Human Antibodies

A human scFv display library has been constructed from peripheral blood lymphocytes of a patient suffering from Hashimoto's thyroiditis. Upon induction of Cre recombinase, the amplified VH and VL genes were recombined via two loxP sites inserted in amplification primers to construct in vitro scFv genes. Either soluble scFvs or scFvs displayed on phage were screened for binding to human thyroglobulin after two pannings with this antigen. Three scFvs were obtained which showed very similar nucleotidic sequences. The VH genes expressed display 96.4% VH251 gene, one of the two functional members of the small VH5 family and are mutated in sites already described as “selectively neutral” mutations and the VL genes are close to the germline DPL8 gene. These scFvs bind not only to human thyroglobulin but also to other self and exogenous antigens.

Immunohistochemical analysis of biopsies, cytology specimens or surgical resection specimens using antibodies directed towards tumor-associated antigens, lineage or differentiation antigens is a technique often used by surgical pathologists to aid in establishing the correct histologic classification of malignant tumors. With the proliferation of experimental approaches to cancer treatment using monoclonal antibodies as targeting agents, it is anticipated that surgical pathologists will increasingly be receiving requests from clinicians to define the antigen profile in biopsy specimens, even when not necessary to render the correct tumor classification. Clinicians may use the immunohistochemically delineated antigen profiles provided by surgical pathologists to plan tailored treatment regimens utilizing monoclonal antibodies to the antigens expressed in the tumor biopsy to target anticancer therapeutic agents. Some of the potential problems in such a process might include the differing sensitivities, and perhaps specificities, of the antibodies used for analyzing the surgical pathology biopsy specimens compared to the monoclonal antibodies actually used {\it in vivo}. Our approach to this dilemma is to develop murine monoclonal antibodies to tumor-associated antigens that can reliably be used to detect antigens in routinely processed surgical pathology specimens as a starting point for further therapeutic monoclonal antibody development.

We previously demonstrated the induction of IFN-γ in polyneuropathy patients associated with monoclonal gammopathy, but not in patients with presumably non-immuno-logical types of neuropathy. We herein examined mechanism involving release of neutralizing autoantibodies (Aabs) to IFN-γ in sera from those pateints. In contrast to polyneuropathy patients with monoclonal gammopathy, patients with polyneuropathy of presumably non-immunological types showed increased production of neutralizing Aabs specific for IFN-γ. These results demonstrate a role for autoimmunity in cytokine regulation. However, their association to the clinical manifestations of the disease requires further investigations, which are necessary for future consideration in therapeutic strategies.

A trispecific F(ab′)3 antibody conjugate (TAC) with specificities for the Fcγ receptor I (FcγRI/CD64), the epidermal growth factor receptor (EGFR) and the HER2/neu antigen has been developed to redirect effector cell-mediated cytotoxicity against cancer cells expressing both or either of the tumor-associated antigens. The TAC was constructed in two steps using the sulfhydryl-specific cross-linker o-phenylenedimaleimide (o-PDM). In step one, a bispecific antibody was prepared by linking the Fab′ fragments of mAb m22 (a murine IgG1 specific for FcγRI) and mAb H425 (a humanized IgG1 antibody recognizing EGFR). The conjugation efficiency was about 60%. the second step, the Fab′ fragment of mAb 520C9, a murine IgG1 specific for HER2/neu, was coupled to the bispecific antibody made in step one. About 40% antibody. The purity of the TAC was more than 90% purification. The TAC was characterized in vitro for its ability to bind specifically to all the three antigens and to kill target cells expressing the tumor antigens. In contrast to bispecific conjugates that can only target cells expressing either of the tumor antigens, the TAC was able to bind both the antigens more efficiently in cell-binding assays and to kill tumor cells expressing EGFR and HER2/neu antigens.

In this study we tried to elucidate further the crossreactivity pattern and binding characteristics of human monoclonal IgM DJ which is an anti-DNA antibody and possesses Y7 natural idiotope.

