Biomedical subjects

R L Ceriani

Publications and source records attributed to R L Ceriani.

At least 19 recordsLinked to original sources

PubMed2001

Structural and functional aspects of three major glycoproteins of the human milk fat globule membrane.

The MUC1 mucin, lactadherin, and butyrophilin are 3 major components of the human milk fat globule membrane. The mucin inhibits binding of S-fimbriated Escherichia coli to buccal epithelial cells, and lactadherin prevents symptomatic rotavirus infection in breast-fed infants. Butyrophilin has been suggested to be a structural component of the human milk fat globule (HMFG) membrane and to have receptor functions, but has no known anti-infective activity. These HMFG glycoproteins also are present in skimmed milk, possibly associated with phospholipid micelles, while mucin is also in a soluble form. Mucin and lactadherin resist digestion in the stomach of milk-fed infants, while butyrophilin is rapidly degraded. The MUC1 mucin is an extended rod-like structure forming part of the glycocalyx on the surface of many epithelial cells and membranes of milk, and may act as a decoy for binding of infective agents. The extracellular segment of butyrophilin has homology to Ig superfamily receptors and an intracellular domain with homology to developmentally regulated proteins. Lactadherin is a laterally mobile cell adhesion molecule that interacts with integrins and has a novel means of membrane-association involving specific binding to phosphatidylserine. The structural and functional aspects of these glycoproteins are discussed with regard to their role in human milk for breast-fed infants.

Anti-Infective Agents↗

PubMed1999

Protective function of human milk: the milk fat globule.

Human milk contains many components that protect the newborn against infection at a time when the infant's own defense mechanisms are poorly developed. Fat is one of the major nutrients in human milk. The fat is contained within milk fat globules composed of a core of triglyceride and a membrane consisting of phospholipids, cholesterol, proteins, and glycoproteins. Both the membrane and the core components can provide protection against microorganisms. The major protective membrane glycoproteins, mucin, and lactadherin are resistant to conditions in the newborn's stomach and maintain their structure and function even at low pH and in the presence of the proteolytic enzyme pepsin. The core triglycerides upon hydrolysis by digestive lipases (especially gastric lipase, which is well developed in the newborn) produce free fatty acids and monoglycerides, amphiphylic substances able to lyse enveloped viruses, bacteria, and protozoa. Therefore, in addition to its nutritional value, the fat in human milk has a major protective function.

Anti-Bacterial Agents↗

PubMed1999

Construction and characterization of a chimeric fusion protein consisting of an anti-idiotype antibody mimicking a breast cancer-associated antigen and the cytokine GM-CSF.

Anti-idiotype antibody, 11D10 mimics biologically and antigenically a distinct and specific epitope of the high molecular weight human milk fat globule (HMFG), a cancer-associated antigen present in over 90% of breast tumor samples. To augment the immunogenicity of 11D10 without the aid of a carrier protein or adjuvant, we made a chimeric 11D10-GM-CSF fusion protein for use as a vaccine. An expression plasmid for 11D10 was made by ligation of the DNA sequences of the 11D10 light-chain variable region upstream of the human kappa constant region. The heavy-chain plasmid carrying GM-CSF was made by ligation of the heavy-chain variable region sequences upstream of the human gamma1 constant region CH1 fused to the DNA fragment encoding the mature GM-CSF peptide 3' to the CH3 exon. NS1 plasmacytoma cells were transfected with the light and heavy-chain vectors by electroporation. Fusion protein secreted in the culture medium was purified and was characterized by gel electrophoresis as well as by determination of the biological activity of the fused GM-CSF. In nonreducing SDS-polyacrylamide gels, a single band approximately 200 Kd reacted with anti-human kappa, anti-human lambda1 and anti-GM-CSF antibodies. In reducing polyacrylamide gels, a approximately 74 kd protein reacted with anti-human lambda1 and anti-GM-CSF antibodies. The fusion protein induced proliferation of GM-CSF dependent NFS-60 cells. These results suggest that the protein is a chimeric anti-idiotype antibody consisting of 11D10 variable domains, human kappa and lambda1 constant domains and that the GM-CSF moiety fused to the constant region lambda1 is biologically active.

