Self-reactive antibody expression by human carcinoma cells engineered with monoclonal antibody genes.

The purpose of this study was to determine if human colon cancer cells transduced with monoclonal antibody (MAb) genes become sensitive to immune destruction through coexpression of both the MAb and its reactive antigen. Murine retroviral expression vectors were constructed with the heavy or light chain genes of an anti-human colon carcinoma MAb, D612, that mediates antibody-dependent cell-mediated cytotoxicity (ADCC). Transduction of D612 MAb genes into the D612 antigen-positive (> 95%) human colon carcinoma cell line, LS-174T, was carried out by sequential cocultivation with PA317 packaging cells producing infectious virions containing the light or heavy chain expression vectors. Six cultures survived drug selection, two of which were found to have elevated levels of both light and heavy immunoglobulin chain activity in their supernatants. IgG secretion levels (24 h) were 1-2 ng/1 x 10(6) cells. Low but definite antigen reactivity was also present in supernatants obtained from these LS-174T transductants. Immunocytochemical staining of transduced tumor cells revealed that > 95% of the cells were positive for IgG expression. Thus, LS-174T transductants were capable of producing both the D612 MAb and D612-reactive antigen. Analysis of transductants by flow cytometry further revealed that > 95% of the cells had murine immunoglobulin on their surfaces. ADCC mediated by human natural killer cells against nontransduced tumor cells was observed when the latter cells were co-cultivated in the presence of transductants producing both D612 heavy and light chains but not in the presence of tumor cells transduced with light chain only. LS-174T cells transduced with both D612 heavy and light chain genes were more sensitive to cytotoxicity mediated by natural killer cells than were light chain gene only transductants. ADCC contributed to the greater sensitivity of the former transductants to cytotoxicity based on its inhibition by anti-FcR gamma III antibody. Thus, these studies demonstrate that tumor cells transduced with genes encoding for MAbs that can participate in ADCC reactions are able to sensitize nontransduced tumor cells to immune destruction as well as to direct killer cells against themselves. These studies may lead to a new immunotherapeutic approach for the treatment of cancer based on MAb gene therapy.

Enhanced expression of the c-myc gene in bovine leukemia virus-induced bovine tumors.

We have detected elevated levels of c-myc gene expression in neoplastic cells from all seven bovine leukemia virus (BLV)-induced bovine tumors examined, but not in BLV-infected, nonneoplastic lymphoid cells. No rearrangement or amplification of the c-myc gene could be demonstrated in any of the BLV-induced tumors. Furthermore, BLV proviral DNA was found to have no preferred site of integration in these tumors. The possible mechanisms of enhanced expression of the c-myc gene in BLV-induced tumors have been discussed.

Correlation of DNA hypomethylation with expression of carcinoembryonic antigen in human colon carcinoma cells.

Using an assay based on the binding of a carcinoembryonic antigen (CEA)-specific monoclonal antibody, we have examined the expression of carcinoembryonic antigen genes in human colon tumor and normal fibroblast cell lines. CEA expression was not detectable in the normal fibroblast cell lines, whereas varying levels of high CEA expression were found in the colon tumor cell lines LS-174T, GEO, and WIDR. We have used a 550-base pair CEA probe derived from cloned complementary DNA to carry out Southern analysis of the DNA isolated from the normal and colon tumor cell lines. At high stringency, the CEA probe detected seven BamHI fragments in all DNAs analyzed. At low stringency, however, 14 BamHI fragments ranging from 1.5 to 23 kilobases were detected. Results of the Southern analysis demonstrate no amplification or rearrangement of the CEA genes in tumor cells. We used methylation-sensitive restriction endonucleases, HpaII and HhaI, to compare the degree of methylation of CEA family of genes in normal and colon tumor cell lines. Our results demonstrate that the CEA family of genes exists in a state of hypermethylation in the normal cell lines. In contrast, the CEA gene(s) are relatively hypomethylated in the tumor cell lines, suggesting a correlation between the state of methylation and degree of expression of the CEA gene(s). A comparison of the state of methylation of the CEA gene(s) in cells before and after treatment with the gamma-interferon (which up-regulates CEA steady-state mRNA levels) showed no detectable difference in the degree of DNA methylation. The segments of CEA genes that are hypermethylated in normal cells, but are hypomethylated in tumor cells, were also identified. Thus, these studies may help identify the sites of methylation that are crucial for the control of CEA gene regulation.

