Human Antibodies

An IgG1kα Fab library was generated on the surface of phage beginning with bone marrow RNA from a healthy 22-year-old human donor. The donor had been immunized to measles in his early childhood but had only a low serum titer to a measles antigen preparation. The resulting library of approximately 10⁷ clones was panned against the measles antigen preparation and three positive Fab-producing clones identified by ELISA. One of the Fabs was found to be specific to measles and to bind with high apparent affinity (10⁸ M⁻¹). The other two bind with lower affinity and show marked cross-reactivity with a number of other antigens. They possess heavy chains derived, with extensive somatic modification, from the single member gene family VH6. The study indicates that both high-affinity specific antibodies and lower affinity polyreactive antibodies can be derived from the library approach under appropriate conditions.

Murine monoclonal antibody (MAb) G3-519 has been shown to recognize a conserved neutralizing epitope in the fourth constant (C4) region of the external glycoprotein gp120 of HIV-1. Inasmuch as this antibody effectively neutralized the infectivity of diverse HIV-1 isolates, it has been selected to be developed for passive immunization against HIV-1 infection in humans. In order to minimize the problem of immunogenicity of murine antibodies and to confer additional accessory immune functions, we have constructed mouse/human chimeric and humanized forms of the antibody. The chimeric antibody was constructed by cloning the murine variable regions and replacing the mouse constant regions with those from human Igγ1,κ. The humanized antibody was constructed using the human KAS variable region framework sequences as template. Engineering was guided by a three dimensional model of the murine variable region. The murine, chimeric and humanized forms of the antibody exhibited similar reactivity with the peptidic antigen in ELISA, and comparably neutralized the infectivity of HIV-1 in vitro. Taken together, our results show that the chimeric and humanized forms of G3-519 essentially retain the binding activity of the mouse parental antibody. Clinical development is planned to assess the prophylactic and therapeutic usefulness of these reshaped antibodies in humans.

A human monoclonal antibody, CB STL-1, against staphylococcal alpha-toxin has been established by hybridoma technology. It is of IgG1 subclass with lambda light chain and possesses a dissociation constant of 8×10⁻¹⁰ mol/l. 1 mg of purified antibody neutralizes the hemolytic activity of 800 μg/ml alpha-toxin in an in vitro hemolysis assay using rabbit erythrocytes. The antibody does not bind to overlapping (7 residues) decapeptides spanning the sequence of alpha toxin, thus it might bind to a conformational epitope. The epitope recognized by the antibody is not accessible in oligomeric toxin. The antibody binds both to the hydrophilic and amphipathic forms of the monomeric toxin Fab fragments of the antibody are stable and show no significant loss of activity. CB STL-1 was able to protect mice in vivo from i.p. challenge with alpha toxin. Thus, the antibody is a candidate for passive immunotherapy. The variable regions of the antibody secreted by CB STL-1 were sequenced and found to be encoded by a VH gene segment belonging to the VH1 family, and a Vlambda segment most likely belonging to the VlambdaIII subgroup. Further analysis concerning the third complementarity determining region (CDR3) of the heavy chain is presented.

Partially desialylated ovine submaxillary mucin plus an immunological adjuvant has been used by us to induce a humoral immune response to Tn antigen and sialylated Tn in colon cancer patients at risk of recurrence. Peripheral blood lymphocytes were purified from one of these patients with a high IgM titer reactive with Tn antigen transformed with Epstein-Barr virus and subsequently fused with a human-mouse heteromyeloma cell line, HMMA2.11TG/O. Clones were screened and sub cloned using an enzyme-linked immunoassay and four stable IgM-secreting clones were tested for reactivity with a variety of natural and synthetic antigens, paraffin-embedded colon carcinomas and normal colonic mucosa to determine their specificity. Four human IgM monoclonal antibodies reactive predominantly with Tn antigen were produced and shown to react with a mucinous human colon carcinoma cell line, LS-174T, and with paraffin-embedded human colon carcinomas. We conclude that modified ovine submaxillary mucin is an effective vaccine for generating a humoral immune response directed to Tn antigen present on human colon cancer.