Adjuvants, Immunologic↗

PubMed1998

Role of human-milk lactadherin in protection against symptomatic rotavirus infection.

BACKGROUND: Human milk contains a 46 kDa mucin-associated glycoprotein, lactadherin, which binds specifically to rotavirus and inhibits its replication. This study tested the hypothesis that lactadherin protects against symptoms of rotavirus infection. METHODS: 200 infants in Mexico City were recruited at birth and monitored by regular stool EIA for rotavirus, serology, and recording of feeding and stool patterns. Milk samples were obtained from the mothers weekly until 4 weeks post partum then monthly. The sample taken immediately before an infant's episode of rotavirus infection was assayed for lactadherin, butyrophilin, mucin, and secretory IgA. An infection was defined as symptomatic if diarrhoea occurred in the 5 days before or after detection of the virus. FINDINGS: 31 infants developed rotavirus infection; 15 were symptomatic and 16 had no symptoms. The median concentration of lactadherin in the milk samples (obtained 4-41 days [median 13] before the infection) was 48.4 (range 5.6-180) microg/mL in the asymptomatic group and 29-2 (6.2-103-4) microg/mL in the symptomatic group. Although these medians did not differ significantly, in logistic regression analysis adjusted for age at infection and secretory IgA concentration there was a significant difference between the groups (p=0O01). No association between symptom status and concentrations of butyrophilin, mucin, or secretory IgA was found. INTERPRETATION: Protection against rotavirus by human milk is associated with the glycoprotein lactadherin. This association is independent of products of the secretory immune system.

Adult↗

PubMed1998

Conjugation of interferon alpha to a humanized monoclonal antibody (HuBrE-3vl) enhances the selective localization and antitumor effects of interferon in breast cancer xenografts.

Human mammary carcinoma xenografts (MCF-7) growing in nude mice were treated with natural interferon alpha (n-IFN-alpha) alone or conjugated to a humanized monoclonal antibody (MoAb) anti-breast mucin (HuBrE-3vl) or to irrelevant human IgG1kappa. The IFN and the conjugates were administered as 20 intra-lesional (i.l.) injections to 1 of 2 xenografts in each mouse, or i.p. The growth inhibitory effects of HuBrE-3vl/nIFN-alpha were significantly greater than those of nIFN-alpha used as a single agent or conjugated to HuIgG1kappa. These effects occurred locally in the tumors receiving i.l. injections and systemically, although to a slightly lesser extent, in the noninjected tumors of mice treated i.l. and in the xenografts of mice treated i.p. Biodistribution studies showed that the uptake of 125I-HuBrE-3vl/nIFN-alpha by the tumors 24 hours after i.l. or s.c. injection was greater than that of 125I-HuIgG1kappa/nIFN-alpha, 125I-nIFN-alpha alone, or by normal tissues, documenting a tumor targeting effect and favorable tumor:normal tissues (T:NT) ratios. The targeting effects and the resulting tumor growth inhibition were favored by the IFN-mediated up-regulation of the HuBrE-3vl reactive antigen, which was more prominent after 3 weeks of treatment with HuBrE-3vl/nIFN-alpha. These results were superior to those we obtained previously with nIFN-alpha conjugated to another MoAb of the same group (Mc5). These studies point out the potential usefulness of HuBrE-3vl/nIFN-alpha for the local and systemic treatment of breast cancer lesions by providing a means of delivering high doses of IFN to the tumors while minimizing the amount of IFN binding to normal tissues.

Animals↗

PubMed1998

Milk fat globule glycoproteins in human milk and in gastric aspirates of mother's milk-fed preterm infants.