Generation and characterization of a recombinant/chimeric B72.3 (human gamma 1).

We report here the generation and characterization of a recombinant/chimeric construct of murine gamma 1 monoclonal antibody (MAb) B72.3, containing the murine variable region and a human gamma 1 constant region [designated cB72.3(gamma i)]. cB72.3(gamma 1) was generated by first isolating functionally rearranged VH and VL genes of B72.3 from partial genomic libraries in phage vectors. Construction of mouse-human chimeric heavy and light chain genes was performed by inserting restriction fragments carrying VL and VH regions of B72.3 into unique sites of expression vectors which contains sequences encoding constant regions of human kappa and gamma 1, respectively. The expression constructs were subsequently electroporated into SP2/0 cells. The transfected SP2/0 murine cell line has been shown to synthesize cB72.3(gamma 1) at a level of 10-20 micrograms/ml. Reciprocal competition radioimmunoassays demonstrated that cB72.3(gamma 1), a previously described cB72.3(gamma 4), and native B72.3 (designated nB72.3) competed similarly. A rat anti-idiotype MAb made against nB72.3 was shown to bind equally well to cB72.3(gamma 1) and to the nB72.3. Immunochemical studies of the nB72.3, cB72.3(gamma 4), and cB72.3(gamma 1) revealed slight differences in size among the three MAb forms on sodium dodecyl sulfate gels and revealed a higher isoelectric point for the cB72.3(gamma 1). Antibody-dependent cell-mediated cytotoxicity experiments using human lymphokine-activated killer effector cells indicated better tumor cell killing by the cB72.3(gamma 1) than the nB72.3 or cB72.3(gamma 4). Dual label studies of coinjected cB72.3(gamma 1) and nB72.3 revealed that both MAbs could efficiently localize human tumor xenografts in athymic mice. Pharmacokinetic studies, analyzing the blood clearance of cB72.3(gamma 1), cB72.3(gamma 4), and nB72.3 in mice, showed that the nB72.3 beta phase of clearance was slower than that of other MAb forms. However, when the pharmacokinetic patterns of these three MAbs forms were analyzed in monkeys, the cB72.3(gamma 1) and the nB72.3 showed similar clearance curves, while the cB72.3(gamma 4) showed a much slower plasma clearance. In view of the binding properties of nB72.3 and its ability to localize a range of carcinomas in clinical trials, the studies reported here demonstrate that the cB72.3(gamma 1) may serve as a potentially useful diagnostic and/or therapeutic reagent.

Biological properties of chimeric domain-deleted anticarcinoma immunoglobulins.

CC49 is a second-generation monoclonal antibody (MAb) that has high affinity for the tumor-associated pancarcinoma antigen tumor-associated glycoprotein-72. In clinical trials using gamma scanning, radiolabeled CC49 has facilitated the detection of more than 90% of carcinomas. We report here the development of a constant heavy-chain 2 (CH2) domain-deleted chimeric (c) CC49 MAb by transfecting an expression construct consisting of the CC49 murine variable region and a CH2 domain-deleted human IgG1 constant region into cCC49 kappa producing SP2/0 murine myeloma cells. As determined by SDS-PAGE, the intact cCC49 delta CH2 has a molecular weight of 153,000 and, under reducing conditions, molecular weights of 43,000 and 27,000. The plasma clearance and tumor-targeting properties of cCC49 delta CH2 were evaluated and compared with those of mouse/human chimeric forms cCC49 delta CH1 and intact cCC49. Previous studies have shown that the in vitro antigen-binding properties of cCC49 delta CH1 are similar to those of cCC49. Biodistribution studies reported here, using 131I-labeled cCC49 delta CH1 and 125I-labeled cCC49 in athymic mice bearing human colon carcinoma xenografts, demonstrated that both cMAbs localized to the tumor and cleared from the normal tissues similarly. However, in comparison with 125I-labeled cCC49, 131I-labeled cCC49 delta CH2 localized to tumors earlier and had a significantly lower percentage of the injected dose of cMAb/g (%ID/g) in normal tissues than cCC49. Immunoscintigraphy of 131I-labeled cCC49 delta CH2 and 125I-labeled cCC49 in athymic mice bearing human tumor xenografts demonstrated a clear image of the tumor by 24 h after i.v. administration of the delta CH2 cMAb versus the 72 h required for cCC49. Biodistribution studies using 177Lu-conjugated cCC49 delta CH1 and cCC49 showed no significant difference between the radiolocalization indices (% ID/g in tumor divided by % ID/g in normal tissue). 177Lu-conjugated cCC49 delta CH2, however, had lower % ID/g values in tumor xenografts and lower radiolocalization indices than either 177Lu-conjugated cCC49 delta CH1 or 177Lu-conjugated cCC49. Pharmacokinetic studies in non-tumor-bearing athymic mice using cCC49 delta CH1 and cCC49 revealed no significant difference between these cMAbs. However, the plasma clearance of cCC49 delta CH2 in non-tumor-bearing mice was significantly faster than that of cCC49. These results were similar when the cMAbs were labeled with either iodine or lutetium. In nonhuman primates, 131I-labeled cCC49 delta CH2 cleared significantly faster than 125I-labeled cCC49. The similar plasma clearance and tumor localization of cCC49 and cCC49 delta CH1 suggest that these two cMAbs may be used in similar clinical settings. However, because of the unique pharmacokinetics and tumor targeting of cCC49 delta CH2 versus cCC49 or cCC49 delta CH1, this chimeric immunoglobulin form may be useful in clinical settings that require efficient tumor targeting and rapid serum and whole-body clearance.

