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.

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.

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.

T-cell killing of heterogenous tumor or viral targets with bispecific chimeric immune receptors.

We have previously described several novel chimeric immune receptors (CIRs) that redirect human T cells to kill malignant or HIV-infected cells. These CIRs comprise a cancer- or virus-specific ligand or single-chain antibody fused to the signaling domain of the T-cell receptor CD3-zeta subunit. Binding of the ligand- or antibody-based CIR to the target antigen (Ag) triggers T-cell-mediated cytolysis of the tumor- or virus-infected cell independent of target cell major histocompatibility complex class I expression. A new type of CIR was developed to mediate the lysis of cells that expressed one or more distinct viral or tumor Ags; three bispecific CIRs (BCIRs) were generated that recognized the carcinoembryonic Ag (CEA) and TAG-72 tumor Ags or, alternatively, distinct epitopes in the HIV envelope (HIVenv). T cells expressing the antitumoral Ag BCIR lysed both CEA- and TAG-72-expressing targets and did not kill Ag-negative targets or target cells expressing other members of the CEA family. Similarly, T cells expressing the anti-HIVenv BCIR lysed target cells expressing both the wild-type HIVenv and a mutant HIVenv that lacked the epitopes recognized by the monospecific CIRs. This approach permits the generation of T cells with a broader spectrum of activity capable of killing virus-infected cells and malignant cells and reduces the potential of progression of disease due to Ag loss variants.

Structural correlates of an anticarcinoma antibody: identification of specificity-determining residues (SDRs) and development of a minimally immunogenic antibody variant by retention of SDRs only.

Clinical utility of murine mAbs is limited because many elicit Abs to murine Ig constant and variable regions in patients. An Ab humanized by the current procedure of grafting all the complementarity determining regions (CDRs) of a murine Ab onto the human Ab frameworks is likely to be less immunogenic, except that its murine CDRs could still evoke an anti-variable region response. Previous studies with anticarcinoma mAb CC49 showed that light chain LCDR1 and LCDR2 of humanized CC49 could be replaced with the corresponding CDRs of a human Ab with minimal loss of Ag-binding activity. The studies reported in this paper were undertaken to dissect the CC49 Ag-binding site to identify 1) specificity determining residues (SDRs), the residues of the hypervariable region that are most critical in Ag-Ab interaction, and 2) those residues that contribute to the idiotopes that are potential targets of patients' immune responses. A panel of variants generated by genetic manipulation of the murine CC49 hypervariable regions were evaluated for their relative Ag-binding affinity and reactivity to sera from several patients who had been immunized with murine CC49. One variant, designated HuCC49V10, retained only the SDRs of CC49 and does not react with the anti-variable region Abs of the sera from the murine CC49-treated patients. These studies thus demonstrate that the genetic manipulation of Ab variable regions can be accomplished by grafting only the SDRs of a xenogeneic Ab onto human Ab frameworks. This approach may reduce the immunogenicity of Abs to a minimum.

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.

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.

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.

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.

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.

The use of a cationic liposome formulation (DOTAP) mixed with a recombinant tumor-associated antigen to induce immune responses and protective immunity in mice.

The cationic liposome DOTAP is a well-known transfection reagent. It has been manufactured and approved for clinical use, is readily available, and can be easily used as an adjuvant. These characteristics prompted us to investigate the effectiveness of DOTAP as an adjuvant to induce immune responses and protective immunity in mice using baculovirus-derived carcinoembryonic antigen (bV-CEA) as a model antigen. Two routes of administration and a dose-response study of bV-CEA were used in BALB/c mice to define the magnitude of the immune response as well as the most effective route of immunization. The results demonstrate differences in antibody titers, immunoglobulin (Ig)G isotype, and T-cell responses between the intravenous (i.v.) or subcutaneous (s.c.) route of immunization. The titer of the anti-CEA antibodies induced by the s.c. immunization was greater than the response by i.v. immunization. The s.c. route enhanced the IgG2a/2b isotype, whereas i.v. immunization elicited primarily IgG1. T-cell proliferation responses and cytokine production paralleled the humoral response (i.e., production was higher in the s.c. immunized animals). No differences in immunological responses were seen using either 25 or 10 microg of bV-CEA three times. An amount of 25 microg of bV-CEA/DOTAP given by s.c. immunization was sufficient in protecting mice from the transplant of syngeneic tumor cells transduced with the human CEA gene. We conclude that the cationic liposome DOTAP may be a useful immunoadjuvant for active anti-tumor immunotherapy in future clinical trials. This study will help to define the most effective way to use such an adjuvant.