Human milk fat globule (HMFG) glycoproteins can prevent infections by microorganisms in breast-fed infants; the MUC-1 mucin inhibits binding of S-fimbriated Escherichia coli to buccal mucosa, and lactadherin may prevent symptomatic rotavirus infections. In this study, the survival of these HMFG glycoproteins in the stomach of human milk-fed preterm infants (gestational age = 27.5 +/- 0.4 wk) was assessed, and levels in their mothers' milk determined, using specific RIAs. Butyrophilin, a major component of HMFG membrane that has no demonstrated antimicrobial activity, was studied for comparison. The levels of mucin, lactadherin, and butyrophilin in 41 milk samples of 20 mothers were 729 +/- 75, 93 +/- 10, and 41 +/- 3 microg/mL, respectively. Mucin and lactadherin were significantly higher in early milk samples (<15 d postpartum) than in later milk samples (15-90 d postpartum), whereas butyrophilin showed no such difference. Significant amounts of mucin and lactadherin were found in almost all gastric aspirates of human milk-fed infants, even 4 h after feeding (mucin, 270 +/- 30 microg/mL; lactadherin, 23.2 +/- 4.4 microg/mL), whereas butyrophilin was rapidly degraded in the majority of aspirates. Western blot analysis demonstrated that the immunoreactive mucin, lactadherin, and butyrophilin in the milk-fed gastric aspirates had the expected native molecular weights. Mucin and lactadherin survived at all gastric pH values, whereas butyrophilin was found only at pH > 4. Neither lactadherin nor butyrophilin were detected in gastric aspirates of formula-fed infants (gestational age = 27.8 +/- 0.5 wk), whereas the very low level of mucin (9.1 +/- 1.1 microg/mL) in this group is presumably cross-reacting gastric mucin. These results demonstrate that two HMFG glycoproteins implicated in prevention of infection, MUC-1 mucin and lactadherin, survive and maintain their integrity in the stomachs of human milk-fed preterm infants.

Anti-Infective Agents↗

PubMed1998

Initial clinical evaluation of radiolabeled MX-DTPA humanized BrE-3 antibody in patients with advanced breast cancer.

To evaluate radiometal-labeled humanized BrE-3 (huBrE-3) monoclonal antibody as a radioimmunolocalization and therapeutic agent in breast cancer patients, tumor localization, pharmacokinetics, radiation dosimetry, and immunogenicity of (111)In-labeled combined 1-p-isothiocyanatobenzyl 3-methyl- and 1-p-isothiocyanatobenzyl 4-methyldiethylenetriamine pentaacetic acid (MX-DTPA) huBrE-3 were studied. Seven women with BrE-3 antigen-positive, metastatic breast carcinoma underwent (111)In huBrE-3 infusion (5 mCi; 50 mg), followed by serial gamma camera imaging and plasma sampling. Region of interest analysis of images was used to make radiation absorbed dose estimates for (111)In huBrE-3. Data were extrapolated to 90Y huBrE-3. Human anti-human antibody (HAHA) response was measured in serum samples obtained up to 3 months after infusion. Patients tolerated infusions well. Seventy-six percent of 105 known sites of disease were identified on planar and single-photon emission computed tomography scans. For six of seven patients, a biexponential model fit the plasma time-activity curve best with an average T1/2alpha=10.6+/-8.5 (SD) h and average T1/2beta=114.2+/-39.2 h. Radiation absorbed dose estimates for (111)In huBrE-3 for whole body averaged 0.53+/-.08 rads/mCi. Dose estimates for 90Y huBrE-3 for marrow averaged 8.4+/-11.9 rads/mCi, and for tumors, 70+/-31.5 rads/mCi. Liver radioactivity uptake averaged 19.7+/-8.8% injected dose at 24 h after infusion, translating into an average radiation absorbed dose 21.1+/-12 rads/90Y mCi administered. Only one of seven patients demonstrated a low level of HAHA response. Although the plasma half-lives are longer and marrow dose higher for radiolabeled huBrE-3 compared with the murine construct, the excellent tumor localization, good tumor dosimetry, and low immunogenicity support the use of 90Y-huBrE-3 antibody for radioimmunotherapy of breast cancer.