No involvement of bovine leukemia virus in childhood acute lymphoblastic leukemia and non-Hodgkin's lymphoma.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine lymphosarcoma. Much speculation continues to be directed at the role of BLV in human leukemia. To test this hypothesis rigorously, a case-control study of childhood acute lymphoblastic leukemia and non-Hodgkin's lymphoma was conducted between December 1983 and February 1986. Cases (less than or equal to 16 years at diagnosis) derived from patients diagnosed at the primary institutions and affiliated hospitals were matched (age, sex, and race) with regional population controls. DNA samples from bone marrow or peripheral blood from 157 cases (131 acute lymphoblastic leukemia, 26 non-Hodgkin's lymphoma) and peripheral blood from 136 controls were analyzed by Southern blot technique, under highly stringent conditions, using cloned BLV DNA as a probe. None of the 157 case or 136 control DNA samples hybridized with the probe. The high statistical power and specificity of this study provide the best evidence to date that genomic integration of BLV is not a factor in childhood acute lymphoblastic leukemia/non-Hodgkin's lymphoma.

Detection of blood-borne cells in colorectal cancer patients by nested reverse transcription-polymerase chain reaction for carcinoembryonic antigen messenger RNA: longitudinal analyses and demonstration of its potential importance as an adjunct to multiple serum markers.

The use of reverse transcription-PCR (RT-PCR) to analyze cells in the blood of cancer patients for the detection of mRNA expressed in tumor cells has implications for both the prognosis and the monitoring of cancer patients for the efficacy of established or experimental therapies. Carcinoembryonic antigen (CEA) is expressed on approximately 95% of colorectal, gastric, and pancreatic tumors, and on the majority of breast, non-small cell lung, and head and neck carcinomas. CEA shed in serum is useful as a marker in only approximately 50% of colorectal cancer patients and rarely is shed by some other carcinoma types. RT-PCR has been used previously to detect CEA mRNA in cells in the blood and lymph nodes of cancer patients. Under the assay conditions validated in the studies reported here, 34 of 51 (67%) patients with different stages of colorectal cancer had blood cells that were positive by RT-PCR for CEA mRNA, whereas none of 18 patients with colonic polyps were positive; 2 of 60 apparently healthy individuals (who were age and sex matched with the carcinoma patients and were part of a colon cancer screening program as controls) were marginally positive. The results of CEA PCR in the blood of the carcinoma patients and the other groups showed strong statistical correlation with the disease (P2 < 0.0001). Analyses were carried out to detect both serum CEA protein levels and CEA mRNA in blood cells of colorectal carcinoma patients by RT-PCR. For all stages of disease, 18 of 51 patients (35%) were positive for serum CEA, whereas 35 of 51 (69%) were positive by RT-PCR. More importantly, only 5 of 23 (20%) of stage B and C colorectal cancer patients were positive for serum CEA, whereas 16 of 23 (70%) were positive by RT-PCR. The use of two other serum markers (CA19.9 and CA72-4) for colorectal cancer in combination with serum CEA scored two additional patients as positive; both were positive by RT-PCR for CEA mRNA. Pilot long-term longitudinal studies conducted before and after surgery identified some patients with CEA mRNA in blood cells that were negative for all serum markers, who eventually developed clinical metastatic disease. The studies reported here are the first to correlate RT-PCR results for CEA mRNA in blood cells with one or more serum markers for patients with different stages of colorectal cancer, and are the first long-term longitudinal studies to use RT-PCR to detect CEA mRNA in blood cells of cancer patients. Larger cohorts will be required in future studies to define the impact, if any, of this technology on prognosis and/or disease monitoring.