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.

A CDR-grafted (humanized) domain-deleted antitumor antibody.

While several murine monoclonal antibodies (MAbs) directed against carcinoma associated antigens have shown excellent tumor targeting properties in clinical trials, the use of radiolabeled MAbs for both diagnostic and therapeutic applications has been hindered by two factors: (a) the induction of host anti-immunoglobulin (Ig) responses and (b) slow plasma clearance of unbound radiolabeled MAb, resulting in bone marrow toxicity for therapeutic application, and long intervals between MAb administration and tumor detection for diagnostic applications. This report describes the development of the first recombinant Ig with properties designed to reduce or eliminate both of the above problems: a complementarity determining region (CDR)-grafted humanized (Hu) MAb with a CH2 domain deletion (delta CH2). The MAb chosen for engineering was CC49, which is directed against a pancarcinoma antigen designated TAG-72 that is expressed on the majority of colorectal, gastric, breast, ovarian, prostate, pancreatic and lung carcinomas. When characterized for antigen binding in solid phase competition radioimmunoassays, the HuCC49 delta CH2 MAb completely inhibited the binding of murine (mu) CC49 and HuCC49 for TAG-72. The relative affinity constants (Ka) of MAbs HuCC49 delta CH2, HuCC49 and muCC49 were 5.1 x 10(-9), 2.1 x 10(-9) and 2.3 x 10(-9), respectively. The plasma clearance of 131I-HuCC49 delta CH2 was significantly faster than that of intact 125I-HuCC49 after either i.v. or i.p. administration in athymic mice (p(2)0.05). Biodistribution studies in athymic mice bearing human colon carcinoma xenografts after i.v. or i.p. administration of 131I-HuCC49 delta CH2 and 125I-HuCC49 demonstrated the efficient tumor localization and substantially lower percent of the injected dose (%ID/g) of the HuCC49 delta CH2 in normal tissues. This is reflected in the significantly higher radiolocalization indices (%ID/g in tumor divided by %ID/g in normal tissue) observed with the HuCC49 delta CH2 for most normal tissues tested (p(2)0.05). The differential between the rate of plasma clearance of HuCC49 delta CH2 and HuCC49 was even more pronounced in SCID mice, which have been shown to be an appropriate model to study the metabolism of human IgG. These studies thus describe the development of a recombinant Ig molecule which, for the first time, combines 1) the properties of more rapid blood clearance than an intact humanized Ig molecule--without loss of antigen binding affinity--and 2) reduced potential for eliciting a human anti-murine antibody (HAMA) response in patients. These studies also demonstrate the potential utility of HuCC49 delta CH2 for i.p. as well as i.v. radioimmunodiagnosis and radioimmunotherapy in patients with TAG-72 positive tumors.

Generation and characterization of a single-gene encoded single-chain immunoglobulin-interleukin-2 fusion protein.

BACKGROUND: Interleukin-2 (IL-2), a potent inducer of cellular immune responses, has been used for biological therapy of human cancer; however, the high doses of IL-2 required to mediate patients' immune responses can cause considerable systemic toxicity. The murine monoclonal antibody (MAb) CC49, which reacts with tumor-associated glycoprotein (TAG)-72, expressed on a variety of human carcinomas, has shown excellent tumor localization in recent clinical trials. OBJECTIVES: Development and characterization of a single-chain immunoglobulin-IL-2 (SCIg-IL-2) fusion protein which, by delivering IL-2 selectively to the tumor site, can serve as an effective reagent for CC49/IL-2 combination therapy. STUDY DESIGN: A single-gene encoding the SCIg-IL-2 fusion protein derived from the chimeric (c) CC49 was designed, generated and inserted in an expression vector. The monomeric single-chain protein consisted of the CC49 heavy and light chain variable domains covalently jointed through a (GGGGS)3 linker peptide. The carboxyl end of the variable domain of the light chain was linked to the amino terminus of the human gamma 1 Fc through the hinge region, and the carboxyl end of the CH3 domain was linked to the amino terminus of the human IL-2 through a GGGSGGG linker peptide. The SCIg-IL-2, expressed from the murine myeloma cells transfected with the expression construct, was characterized for its antigen-binding specificity, antibody effector functions and IL-2 biological activity. RESULTS AND CONCLUSION: Transfection of murine myeloma cells with the single-gene expression construct SCIg-IL-2 expressed a single-chain protein of approximately 70 kD, which was secreted into tissue culture fluid as a homodimer of approximately 140 kD. SCIg-IL-2 competed completely with cCC49 for binding to the TAG-72 antigen, but approximately three- to four-fold more of the SCIg-IL-2 was required to achieve levels of competition similar to those observed with the murine or chimeric CC49. With human effector cells, the fusion protein mediated lysis of TAG-72-positive human carcinoma cells. Prior treatment of human effector cells with 100 U/ml of human IL-2 enhanced the fusion protein-mediated cytolysis from 32 to 65%. At doses of > or = 1 ng/ml, the stimulatory effect of SCIg-IL-2 on IL-2 dependent murine HT-2 cell proliferation was comparable to that of the recombinant human IL-2. The single-gene construct may also facilitate inoculation of the gene in animal tissue for in vivo expression of the fusion protein.