Adult↗

PubMed1997

Effect of multiple, repeated doses of radioimmunotherapy on target antigen expression (breast MUC-1 mucin) in breast carcinomas.

The effect of radioimmunotherapy (RIT) on target antigen expression was studied in breast carcinomas transplanted in immunodeficient mice. In nine separate experiments, a single dose of 1500 microCi of 131I-labeled monoclonal antibody (MAb) Mc5 was given to groups of mice carrying well-established, vascularized, transplantable breast tumors (MX-1). Mc5 recognizes an epitope on the tandem repeat of the breast epithelial MUC-1 mucin. This dose suppressed tumor growth for at least 20 days, after which the tumors began to regrow. At various times thereafter, tumors were removed and analyzed for target antigen expression by flow cytometry and immunohistochemistry. In no case was there any significant decrease in antigen content/cell in the tumors of treated mice compared to tumors in control untreated mice. Similar results were obtained with four other breast carcinomas (MCF-7, MDA-MB-331, MDA-MB-435, and MX-2A). To assess the effect of repeated RIT doses on target antigen expression, groups of mice with MX-1 tumors were given 2, 3, and 4 consecutive doses of 1200 microCi of 131I-labeled Mc5. One mouse each at 2, 3, and 4 doses (3 of 18) was cured of its tumor. Control mice were sacrificed after 50 days due to the excessive size of their tumors. Tumors from four mice from each group (2, 3, and 4 doses), after they began to regrow, were excised and analyzed for mucin content and compared to tumors from untreated mice with similar-size tumors transplanted at later dates. In none of the treated groups was there any decrease in mucin content. These results demonstrate that RIT with an anti-breast mucin MAb does not result in the appearance of antigen-negative tumor cells, thus indicating that repeated fractionated doses, which will most likely be necessary for an eventual cure of breast cancer with MAb therapy, are possible.

Animals↗

PubMed1997

Lactadherin (formerly BA46), a membrane-associated glycoprotein expressed in human milk and breast carcinomas, promotes Arg-Gly-Asp (RGD)-dependent cell adhesion.

Lactadherin, a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and expressed in human breast carcinomas. Previously, we have shown that the mature protein, formerly known as BA46, has three domains: an epidermal growth factor (EGF)-like domain containing an Arg-Gly-Asp (RGD) cell adhesion sequence and C1 and C2 domains similar to those found in coagulation factors V and VIII. An alignment of lactadherin with its bovine (MGP57/53) and murine (MFG-E8) homologs shows that the RGD sequence has been conserved during evolution, suggesting that the RGD sequence is not fortuitous. We demonstrate that lactadherin purified using Triton X-114 phase partitioning promotes RGD-dependent cell attachment of green monkey kidney cells (MA104), mouse fibroblast cells (3T3-L1), and breast carcinoma cells (ELL-G). A lactadherin-specific monoclonal antibody, Mc3, inhibits attachment to purified lactadherin, suggesting that contaminants in the purification are not responsible for binding. In addition, the anti-integrin alpha(v)beta3 monoclonal antibody LM609 inhibits cell attachment of MA104 cells to lactadherin. These results demonstrate that lactadherin promotes RGD-dependent cell adhesion via integrins. Denaturation of lactadherin with heat and reducing conditions diminished cell attachment, suggesting that optimal cell attachment to RGD is dependent on the structural presentation of the sequence.

3T3 Cells↗

PubMed1997

Radioimmunotherapy for breast cancer using indium-111/yttrium-90 BrE-3: results of a phase I clinical trial.