Evidence of tandem duplication of genes in a merodiploid region of Pneumococcal mutants resistant to sulfonamide.

A Pneumococcal mutant, sulr-c, resistant to sulfonamides, and three transformants bearing associated d or d+ resistance markers have earlier been reported to be unstable and show distinct patterns and frequencies of segregating stable progeny lacking the c marker. Each of the four strains showed a characteristic dosage of the genes involved in the merodiploidy. Complementary strands of DNA's from these stable and unstable strains were resolved and homoduplex and heteroduplex hybrids made from the separated DNA strands were used as donors in genetic transformations. Activities of a normal marker (streptomycin resistance) and those involved in the heterozygosity (c, d and d+) were quantitatively measured. From those heteroduplexes made up of opposite strands derived from a heterozygote and a stable strain, the normal marker is transferred efficiently, but the heterozygous markers are not. On the other hand, if both strands of a heteroduplex are derived from different heterozygotic strains, all markers can be transferred with usual efficiency to a stable recipient strain. The lowered efficiency in the former type of heteroduplex is attributed to an inhomology resulting from a tandem duplication in the merodiploid strains, and a postulated DNA repair process stimulated by it while in the form of the donor duplex. The inhomology probably includes (a) a microheterogeneity between the c site and the wild type locus, and (b) a more extensive incompatibility attributable to an extra segment of genome in a tandem duplication covering the c and d sites. The first of these inhomologies produces a lowered efficiency of transfer from all configurations of the particular d allele associated with the mutant c marker, and therefore accounts for the characteristic transfer patterns even from the native merodiploid DNA's.

A study of Pneumococcal merodiploids at the molecular level.

The DNA of a sulfonamide-resistant Pneumococcal strain (heterozygous for sulr-c) and that of three highly resistant and persistently heterozygous cd transformants, derived by introducing sulr-c marker into a stable sulfonamide resistant strain (sulr-d), were studied to analyze the genetic basis of their merodiploidy. The physical properties of the native and denatured DNA from the heterozygotes and the nonheterozygous strains were not distinguishable. The denaturability and the renaturability of biological activity for the heterozygous markers were essentially identical to those of the normal markers. The heterozygosity extends to the closely linked locus giving rise to four different configurations of cd and cd+ transformants, characterized by their frequencies of segregation and donor-marker activities. The marker-activity ratios and the frequency of co-transfer of heterozygous markers were found to remain the same in each when the donor DNA was native, denatured or reannealed without fractionation or reannealed after remixing of resolved strands. Possible models were weighed against these observations and these considerations led to the suggestion that tandem duplication of a gene region may be responsible for the heterozygosity and instability of this region. A more detailed examination of this model will be presented in an accompanying paper.

Generation and characterization of a single gene-encoded single-chain-tetravalent antitumor antibody.

Monoclonal antibody (mAb) CC49, a murine IgG1, reacts with the tumor-associated glycoprotein-72 expressed on a variety of carcinomas. In clinical trials, radiolabeled CC49 has shown excellent tumor localization to a variety of carcinomas. To minimize the immunogenicity of CC49 mAb in patients, a humanized CC49 (HuCC49) was generated by complementarity-determining region (CDR) grafting. The relative affinity of HuCC49 was 2-3-fold less than that of the murine mAb. With the aim of improving tumor targeting, attempts have been made to enhance the avidity of the HuCC49 mAb. Previous research has yielded a single gene-encoded immunoglobulin, SCIgcCC49deltaCH1, which is a dimer of a single chain consisting of CC49 single-chain Fv linked to the NH2 terminus of the human gamma1 Fc through the hinge region. This molecule is comparable to the mouse-human chimeric CC49 in terms of in vitro antigen binding properties, cytolytic activity, and rate of plasma clearance in athymic mice bearing human tumor xenografts. Recently, a single gene encoding a single-chain immunoglobulin consisting of a HuCC49 diabody attached to human gamma1 Fc via the hinge region was constructed. The diabody, a bivalent antigen-binding structure, is made up of variable heavy (V(H))/variable light (V(L)) domains and V(L)/V(H) domains. In each of the variable domain pairs, the V(H) and V(L) domains are linked through a short linker peptide. Meanwhile, the two pairs are linked via a 30-residue Gly-Ser linker peptide to yield two antigen-binding sites by lateral and noncovalent association of the V(L) of one pair with the V(H) of the other. Transfectomas expressing the single-gene immunoglobulin secrete a homodimer of about Mr 160,000 that reacts to tumor-associated glycoprotein-72. This tetravalent humanized antitumor immunoglobulin molecule may potentially be an efficacious therapeutic and diagnostic reagent against a wide range of human carcinomas.