Cytokines such as interleukin-2(IL-2), gamma interferon (IFN-γ) and alpha tumor necrosis factor (TNF-α) are important mediators in immune responses against tumors. However, their therapeutic efficacy and clinical utilities in treatment of human malignancies are in large part limited due to the low concentrations of cytokine in tumors and the severe toxic side-effects derived from high-dose administration of cytokines. One critical issue to improve therapeutic efficacy is how to increase the local concentration of cytokine in tumors without causing severe side-effects. A series of recent reports demonstrated that the introduction of cytokine genes into tumor cells and subsequent local secretion can circumvent the limitations associated with the systemic cytokine administration. An alternative means of cytokine delivery is to target cytokines to tumor cells with tumor specific antibodies. Thereby, effective local cytokine concentrations can be achieved at the tumor sites without resorting to patient-specific therapy. With the advance in biotechnology, two structurally disparate domains of immunoglobulin and cytokine can be brought together into one fusion protein molecule by protein engineering These engineered antibody-cytokine fusion proteins combine ihe unique targeting ability of tumor-specific antibodies with the multifunctional activity of cytokines. In general, there are two commonly engineered fusion proteins, the F(ab′)tsub2/cytokine expressed in mammalian cells and the single-chain FV/cytokine expressed in Escherichia coli. Both the tumor-binding reactivity and the functional cytokine activity are maintained in most of fusion proteins. Therefore, these fusion proteins may be useful in targeting cytokine to tumors to stimulate immune destruction of tumors, while limiting severe toxic side-effects by the high dose of cytokine administration. Recent preclinical studies have shown that these fusion proteins are able to target cytokines to tumors expressing the tumor-associated antigen in vivo, and to inhibit both the primary and metastatic tumors in an immune competent animal model. Therefore. these recombinant fusion proteins may represent a new generation of novel immunotherapeutic reagents for the treatment of human malignant diseases.

Cocktails of human monoclonal antibodies (HuMAbs) have been used to increase the likelihood of identifying heterogeneous cancer cells. We utilized 3 HuMAbs termed SK-1, GM4, and GMA1, which recognized a 42–46kDa two chain structure, a 57kDa antigen, and the ganglioside GD3, respectively. An estimated two dozen cell lines were tested for the coexpression of these antigens and this was found to be present only on pancreatic carcinoma cell line, PANC-1; A 24 hr treatment of PANC-1 cells with interferon gamma (IFNγ; 100 units), interferon beta (IFNβ; 1000 units), as well as interferon alpha (IFNα; 1000 units) resulted in roughly a four fold increase in the co-expression of the 42–46 kDa/GD3 antigens as well as the 42–46 kDa/ 57 kDa antigens. After a 4 day incubation the co-expression of these antigens progressed and IFN α treatment had the most pronounced effect, which was 8 fold higher than background for the 42–46kDa/57kDa antigens, whereas IFN β resulted in a five fold antigen upregulation. The pronounced effect of vinblastine on the co-expression of the 42–46 kDa/GD3 antigens (4 fold on day 1 and , 10 fold on day 4) and 42–46 kDa/57kDa antigens on PANC-1 (5 fold on day 1 and 7 fold over background on day 4) cells can be seen at concentrations as low as 10^-7M. Colchicine and vincristine dramatically enhanced co-expression of these tumor antigens on day 4 but not on day 1 PANC-1cells. The expression of these antigens was also found to be cell cycle dependent.

IL-11, a less identified cytokine, possesses some overlapping functions with IL-6 that are able to facilitate the growth and antibody secretion of B lymphocyte hybridomas. In this report, a DNA fragment encoding human IL-11 was transduced into fusion partners (mouse myelomas Ag8.653 and SP2/0, and human lymphoblastoid cell line HF2) mediated by lipofection. The transfected cells selected with G418 secreted IL-11 constitutively over the range of 32.4±10.5units/ml to 76.6±18.4units/ml, which could be inhibited by an IL-11 neutralizing MAb up to 80%. doubled, while that of LCLs displayed a 2.4- or 3.3- fold increase, when fused with the transfected fusion partners, respectively. The derived hybridomas from IL-11 secreting fusion partner secreted 3 or 4 times as many immunoglobulins as that from its ancestor. Our data indicate that IL-11 gene transfected fusion partners are improved cell lines for generation of human B lymphocyte hybridomas, and IL-11 may contribute to the increased fusion frequency and antibody secretion of B lymphocyte hybridomas.