UNLABELLED: BrE-3 is a murine IgG1 monoclonal antibody that binds to 97% of human ductal breast cancer specimens. A previous study documented the ability of 111In-labeled 1,4-methyl-benzyl isothiocyanate diethylenetriamine pentaacetic acid (111In-MX-DTPA) BrE-3 to specifically target breast cancer tissue in patients, and the dosimetry derived from the pharmacokinetics suggested that a useful therapeutic index could be obtained with 90Y-MX-DTPA BrE-3. A Phase I maximum tolerated dose study was, therefore, initiated. METHODS: Six patients received 111In/90Y-MX-DTPA BrE-3, three of them receiving 6.25 and the other three receiving 9.25 mCi/m2 of 90Y. Pharmacokinetics, dosimetry, human anti-mouse antibody (HAMA), toxicity and clinical responses were evaluated. RESULTS: Three of six patients demonstrated a minor and transient, but objective tumor response, and none of the patients had significant toxicity. Tumor dosimetry ranged from 39 to 167 rad/mCi of 90Y (442-1887 rad/ dose). HAMA response occurred in five of six patients. CONCLUSION: Minimal toxicity, dosimetric calculations and clinical assessment indicate that a useful therapeutic index can be achieved with this therapy. Indium-111/yttrium-90-MX-DTPA BrE-3 can be safely administered to patients with metastatic breast cancer, and therapy doses yielded pharmacokinetics similar to those of tracer doses. Clinical responses, albeit transient, were achieved with single-dose therapy. Rapid onset of the HAMA response will hinder multicycle therapy, unless it is prevented with immunosuppressive drugs or the use of a "humanized" antibody. Further studies are needed to determine the optimal use of BrE-3 for radioimmunotherapy.

Breast Neoplasms↗

PubMed1996

Cloning and sequence analysis of human butyrophilin reveals a potential receptor function.

Human butyrophilin was cloned and sequenced from a human breast cDNA library. The derived amino acid sequence shows 84% sequence identity and identical domain arrangements with the previously reported bovine sequence. Sequence analysis reveals an immunoglobulin constant (IgC) domain that was not previously identified in the bovine sequence. The extracellular domain composition of butyrophilin suggests a cell surface receptor function.

Amino Acid Sequence↗

PubMed1996

Inverted Fab2s (IFab2s): engineering and expression of novel, dimeric molecules, with a molecular weight of 100 000.

The IFab2 molecule described is expressed by a single gene construct that encodes the VH-CH1 and the Vk-Ck modules of a humanized version of the anti-breast cancer antibody BrE-3, joined via a short peptide linker. It can be shown that this basic genetic construct can lead to the expression of monomers, dimers (IFab2s) and other multimer species, depending on the length of the linker. A 21-residue linker promotes the principal formation of the desired dimer IFab2 molecules. It was also shown that in binding competition assays, purified IFab2 completely retained the affinity and specificity of the original monoclonal antibody.

Amino Acid Sequence↗

PubMed1996

Cloning and sequence analysis of human breast epithelial antigen BA46 reveals an RGD cell adhesion sequence presented on an epidermal growth factor-like domain.

The BA46 antigen of the human milk fat globule (HMFG) membrane is expressed in human breast carcinomas and has been used successfully as a target for experimental breast cancer radioimmunotherapy. To characterize this antigen further, we obtained the entire cDNA sequence and focused on its possible role in cell adhesion. The derived protein sequence of BA46 encodes a 387-residue precursor composed of a putative signal peptide, an amino-terminal epidermal growth factor (EGF)-like domain containing the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD), and human factor V and factor VIII C1/C2-like domains. The EGF-like domain of BA46 is similar to the calcium-binding EGF-like domains of several coagulation factors, but the BA46 domain lacks a residue required for calcium binding and the coagulation factor domains do not include an RGD sequence. Assuming that all EGF-like domains fold into a similar structure, the RGD-containing sequence in BA46 is inserted between two antiparallel beta strands. This positioning suggests a novel function for the EGF-like domain as a scaffold for RGD presentation.