Targeting strategies for cancer radiotherapy.

Novel strategies to increase the therapeutic ratio in clinical radioimmunotherapy studies are needed. Limitations to radioimmunotherapy include bone marrow suppression due to the long circulating half-life of radiolabeled monoclonal antibodies (mAbs) and heterogeneous tumor penetration of the high-molecular-weight mAb. An approach to overcome these problems is the use of genetically engineered mAbs. The engineered mAb discussed in this paper contains a deletion in the constant region of the mAb that increases its tumor penetration and blood clearance compared with the intact mAb. Radiolabeling of this mAb should lead to a similar radiation-absorbed dose to tumor compared with the intact mAb, but reduce the radiation absorbed dose to bone marrow. In addition, low or variable expression of tumor-associated target antigens or receptors may lead to low or heterogeneous tumor uptake of radiolabeled mAbs. This report also discusses a novel approach toward systemic radiotherapy that combines gene transfer techniques (to increase tumor receptor expression) with radiolabeled peptides that target the induced receptor. The radiolabeled peptides achieve good tumor uptake, rapid tumor penetration, and rapid blood clearance. A humanized construct of the CC49 (HuCC49) high-affinity anti-TAG-72 mAb, as well as a construct with the CH2 region deleted (HuCC49deltaCH2), were labeled with 131I and 177Lu. Biodistribution of the radiolabeled constructs was evaluated 24 h after regional i.p. injection in athymic nude mice bearing i.p. LS174T human colon cancer xenografts. The 131I-HuCC49deltaCH2 showed a median tumor uptake of 5.5% ID/g which was similar to that of 131I-HuCC49 at 5.2% ID/g. However, the median blood concentration of 131I-HuCC49deltaCH2 was 0.2% ID/g which was significantly lower than 0.8% ID/g for 1311-HuCC49. The uptake of the constructs in other normal tissues were similar. The 177Lu-HuCC49deltaCH2 showed a median tumor uptake of 9.4% ID/g, which was slightly higher than that of 177Lu-HuCC49 at 7.9% ID/g. The median blood concentration of 177Lu-HuCC49deltaCH2 was 0.2% ID/g, which was significantly lower than 0.4% ID/g for 177Lu-HuCC49. The uptake of the antibody constructs in other normal tissues were similar except for the kidney. The tumor:blood ratios of 177Lu-HuCC49 and 177Lu-HuCC49deltaCH2 were 19.4 and 60.2, respectively, at 24 h after injection. The purpose of the second aspect of the study was to determine the biodistribution of 64Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA)-octreotide in a human ovarian cancer model induced to express human somatostatin receptor subtype 2 (SSTr2) using gene transfer techniques as a prelude to future therapy studies. Mice bearing i.p. SKOV3.ip1 tumors transduced with an adenoviral vector encoding the cDNA for SSTr2 (AdSSTr2) and injected i.p. with 64Cu-TETA-octreotide showed a median uptake of 24.3% ID/g in tumor at 4 h postinjection compared with 4.9% ID/g at 18 h after injection. Also, tumor uptake of 64Cu-TETA-octreotide at 4 h was not significantly different when administered either 2 or 4 days after injection of AdSSTr2 (P = 0.076). 64Cu-TETA-octreotide should be useful for targeted radiotherapy against tumors that are genetically induced to express high levels of SSTr. These two novel targeting strategies show promise for improved cancer radioimmunotherapy.

Serological and biochemical characterization of recombinant baculovirus carcinoembryonic antigen.