A series of human hybridomas were derived by the fusion of the immature T cell line, JM, and lymphocytes from the peripheral blood of a colon carcinoma patient in long term remission who was shown to produce anti-tumor antibody. Five of the hybridomas have been selected for further study. These hybridomas express the T cell surface markers CD2, CD3, CD4, and CD8. The human IgG2/kappa antibody produced by these clones has been purified using affinity chromatography and has been used in immunohistiologic staining procedures. Biotinylated monoclonal antibody (MAb) stained colon carcinoma tissue but did not stain normal colon. Positive immunostaining was also seen utilizing colon carcinoma cell lines but not with other cell lines tested. These hybrids were constructed without using the conventional fusion technology which employs 8-azoguanine resistant, HAT sensitive malignant fusion partners. Because the fusion partner was a T cell, we were able to select hybrids on the basis of surface immunoglobulin. This approach was accompanied by stringent selection of patients as lymphocyte donors. Utilizing these unique methods, we have successfully produced hybridomas with T cell surface markers that produce human MAb with reactivity to colon carcinoma.

OKT3 is a murine monoclonal antibody which recognizes an epitope on the e-subunit within the human CD3 complex. OKT3 possesses potent immunosuppressive properties in vivo and has been proven effective in the treatment of renal, heart and liver allograft rejection. Despite its efficacy, significant problems remain associated with OKT3 therapy, i.e. T-cell activation and the anti-murine antibody response. To address the problem of the anti-murine antibody response we have constructed humanized versions of OKT3. One of the humanized derivatives, gOKT3-7 incorporating the OKT3 complementarity determining regions plus a small number of alterations to the human framework, has an affinity of 1.4×10⁹ M⁻¹ compared with 1.2×10⁹ M⁻¹ for the murine OKT3. A humanized antibody (gOKT3-1) incorporating only the CDRs from OKT3 was found to be functionally inactive, confirming the requirement for nonCDR substitutions. gOKT3-7 retains the ability of mOKT3 to induce T cell proliferation in vitro and appears to be a good candidate for further development for in vivo therapy.

Phagemid vectors have been developed which promise to supersede hybridoma technology for the selection and production of human antibodies. We have modified an existing phagemid vector to improve the stability of synthesized soluble antibody fragments. The vector allows the antibody fragment to be produced: i) as a soluble protein incorporating a stable carboxyl terminal octapeptide (FLAG) or, ii) on the surface of a bacteriophage fused to a minor coat protein (the gene III protein). The antibody gene encoding the well characterized monoclonal antibody NC10 (an antibody that recognizes the neuraminidase of the influenza strain N9) was inserted as a single chain Fν construct into the phagemid vectors pHFA and pHFA/SacII. Western blotting, ELISA and electron microscopy studies showed that recombinant clones could be manipulated to either synthesize soluble protein into the peri plasm or present the protein on the surface of bacteriophage. Cosynthesis of GroEL and GroES chaperon ins resulted in complete proteolysis of the scFνNC10-FLAG-gIIIp fusion product and did not improve total phage production. Coexpression of chaperonins should be used with caution for library construction due to the expected selection pressure for protease resistant gene III fusions.

We have developed PCR primers for the amplification and cloning of the genes encoding human antibody fragments. Two sets of primers were designed to amplify the coding sequence of the complete variable region and the first constant domain of immunoglobulin heavy chains. One set of primers was designed to amplify the coding sequence of complete kappa light chains. These three sets of primers were used in PCR amplifications with cDNA prepared from EBV transformed B cells as the template. The amplified fragments were cloned and their nucleotide sequences were determined. Analysis of the DNA sequences of such PCR generated antibody fragments suggested that our primer sets were able to amplify the major subsets of the heavy and light chain variable region genes. Comparison between the estimated frequency of the different subsets of heavy chain variable region genes and the distribution of our subclones further indicated that one set of our primers was able to proportionally amplify the subsets of the heavy chain family.

We report a detailed comparison of two commonly used stable, amplifiable mammalian expression systems (Chinese Hamster Ovary cells/dihydrofolate reductase and Mouse NSO myeloma/glutamine synthetase) used to express a humanized IgG1 monoclonal antibody. We compare copy number and steady state mRNA levels of both the selectable marker and heavy chain of the antibody throughout the selection and amplification process. In both cell lines, copy number and steady state levels of heavy chain and selectable marker increased during selection and were further increased during amplification. As expected, an increase in steady state mRNA levels of heavy chain correlated with an increase in expression of antibody whilst an increase in the steady state levels of mRNA of the selectable marker correlated with increased resistance to the selective agent. In NSO and CHO cells producing equivalent amounts of antibody, the copy number of the antibody genes and selectable marker was significantly higher in the CHO cells than in the NSO cells. However, the steady state mRNA levels of the heavy chain of the antibody were virtually identical. Rates of protein secretion in the two cell lines were also compared and found to be very similar. When the antibody purified from both systems was compared in a number of functional assays they behaved identically.

