Moloney murine leukemia virus tolerance in anti-CD4 monoclonal antibody-treated adult mice.

Adult mice injected with Moloney murine leukemia virus (M-MuLV) 1 day after a short term treatment with anti-CD4 mAb developed T cell lymphomas, or sarcomas when rechallenged with Moloney murine sarcoma virus (M-MSV). Neoplastic development was correlated with virus spread, as thymic and peripheral T and B lymphocytes promptly expressed M-MuLV-induced Ag after virus introduction; no virus-specific CTL generation was detected in MLTC. This failure, which was selective for M-MuLV-induced Ag, persisted throughout the life span of the mice, and was not sustained by suppressor cell activity. The frequencies of splenic virus-specific CTL precursor varied in relation to time after virus injection; in the first postinjection month, frequencies were similar to those in nonimmune control mice, while at 4 mo post-virus exposure, they showed a striking reduction. These findings indicate that a transient functional impairment of CD4+ cells at the time of retrovirus injection provided the appropriate milieu for tolerance induction in both the peripheral mature and intrathymic T cell compartments.     

Amelioration of experimental lung metastasis in mice by therapy with anti-CD3 monoclonal antibodies

Summary Binding of CD3-specific antibodies to the TcR-CD3 complex results in T cell activation without the need for occupation of the T cell receptor (TcR) by its ligand. Murine T cells activated in this manner will kill a broad range of tumor targets but not normal lymphoblasts. We report here that non-specific cytolytic activity can be induced in vivo by a single i.p. injection of nonlytic 145-2C11 anti-CD3 monoclonal antibody. At least three populations of effector cells are activated in these mice. These are non-MHC(major histocompatibility complex) restricted cytotoxic T lymphocytes, activated natural killer cells, and lymphokine-activated killer cells. Anti-CD3 treatment is effective in significantly reducing the number of lung tumor nodules which form in mice inoculated with oncogenic ras-transfected syngeneic 10T1/2 fibroblasts. Anti-CD3-activated killer cells may, therefore, find a future role in cancer immunotherapy.     

Granulysin-mediated tumor rejection in transgenic mice

Granulysin (GNLY) is a cytolytic molecule expressed by human CTL and NK cells with activity against a variety of tumors and microbes, including Mycobacterium tuberculosis. Although the molecular mechanism of GNLY-induced apoptosis of Jurkat T cells is well defined in vitro, no direct evidence for its in vivo effects has been demonstrated. Because there is no murine homologue of GNLY, we generated mice expressing GNLY using a bacterial artificial chromosome containing the human GNLY gene and its 5' and 3' flanking regions. GNLY is expressed in leukocytes from transgenic mice with similar kinetics as in PBMC from humans: GNLY is constitutively expressed in NK cells and, following stimulation through the TCR, appears in T lymphocytes 8-10 days after activation. Both forms of GNLY (9 and 15 kDa) are produced by activated T cells, whereas the 15-kDa form predominates in freshly isolated NK cells from transgenic animals. GNLY mRNA is highest in spleen, with detectable expression in thymus and lungs, and minimal expression in heart, kidney, liver, muscle, intestine, and brain. Allospecific cell lines generated from GNLY transgenic animals showed enhanced killing of target cells. In vivo effects of GNLY were evaluated using the syngeneic T lymphoma tumor C6VL. GNLY transgenic mice survived significantly longer than nontransgenic littermates in response to a lethal tumor challenge. These findings demonstrate for the first time an in vivo effect of GNLY and suggest that GNLY may prove a useful therapeutic modality for the treatment of cancer.     

Remission of established disease in diabetic NOD mice induced by anti-CD3 monoclonal antibody]

Anti-CD3 monoclonal antibodies are potent immunosuppressants widely used in clinical transplantation to prevent or treat acute allograft rejection. We have used a hamster monoclonal antibody (145 2C11) specific for the epsilon chain of the murine CD3 complex to treat autoimmune insulin-dependent diabetes appearing spontaneously by 15 to 30 weeks of age in female Non Obese Diabetic (NOD) mice. Mice showing overt disease (glycosuria and glycemia > or = 4 g/l) were randomized in two groups receiving either anti-CD3 (5 micrograms/day i.v. for 5 consecutive days) or an identical dose of hamster polyclonal immunoglobulins. Progressive remission of disease was observed, 3 to 6 weeks after the end of treatment, in 80% of anti-CD3 treated mice as compared to 6% of mice in the control group. This remission was maintained long term namely, during the 4 to 5 months (after the end of treatment) observation period. These results open interesting perspectives on the possibility to treat recently diagnosed diabetic patients with therapy showing long term efficacy and no chronic toxicity.     

Differential immunomodulation by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell activation: dependence upon the individual anti-CD3 monoclonal antibody used for activation

Two monoclonal antibodies (mAb) recognizing different CD2 epitopes each inhibited anti-CD3-induced proliferation and anti-CD3-induced increase in surface CD2 expression. The magnitude of inhibition by either anti-CD2 mAb was dependent upon which anti-CD3 mAb was used as the stimulus, being more pronounced when the anti-CD3 mAb 454 was used as the stimulus than when either anti-CD3 mAb 147 or 446 was the stimulus. The effects of neuraminidase-treated sheep erythrocytes (which bind to CD2) were also more pronounced on mAb 454-induced proliferation than on mAb 147- or 446-induced proliferation. Furthermore, the effects of preincubation with anti-CD2 mAb depended upon the responder status of the donor to IgG1 anti-CD3 mAb. Preincubation of high-responder cells with anti-CD2 mAb had little effect on subsequent IgG1 anti-CD3-induced proliferation. In contrast, preincubation of low-responder cells with anti-CD2 mAb usually augmented the otherwise small proliferative response to IgG1 anti-CD3 mAb. Taken together, these observations suggest that interaction of surface CD2 with ligand alters the response of T cells to anti-CD3 mAb, but these effects depend upon the individual anti-CD3 mAb used for stimulation. These studies raise the possibility that perturbation of different parts of the CD3-T cell antigen receptor complex may lead to different sequelae, and, as a result, the T cell may respond to a given immunomodulator in different ways.     