Carcinoembryonic antigen (CEA), a glycosylated protein of M(r) 180 kDa, is one of the most widely used human tumor markers. A majority of gastrointestinal cancers as well as breast and nonsmall cell lung carcinomas express CEA. We have previously described a recombinant baculovirus BVCEA-140 expressing the full-length human CEA and a variant, BVCEA-16, that encodes only the NH2-terminal domain, as well as a recombinant (BVNCA) expressing the closely related molecule nonspecific cross-reactive antigen (NCA). We have now compared a panel of 24 anti-CEA and anti-NCA monoclonal antibodies (MAbs) for their ability to bind to these recombinant CEA and NCA proteins, as well as with a new 60 kDa subgenomic form designated BVCEA-60. The epitope mapping studies indicate that all the CEA specific MAbs can recognize BVCEA-140. We also compared the sugar composition of BVCEA-140 to native CEA, using a lectin-linked immunoradiometric assay. The results demonstrated that both the native and recombinant baculovirus CEA contain simple high-mannose carbohydrates as well as biantennary and biantennary hybrid complexes. However, native CEA also contains triantennary and tetraantennary complex sugars, while the recombinant CEA molecule does not. Immunogenicity of the recombinant CEA molecules was demonstrated in mice. ELISA and Western blot analyses were used to determine the cross-reactivity of the anti-CEA sera. Mice immunized with BVCEA-140 elicit antibodies that are reactive to native CEA. When the BVCEA-16 was used as an immunogen, the antisera failed to detect native CEA or BVCEA-140. These studies demonstrate that minor sugar differences exist between native and baculovirus-derived CEA. However, epitope mapping with a panel of 24 anti-CEA MAbs (recognizing at least 10 CEA epitopes) stowed virtual immunologic identity between these two molecules. Moreover, BVCEA-140 appears to be a more potent humoral immunogen in mice than native CEA. These purified recombinant proteins can thus serve as standards in CEA serum assays for the possible detection and characterization of cell-mediated immune responses to CEA and as a potential source of immunogen (primary or for boosting) for active specific immunotherapy protocols of human carcinomas.

Generation and characterization of a novel tetravalent bispecific antibody that binds to hepatitis B virus surface antigens.

Hepatitis B virus (HBV) infection is a worldwide public health problem affecting about 350 million people. HBV envelope contains three surface antigens, called pre-S1, pre-S2 and S. For the prophylaxis of HBV infection, only an anti-S monoclonal antibody was tested for the protective efficacy against HBV infection, but it was shown to be incomplete. In addition, some immune escape mutants carrying mutations on the S antigen were reported. Therefore, a multivalent bispecific antibody rather than a single monoclonal antibody would be more beneficial for the prophylaxis of HBV infection. We have generated a novel tetravalent bispecific antibody with two binding sites for each of the S and pre-S2 antigens. Each of the antigen-binding sites was composed of a single-chain Fv (ScFv). The tetravalent antibody was generated by constructing a single gene encoding a single-chain protein. This protein consisted of an anti-S ScFv whose carboxyl end was tethered, through a 45 amino acid linker, to the amino terminus of anti-preS2 ScFv that in turn was joined to the hinge region of human gamma1 constant region. The single-chain protein was expressed in Chinese hamster ovary cells and secreted in culture supernatant as a homodimeric molecule. The tetravalent bispecific antibody showed both anti-S and anti-pre-S2 binding activities. In addition, the binding affinity of the bispecific antiboy for HBV particles was greater than that of either parental antibody. The tetravalent bispecific antibody is a potentially useful reagent for the prevention and treatment of HBV infection.

CDR substitutions of a humanized monoclonal antibody (CC49): contributions of individual CDRs to antigen binding and immunogenicity.

One of the major obstacles in the successful clinical application of monoclonal antibodies has been the development of host immune responses to murine Ig constant and variable regions. While the CDR grafting of MAbs may alleviate many of these problems, the potential remains that one or more murine CDRs on the human Ig backbone of a "humanized" MAb may still be immunogenic. Studies were undertaken employing a MAb of potential clinical utility, CC49, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We previously developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto frameworks of human MAbs. To identify those CDRs essential for binding, a panel of variant HuCC49 MAbs was generated here by systematically replacing each of the murine CDRs with their human counterparts. The relative affinity constant of each variant was determined. Serum from a patient who received murine CC49 was used to determine the potential immunogenicity of each CDR in humans. The serum was shown to react with the anti-CC49 variable region. Results showed that patients' anti-idiotypic responses are directed mainly against LCDR3 and moderately against LCDR1 and HCDR2. These studies demonstrate for the first time that variants containing individual CDR substitutions of a humanized MAb can be constructed, and each CDR can be defined for the two most important properties for potential clinical utility: antigen binding and immunogenicity.

Minimizing immunogenicity of the SDR-grafted humanized antibody CC49 by genetic manipulation of the framework residues.