In this study we demonstrated that fetal calf serum (FCS) depleted of IgG by protein G affinity chromatography (G-FCS) is superior to whole FCS or serum-free culture media as a culture supplement for the production of purified IgG monoclonal antibodies (MAb). One hundred ml FCS was applied to a 25 ml protein G Sepharose 4 Fast Flow Column, which was shaken gently for 2 days at 4° C. The procedure was repeated using a protein G column for an additional day. G-FCS was used at a concentration of 5% in RPMI 1640 medium to grow the mouse myeloma cell line P3X63.Ag8.653, which secretes an IgG-1 mouse-human chimeric monoclonal antibody (TVE-1). Cell density, viability, doubling time, and antibody production were used as indices to compare the efficacy of this medium with that of whole FCS medium, AIM-V (Gibco, USA) and other serum-free media. The results demonstrate that cell growth and antibody production in -GFCS medium did not differ significantly from that in FCS medium, but were significantly better than in the serum-free media (p<0.001). TVE-1 antibody in the spent tissue culture media was purified by 50% ammonium sulfate precipitation and Protein A affinity chromatography. An antibody that was more than 99% pure was obtained. Endotoxin analysis revealed that the IgG depletion process does not generate a significant level of endotoxin in FCS (<0.06 EU/ml). Because the IgG depletion procedure is simple and economical, and the purification procedure is simple and rapid, -GFCS medium may be an ideal serum supplement when a large quantity of purified IgG monoclonal antibody is required for in vivo clinical application.

Human monoclonal antibodies directed against human cytomegalovirus were generated by fusion of in vitro stimulated human spleen lymphocytes from an HCMV-seropositive 53-year-old organ donor with the mouse myeloma cell line Ag8.653. Fourteen human/mouse hybridomas producing anti-cytomegalovirus IgG were screened by an ELISA technique and four selected clones have been estabished since March 1988, generating about 5–40 μg/24 h IgG per ml culture supernatant. Reference and local cytomegalovirus strains were stained by the antibodies without showing cross-reactivity to other herpes viruses. Three monoclonal antibodies, A4B4 (IgG11), A6B3 (IgG1k) and A6A2 (IgG1k), immunoprecipitated a 68 kDa early viral protein which appears during the infectious cycle, first in the nucleus (18–24 h) and then also in the cytoplasm (24–96 h) of infected cells. Inhibition of DNA replication restricted the detection of the 68 kDa viral protein to the nucleus of infected cells. Staining of unfixed infected cells showed that two of the antibodies bound at the surface of a few cells. The fourth monoclonal antibody A3C5 (IgG11) immunoprecipitated a 34/38 kDa late viral protein which appears in the nucleus (48–72 h) of infected cells. These antibodies enable us to study the human host response to human cytomegalovirus and to elucidate the functions of human antibodies especially in their interaction with the T-cell response.

To study the human fetal B cell repertoire, liver and spleen lymphocytes were fused with the human × mouse heteromyeloma line CB-F7. Initially, 2310 IgM and 181 IgG producing hybridoma lines were established. Culture supernatants were analysed for binding activity to antigens from both the exogeneous and the endogeneous environments. For IgG secreting cell lines no antigenetic specificity has been detected. However, independently from the gestational age, monoclonal IgM antibodies binding to bacterial antigens (tetanus toxoid, lipid A, N. meningitidis antigens) were found. In particular, nearly 10% of the hybridomas obtained from fetal liver produced antibodies binding to lipid A (endotoxin). Among the IgM antibodies with anti-bacterial specificities approximately 30% were found to be polyspecific, i.e. these antibodies recognized auto-antigens of different molecular origin as well (ssDNA, keratin, myosin, actin). We conclude that polyreactive natural IgM antibodies may be generated during feta/life, which may take part in the formation of a humoral anti-infectious first line defense barrier in the neonate.