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.

Baculovirus expression of a functional single-chain immunoglobulin and its IL-2 fusion protein.

The baculovirus expression system has been used for the production of a variety of proteins, including antibodies. Two single-gene constructs encoding single-chain immunoglobulins have recently been developed. The antibody employed was monoclonal antibody (MAb) CC49 which reacts with the pancarcinoma antigen, tumor associated glycoprotein, TAG-72. One, single-chain construct designated SCA delta CLCH1 (SCIg), consists of the CC49 sFv covalently joined to the human Fc (gamma 1) through the hinge region. The other, SCA delta CLCH1-IL-2 (SCIg-IL-2), has a human IL-2 molecule attached to the carboxyl end of the SCIg. These constructs have been used to test the feasibility of producing biologically active antibodies using the baculovirus expression system. Both constructs have been successfully expressed in insect cells and purified. The baculovirus recombinant single-chain antibodies have been designated, bV-SCA delta CLCH1 (bV-SCIg) and bV-SCA delta CLCH1-IL-2 (bV-SCIg-IL-2) they have been shown to be secreted in the culture supernatant as dimeric molecules of approximately 115 kDa and 140 kDa, respectively. The specificity and antibody dependent cellular cytolytic activity of the baculovirus recombinant single-chain antibodies were shown to be similar to that of the myeloma derived molecules. Glycosylation analysis showed that baculovirus derived proteins were N-glycosylated, but carried few if any high mannose residues. The biological activity of the IL-2 moiety was retained in bV-SCIg-IL-2, as evidenced by its stimulatory effect on the proliferation of the IL-2 dependent cell line HT-2. The observation that a significantly shorter time is required to develop baculovirus recombinant molecules as compared to myeloma derived molecules and that insect cells express single chain MAbs at acceptable levels may have implications for the production of these molecules for clinical use.

Generation, characterization, and in vivo studies of humanized anticarcinoma antibody CC49.

Monoclonal antibody (MAb) CC49 reacts with tumor-associated glycoprotein (TAG)-72, a human pancarcinoma antigen. In clinical trials, radiolabeled CC49 has shown excellent tumor localization; however, many of the patients receiving MAb CC49 develop a human antimouse antibody response. In an attempt to prevent this antiimmunoglobulin response, we have developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto the variable light (VL) and variable heavy (VH) frameworks of the human MAbs LEN and 21/28' CL, respectively, while retaining those murine framework residues that may be required for the integrity of the antigen combining-site structure. The HuCC49 MAb was compared with native murine CC49 (nCC49) and chimeric CC49 (cCC49), using a variety of assays. SDS-PAGE analysis under nonreducing conditions showed that the HuCC49 MAb has virtually identical mobility to that of cCC49. Under reducing conditions, the HuCC49 yielded two bands of approximately 25-28 and approximately 50-55 kDa, characteristic of heavy and light immunoglobulin chains. In competition radioimmunoassays, HuCC49 completely inhibited the binding of 125I-labeled nCC49 to TAG-72, although 23- to 30-fold more HuCC49 was required to achieve a level of competition similar to those of cCC49 and nCC49. The relative affinity of HuCC49 was 2- to 3-fold less than those of the cCC49 and nCC49 MAbs, respectively. The plasma clearance in mice of HuCC49 was virtually identical to that of cCC49. Biodistribution studies demonstrated equivalent tumor-targeting of HuCC49 and cCC49 to human colon carcinoma xenografts. These studies thus suggest that HuCC49 and genetically modified molecules, such as sFv and domain-deleted immunoglobulins developed by using the HuCC49 variable region as a cassette, may be potentially useful in both diagnostic and therapeutic clinical trials in patients with TAG-72-positive tumors.

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.