The murine mAb CC49 specifically recognizes a tumor-associated glycoprotein (TAG)-72, which is expressed on the majority of human carcinomas. This Ab has potential applications in the diagnosis and treatment of human carcinomas. However, patients receiving murine CC49 generate human anti-murine Ab (HAMA) responses, preventing repeated administration of the Ab for effective treatment. To minimize the HAMA response, two versions of humanized CC49 (HuCC49) were developed: (a) HuCC49 and (b) HuCC49V10 (V10). HuCC49 was developed by grafting the CC49 CDRs, while V10 was generated by grafting only the specificity determining residues (SDRs) of the CC49 onto the frameworks of the human Abs. During the generation of both HuCC49 and V10, a few murine framework residues that were believed to be essential for the integrity of the Ag-binding site were retained. However, the indispensability of these residues for the Ag-binding activity of CC49 has not been experimentally validated. In this study, an array of V10 variants were generated by replacing, by site-specific mutagenesis, the murine framework residues that were retained in the humanized Ab with their counterparts in the human templates. The variants were tested for their (a) Ag-binding activity and (b) reactivity to sera from patients who were previously administered murine CC49 in a clinical trial. One such variant, V59, compared to the parental V10, shows a significant decrease in its reactivity to the anti-variable region Abs present in the patients' sera, while it binds to the TAG-72 Ag with a slightly higher affinity. Variant 59, which is expected to be minimally immunogenic because of its low sera reactivity, is a potentially useful clinical reagent against human carcinomas. In this study, we show for the first time that experimental validation rather than reliance on the protein data bank (PDB) should be the criterion for the indispensability of framework residues for the humanization of any murine Ab to retain its Ag-binding property and reduce its immunogenicity in patients.

Generation and characterization of a novel single-gene-encoded single-chain immunoglobulin molecule with antigen binding activity and effector functions.

Monoclonal antibody (MAb) CC49 is a murine IgG1 that reacts with tumor-associated glycoprotein (TAG)-72, a pancarcinoma antigen. Clinical trials using radiolabeled CC49 for diagnostic imaging have demonstrated specific localization of more than 90% of carcinomas. The feasibility of adopting in vivo gene inoculation methods for antibody-based immunotherapy requires introduction and expression of two genes, encoding immunoglobulin (Ig) heavy and light chains, in a single cell to generate a functional antibody. To circumvent the problems inherent in this approach, we have constructed a single-gene encoding a single-chain immunoglobulin (SCIg) that, unlike previously developed SCIgs, contains all IgG domains. To construct the novel SCIg, the carboxyl end of the constant region of the chimeric (c) CC49 kappa chain is joined, via a 30 residue Gly-Ser linker peptide, to the amino terminus of the CC49 heavy chain. To our knowledge, neither a linker peptide this long nor a linkage between the constant light (C(L)) and variable heavy domains has been reported previously. Transfectomas developed by introducing the expression construct of the amplifiable gene in dihydrofolate reductase-deficient Chinese hamster ovary (CHO dhfr-) cells secrete a 160 kDa homodimeric molecule, SCIgcCC49. The in vitro antigen binding properties of SCIgcCC49 are comparable to those of cCC49 and SCIgcCC49deltaC(H)1, a single-chain Ig deficient in constant heavy chain-1 (C(H)1) and C(L) domains. The antibody-dependent cellular cytotoxicity (ADCC) of SCIgcCC49 and cCC49 were also comparable. This single-gene approach for generating an immunoglobulin molecule may facilitate in vivo gene inoculation as well as ex vivo transfection of patients' cultured tumor-infiltrating lymphocytes for immunotherapy protocols for a variety of diseases, including cancer.

Anti-tumor activity of human T cells expressing the CC49-zeta chimeric immune receptor.

A chimeric immune receptor consisting of an extracellular antigen-binding domain derived from the CC49 humanized single-chain antibody, linked to the CD3zeta signaling domain of the T cell receptor, was generated (CC49-zeta). This receptor binds to TAG-72, a mucin antigen expressed by most human adenocarcinomas. CC49-zeta was expressed in CD4+ and CD8+ T cells and induced cytokine production on stimulation. Human T cells expressing CC49-zeta recognized and killed tumor cell lines and primary tumor cells expressing TAG-72. CC49-zeta T cells did not mediate bystander killing of TAG-72-negative cells. In addition, CC49-zeta T cells not only killed FasL-positive tumor cells in vitro and in vivo, but also survived in their presence, and were immunoprotective in intraperitoneal and subcutaneous murine tumor xenograft models with TAG-72-positive human tumor cells. Finally, receptor-positive T cells were still effective in killing TAG-72-positive targets in the presence of physiological levels of soluble TAG-72, and did not induce killing of TAG-72-negative cells under the same conditions. This approach is being currently being utilized in a phase I clinical trial for the treatment of colon cancer.