Amino Acid Sequence↗

PubMed1996

Cloning and expression of cDNAs encoding the variable domains of the antibreast carcinoma antibody Mc5.

Mc5, a murine monoclonal antibody that binds to human breast epithelial mucin (BEM), has been shown to be a promising reagent in the diagnosis of breast cancer. We have cloned cDNAs encoding both variable regions of Mc5 (VL and VH) as well as the CL and CH1 constant regions. Mc5 is an IgG1, kappa antibody. We have constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine VH and VL-encoding cDNAs into plasmids encoding human constant domains), and expressed it in SP2/0-Ag14 mouse myeloma cells. The affinity of chimeric Mc5 (chMc5) for BEM is 4.4 x 10(8) M-1. Mc5 binds BEM with an affinity constant of 2.8 x 10(8) M-1. Purified chMc5 and purified Mc5 gave similar competition curves when tested against either 125I-labeled Mc5 or 125I-labeled chMc5 for binding to BEM in a competition radioimmunodetection format. Additionally, chMc5 used in breast carcinoma tissue staining stained as well as the original Mc5.

Amino Acid Sequence↗

PubMed1995

Selection of tumor-specific epitopes on target antigens for radioimmunotherapy of breast cancer.

Evidence is presented for two different breast epithelial antigens that some epitopes have greater tumor specificity and are more effective targets for radioimmunotherapy than others. The two antigens, which are major components of the human milk fat globule membrane, are breast mucin and a M(r) 46,000 glycoprotein (BA46). Of five monoclonal antibodies (Mc5, Mc1, BrE-1, BrE-2, and BrE-3) against breast mucin, all recognize overlapping amino acid epitopes on the tandem repeat domain. However, each have unique and different tissue and tumor specificities and unique epitope structures on the fully glycosylated breast mucin. In preclinical studies, radioimmunoconjugates of all five monoclonal antibodies inhibit growth of transplantable breast tumors in immunodeficient mice. In human clinical trials, radioiodinated Mc5 was very poor in localizing breast tumor metastases. On the other hand, 111In-labeled BrE-3 imaged almost 90% of breast tumors and showed promise in radioimmunotherapy when labeled with 90Y. The failure of Mc5 in clinical trials may be partly attributed to the high levels of its epitope on circulating mucin compared to the epitope of BrE-3. The Mc5 binding affinity increased significantly with glycosylation, while the BrE-3 epitope was masked by glycosylation. The BA46 glycoprotein is a breast tumor-associated membrane antigen containing an NH2-terminal, epidermal growth factor-like domain into which a cell adhesion sequence (RGD) is inserted and a COOH-terminal domain with homology to the phospholipid binding C1/C2 domain of coagulation factors V and VIII. It promotes cell attachment in an RGD-dependent manner. Monoclonal antibody Mc8, which binds to the C2-like domain, is only moderately effective in experimental radioimmunotherapy, while Mc3, which binds an epitope in the EGF-like RGD domain, was highly effective in destroying breast tumors in nude mice. With 90Y-labeled Mc3, 6 of 7 mice are cured of the tumors. These results indicate that by selecting appropriate monoclonal antibodies, a normal antigen can be used as a target for radioimmunotherapy.

3T3 Cells↗

PubMed1995

Biological activity of two humanized antibodies against two different breast cancer antigens and comparison to their original murine forms.

