in a highly conserved peptide domain that has previously been implicated in antibody binding and neutralizing antibody epitopes. In independent patients infected with either adw or ayw hepatitis B virus subtypes, single nucleotide changes resulted in one to two amino acid differences for each variant allele (residues 124, 129, 131, 137, 140 and/or 145) when compared with pretherapy viral DNA. Administration of serum containing one of these variant viruses to a single hepatitis B-naive chimpanzee resulted in subclinical hepatitis and detectable levels of circulating anti-HBs and anti-HBc antibodies 49 and 70 days after virus administration, respectively. Hepatitis B virus DNA was recovered on liver biopsy between 6 and 8 wk after inoculation, although the animal remained persistently seronegative for HBsAg. DNA sequence analysis of both primate and patient liver hepatitis B virus confirmed the presence of the DNA encoding the S protein variant and associates this DNA with the predominant hepatotropic virus in liver infection.

Human and primate monoclonal antibodies for in vivo therapy.

Human monoclonal antibodies, owing to their decreased immunogenicity, are expected to be an improvement over mouse monoclonal antibodies for in vivo therapy. Human and primate monoclonal antibodies are best produced with a human x mouse heteromyeloma. Several human chromosomes are stable in the human x (human x mouse) hybrids. Chimpanzee anti-digoxin monoclonal antibodies were prepared and characterized. Because they are structurally very similar to human antibodies, they should be well tolerated in humans. The anti-digoxin antibodies can be used for therapy of extreme overdoses or as an in vivo diagnostic tool for slight overdoses. Because the advantage of using human monoclonal antibodies is their lack of immunogenicity, preparation of the antibody must be scrupulous so as not to introduce extraneous immunogens. Analysis to ensure the purity of the preparation can be complicated by the presence of high concentrations of the antibody and the low levels of contamination that must be detected. We describe a Western blot assay for Protein A that is sensitive even in the presence of human IgG.