Sarah M. Andrew

The effect of temperature on the binding kinetics and equilibrium constants of monoclonal antibodies to cell surface antigens

The effect of temperature on the kinetic association and dissociation binding parameters, and equilibrium constants of four monoclonal antibodies to the murine Ly-2.1 and Ly-3.1 antigens has been studied using flow cytometry. All four monoclonal antibodies were conjugated to FITC and their association to, and dissociation from, the surface of murine thymoma cells was observed at 15 sec intervals, at temperatures between 1 and 37 degrees C. The initial association rate constant and the dissociation rate constant for each antibody at each temperature were calculated from graphs of the first-order reactions and it was demonstrated that an increase in temperature caused an increase in both association rate and dissociation rate of the antibodies. Generally the increase in association rate with temperature was less than the increase in dissociation rate. Differences between antibodies to the same antigen (Ly-2.1) suggest that changes in membrane fluidity were not solely responsible for the changes in association rate. However, the equilibrium constants (Keq) did not always show a simple relationship of increasing temperature causing decreasing values for Keq. For one antibody the highest value for Keq was seen at 17 degrees C rather than at 37 degrees C and differences in Keq between individual antibodies were greater at 1 degree C than at 37 degrees C. Kinetic rate constants are usually measured at 4 degrees C or room temperature, therefore for antibodies under consideration for in vivo use, measurements at 37 degrees C are more appropriate.

Targeting of anti-tumor responses with bispecific antibodies

T cells can be induced to specifically lyse tumor cells with bispecific antibodies containing anti-T cell receptor mAbs crosslinked to anti-tumor mAbs. Such targeted cytolysis requires that the target cell be bound directly to the cytotoxic cell. In addition, targeted T cells mediate a second activity, the secretion of factors that can block the growth of both tumor target cells and bystander tumor cells. When given to nude mice bearing intraperitoneal human ovarian carcinoma, targeted human T cells cause the rapid removal of most tumor cells from the peritoneum, and markedly prolong the times of survival of treated mice. The efficacy of targeted T cells for treating human cancer is currently being tested in clinical trials.

Human non-lineage antigen, CD46 (HuLy-m5): purification and partial sequencing demonstrates structural homology with complement-regulating glycoproteins.

CD46, until recently known as HuLy-m5, is a non-lineage restricted surface antigen ubiquitously expressed by almost all human cells except erythrocytes. The CD46 antigen is identified by the E4.3 monoclonal antibody (mAb) and exists at the surface of human peripheral blood lymphocytes (PBLs) as two acidic, non-disulfide bonded chains, α and β, ofMr 66 000 and 56 000. Receptor density analysis showed that CD46 was of moderately low abundance on PBLs with 7.5×103 molecules present on each cell. The two chains of CD46 were purified (144 000-fold) by immunoaffinity-chromatography with E4.3 mAb from the plasma membranes of a human spleen infiltrated with chronic myelogenous leukemia cells. Amino acid sequence analysis of the NH2-terminal of both α and β chains yielded the same sequence; XEEPPQ/TFEAMELIGKPKPYYEIGE. Peptide mapping studies confirmed that both CD46 chains were closely related, except for one peptide fragment. This amino acid sequence is identical to that of the NH2-terminal of the recently cloned membrane co-factor protein (MCP), a membrane protein that binds the C3b and C4b fragments of complement and acts as a co-factor for I protein-mediated decay of the complement convertases. CD46 shares a cross-reactive epitope with some primate retroviruses, and this may indicate that some retroviruses mimic the mechanisms used by autologous human cells to evade complement-mediated immune clearance.

Bispecific antibodies and retargeted cellular cytotoxicity: novel approaches to cancer therapy

SummaryWe have used a relatively new technology to increase the number of human lymphocytes that will react with human ovarian carcinoma cells. This technology, often called “retargeting of the immune system,” can temporarity redirect the activity of immune cells that were originally committed to react with foreign substances other than cancer cells. In the example presented here, the antitumor effects of retargeted human T lymphocytes, collected from normal donors, were tested in immunodeficient mice with a human ovarian carcinoma line growing intraperitoneally. We retargeted T cells in vitro with a bispecific antibody that reacted with the T cell receptor complex and with a cell-surface antigen expressed by the ovarian carcinoma cells. Retargeted lymphocytes, injected intraperitoneally into mice 4 days after intraperitoneal injection of the tumor cells, impeded tumor growth and doubled the host survival time. These findings provide support for the concept that treatment of ovarian cancer patients with retargeted T cells could prove beneficial.

Immunochemotherapy of human colon carcinoma xenografts in nude mice using combinations of idarubicin-monoclonal antibody conjugates

Tumour cell heterogeneity is probably a principal cause of treatment failure and represents a formidable barrier for effective antibody‐targeted chemotherapy. Idarubicin (Ida), a more potent and less cardiotoxic analogue of daunomycin, has been demonstrated to specifically target and eradicate homogeneous, cloned, murine tumour cell populations in vitro and in vivo when coupled to monoclonal antibodies (MoAb); however, the antitumour activity of Ida‐MoAb conjugates against human tumour xenografts remains to be established. In this study, the value of cotargeting conjugates to different human tumour‐associated antigens within a solid tumour has been assessed by comparing the effects of combinations of Ida‐anti‐colon carcinoma MoAb conjugates with any one Ida‐anti‐colon carcinoma MoAb conjugate used alone. Individual Ida‐MoAb conjugates have previously been evaluated for their specific binding and cytotoxicity to one of two different human colon carcinoma xenografts (Colo 205 or LIM2210) in vitro, although their efficacy alone or in combination required assessment in vivo. Combinations of the most effective Ida‐MoAb conjugates were demonstrated to enable a greater number of complete tumour regressions than the most efficacious Ida‐MoAb conjugate administered alone in vivo; some combinations inhibited control tumour growth by up to 95%. This study suggests that Ida‐MoAb conjugates can be effective against subcutaneous human tumours in nude mice, although it is unlikely that any single conjugate will eradicate all the tumour cells in a solid tumour, and the value of ‘cocktails’ of drug‐MoAb conjugates against some xenografts (i.e. LIM2210) appears to be limited.

Redirection of cellular cytotoxicity: a two step approach using recombinant single-chain Fv molecules.

In this article the authors discuss an indirect system for redirecting cellular cytotoxicity, which utilizes a “universal” bispecific antibody to redirect T-cells to kill cells targeted with single-chain Fv (sFv) fusion proteins that carry a peptide tag recognized by the bispecific antibody. This approach has a number of theoretical advantages in the immunotherapy of cancer.