We have humanized two monoclonal antibodies (MoAbs), hu-BrE-3 and hu-Mc3, that are bound to two different antigens of the breast epithelial cell. They bind to the breast epithelial mucin (M(r) 400,000) and the BA46 antigen (M(r) 46,000). They could participate in a joint radioimmunotherapy strategy administering repeated or fractionated dosages, where increased irradiation could be delivered by their simultaneous administration. Both antibodies, hu-BrE-3 and hu-Mc3, had similar reactivity to their antigens and similar binding affinity as those of their original murine forms. However, because humanized MoAbs could have different pharmacokinetic and radioimmunotherapeutic characteristics than their original murine forms, the experimental biodistribution in vivo of both of these two humanized anti-breast tumor MoAbs was compared to their original murine forms. Biodistributions in immunodeficient mice grafted with transplantable human breast tumors, both after radioiodination and 111In labeling via 1-p-isothiocyanatobenzyl-methyl-diethylene-triaminepenta-ace tic acid (MXDTPA), demonstrated comparable tumor:normal tissue ratios for the humanized and murine forms. In radioimmunotherapy, the humanized forms for both MoAbs showed also similar tumoricidal activity as that of the original murine MoAbs. These results show that the new humanized forms are amenable to conjugation and radioisotope labeling without loss of biological activity. Furthermore, they demonstrate that these engineered molecules kept intact, both qualitatively and quantitatively, their binding ability, pharmacokinetics, and radioimmunotherapeutic characteristics after the humanization process.

Animals↗

PubMed1995

Designing human consensus antibodies with minimal positional templates.

A humanized antibody retains from the original murine antibody the variable region amino acid residues that are required for antigen binding. These generally include the grafted complementarity determining regions, as well as a few key framework residues. Although the remainder of the framework sequences are imported from a human antibody, they nevertheless differ at a few positions from the human consensus sequences. These atypical residues, which arose by somatic mutation during the affinity maturation of the chosen human antibody, could elicit an immune response in some of the patients receiving the humanized antibody. Thus, ideally one should, instead, choose human consensus frameworks for humanizing murine antibodies. Because there is a different consensus sequence for each of the subclasses of variable light and heavy chains, a method is needed to choose the most appropriate one. We are developing a minimal positional template for such a purpose. A minimal positional template indicates which positions in the variable region frameworks are absolutely required for maintaining the integrity of the binding domains. Therefore, to choose a human framework for humanization, one screens the available human consensus sequences for those that are most similar to the original murine sequence, specifically at the positions indicated by the template. In the subsequent humanization protocol, one then retains all of the murine residues found in the positions indicated in the template while humanizing the residues found at all other positions. A conservative positional template has been applied to the humanizations of the antibreast epithelial mucin antibodies BrE-3 and KC4-G3 without loss of binding affinity. Now we are using progressive cycles of computer modeling and laboratory testing to develop a minimal template. The first of such cycles produced template B, which has been used successfully in the humanization of the antibreast epithelial antigen BA46 antibody Mc3. This prompted us to design template C, which further approaches the desired minimal template. Future constructs will test the validity of this template as well as the validity of this novel humanization approach.

Amino Acid Sequence↗

PubMed1995

Anti-BA46 monoclonal antibody Mc3: humanization using a novel positional consensus and in vivo and in vitro characterization.

Mc3 is a murine mAb that is highly effective in treating breast tumors in experimental radioimmunotherapy. Mc3 binds to BA46, a 46-kDa glycoprotein of the human milk fat globule membrane that is also expressed in breast carcinoma cells. We cloned and sequenced cDNAs encoding the variable regions of Mc3 and constructed an IgG1, kappa human/mouse chimeric antibody. We then humanized the variable regions of Mc3 using a positional consensus method and retaining residues that might either contact the complementarity-determining regions or the opposite chain. This positional consensus is novel in that it does not include residues with buried side chains. Humanized Mc3 retained full binding affinity, and fully competes with murine Mc3 for antigen binding. Humanized and murine 131I-labeled Mc3 behaved identically in athymic nu/nu mice biodistribution studies. The tumor uptake levels for both antibodies increased over a period of 4 days within a range of 13-20% of the injected dose per g with extremely favorable tumor:normal ratios. Also, a single therapeutic dose of 131I-labeled humanized Mc3 in the same animal model reduced the average tumor size and produced one of five cures while in the uninjected control tumor growth continued unabated. We believe that these results justify the implementation of Phase I human clinical trials for imaging and radioimmunotherapy of breast cancer.

Amino Acid Sequence↗