Development of a minimally immunogenic variant of humanized anti-carcinoma monoclonal antibody CC49.

Monoclonal antibody (MAb) CC49 reacts with a pancarcinoma antigen, tumor associated glycoprotein (TAG)-72. To circumvent human anti-murine antibody (HAMA) responses in patients, we earlier developed a humanized CC49 (HuCC49) by grafting the complementarity-determining regions (CDRs) of MAb CC49 onto variable light (VL) and variable heavy (VH) frameworks of the human MAbs LEN and 21/28'CL, respectively. With the aim of minimizing its immunogenicity further, we have now generated a variant HuCC49 MAb by grafting the specificity-determining residues (SDRs) of MAb CC49 onto the frameworks of the human MAbs. Based on the evaluation of its binding affinity for TAG-72 and its reactivity with anti-idiotypic antibodies present in sera from patients who have been treated with murine CC49, this variant retains its antigen-binding activity and shows minimal reactivity with anti-idiotypic antibodies in patients' sera. Development of this variant, which is a potentially useful clinical reagent for diagnosis and therapy of human carcinomas, demonstrates that for humanization of a xenogeneic antibody grafting of the potential SDRs should be sufficient to retain its antigen-binding properties.

Expression of a tumor-reactive antibody-interleukin 2 fusion protein after in vivo particle-mediated gene delivery.

We have used a particle-mediated gene transfer method to analyze the posttransfection expression pattern of an antibody-cytokine fusion protein (FP) in vivo. The FP, denoted CC49-IL2, consists of a single-chain antibody containing the antigen recognition domain from the murine monoclonal antibody CC49 (recognizing the tumor-associated antigen TAG-72), a human IgG1 constant heavy chain, and human interleukin-2 (IL-2). This FP can bind to TAG-72-expressing tumor cells and exhibits IL-2 activity. To induce systemic levels of this FP in vivo, we have transferred the FP gene into murine epidermal cells by direct delivery of DNA-coated gold particles using a transcutaneous "gene gun." After the pericutaneous delivery of the FP gene via gold particles, production of the exogenous FP was detected at the epidermal target site. The FP produced in vivo at the site of gene delivery has cytokine activity and antigen recognition capabilities similar to those present in CC49-IL2 FP purified from hybridoma culture supernatants in vitro. FP was also detectable in the serum from test animals treated with particle-mediated gene transfer. Time course experiments indicated that serum levels of FP reached a peak level within 8 hours after DNA delivery, whereas the epidermal target tissue levels continued to increase for 24 hours before plateauing. Our results indicate that exogenous protein levels consistent with immunotherapeutic effects of the FP can be readily achieved at the skin tissue site of gene delivery, with the potential for achieving therapeutic levels systemically.

In vivo gene inoculation of a recombinant single-chain antitumor antibody induces anti-immunoglobulin response.

While in vivo gene inoculation is being increasingly exploited to express genes of choice and elicit specific immune responses in animal models, the utility of this method has not been explored extensively for the expression of antibody genes. The primary constraint of this method is the need to deliver to, and express in, a single cell two functional genes, i.e., those encoding heavy and light chains of an antibody molecule. Several single-gene constructs encoding variants of the monoclonal antibody (MAb) CC49 have been developed, MAb CC49 recognizes a tumor-associated glycoprotein, TAG-72. SP2/O myeloma cells, transfected with the CC49 single gene, express a single-chain protein which is secreted by the transfectoma as a homodimer. Following intramuscular injection of mice with the expression plasmids of the single-gene constructs, the encoded CC49 antibody (AB1) was detected in the plasma of the host. In addition, cellular and humoral immune responses to AB1 have been demonstrated. Antibodies (AB2) to the in vivo-produced variable region of AB1 have been detected and persisted for at least 70 days post-inoculation of the recombinant plasmid. Thus, in vivo gene inoculation of single-chain immunoglobulins may be an alternative or complimentary approach to the induction of anti-idiotypic responses in immunotherapy protocols.