Activation and expansion of tumour-infiltrating lymphocytes by anti-CD3 and anti-CD28 monoclonal antibodies

Summary Cytotoxic T lymphocytes from healthy donors can be expanded to high numbers from the peripheral blood using combinations of anti-CD3 and anti-CD28 monoclonal antibodies (mAb). We investigated whether these antibodies could also be used to induce outgrowth of tumour-infiltrating lymphocytes (TIL) from tumour tissue. In the initiation phase of TIL culture immobilized anti-CD3 antibodies together with anti-CD28 mAb and low-dose interleukin-2 induced a rapid expansion of T cells from various human tumour tissues. The cultured cells showed high levels of cytotoxic T lymphocyte activity, but low levels of lymphokine-activated killer cell activity were generated. This study shows that TIL can be efficiently expanded from tumour tissue by combinations of anti-CD3 and anti-CD28 antibodies. This protocol for cell expansion in vitro may substantially reduce the time required to reach sufficient numbers of TIL for re-infusion to the patient.     

Mechanism of indoleamine 2, 3-dioxygenase inhibiting cardiac allograft rejection in mice

Indoleamine 2, 3-dioxygenase (IDO)-mediated regulation of tryptophan metabolism plays an important role in immune tolerance in transplantation, but it has not been elucidated which mechanism specifically induces the occurrence of immune tolerance. Our study revealed that IDO exerts immunosuppressive effects through two pathways in mouse heart transplantation, ‘tryptophan depletion’ and ‘tryptophan metabolite accumulation’. The synergism between IDO⁺DC and TC (tryptophan catabolic products) has stronger inhibitory effects on T lymphocyte proliferation and mouse heart transplant rejection than the two intervention factors alone, and significantly prolong the survival time of donor-derived transplanted skin. This work demonstrates that the combination of IDO⁺DC and TC can induce immune tolerance to a greater extent, and reduce the rejection of transplanted organs.     

Anti-tumor effects of depleting and non-depleting anti-CD27 monoclonal antibodies in immune-competent mice

CD27 plays an important role in T-cell co-stimulation and is also expressed on lymphomas. In the present study, we generated novel depleting and non-depleting monoclonal antibodies (mAbs) against mouse CD27 and characterized their co-stimulatory activity in vitro and anti-tumor effects in immune-competent mice bearing syngeneic T-cell lymphoma (EG7) expressing or lacking CD27. A profound anti-tumor effect was observed with a non-depleting mAb (RM27-3E5), but not with a depleting mAb (RM27-3C1), against either EG7/CD27(+) or EG7/CD27(−) tumors, which was associated with the induction of EG7-specific cytotoxic T lymphocytes (CTLs). Consistently, the anti-tumor effect of RM27-3E5 was abolished in T cell-deficient nude mice. These results indicate that a non-depleting agonistic mAb against CD27 is promising for cancer therapy by co-stimulating tumor-specific CTL induction.     

Inhibition by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell-dependent B cell activation

Anti-CD3 mAb can activate T cells to help in B cell activation as detected by late events, such as maturation of B cells into Ig-secreting cells (IgSC), or by early events, such as B cell surface expression of the activation marker CD23. Two different anti-CD2 mAb each inhibited anti-CD3-induced T cell-dependent B cell activation in a dose-dependent fashion. Neither irradiation of the T cells prior to culture nor depletion of CD8+ cells abrogated the inhibitory effects of anti-CD2 mAb. Despite the ability of these anti-CD2 mAb to inhibit anti-CD3-induced IL2 production, addition of exogenous IL2 to anti-CD2 mAb-containing cultures could not fully reverse the inhibitory effects on IgSC generation. Furthermore, addition of various combinations of IL1, IL2, IL4, and IL6 or crude PBMC or monocyte culture supernatants also could not reverse anti-CD2-driven inhibition. In T cell-depleted cultures, anti-CD2 mAb had no effect on the ability of IL4 to induce B cell CD23 expression, confirming that anti-CD2 mAb had no direct effect on B cells. However, in cultures containing T+ non-T cells, anti-CD2 mAb did partially inhibit IL4-induced B cell CD23 expression. Taken together, these observations demonstrate that certain CD2 ligands can modulate T cell-dependent B cell activation by a mechanism which, at least in part, involves a direct effect by the CD2 ligand on the T cell itself.     

A functionally unique anti-CD3 monoclonal antibody cross-reactive with basal keratinocytes

Monoclonal antibodies (mAb) 147, 446, and 454 each recognize different epitopes of CD3. The CD3 epitope recognized by mAb 446 is functionally unique for the T cell. In contrast to mAb 147 and 454, mAb 446 induces modulation of surface CD3 with delayed kinetics and, hence, is impaired in inducing a refractory state in the T cell to subsequent anti-CD3-induced helper function. MAb 446 (but not other anti-CD3 mAb, including mAb 147, 454, OKT3, and anti-Leu4) recognizes a cytoplasmic determinant within basal keratinocytes. Extraction of keratinocytes with nonionic detergent and 2 M NaCl abolished subsequent staining with mAb 446 but enhanced subsequent staining with anti-keratin mAb, suggesting that this cross-reactive determinant is not keratin. Immunoprecipitation of internally labeled keratinocytes with the anti-CD3 mAb 147 and 446 failed to reveal specific bands, whereas these same mAb immunoprecipitated specific bands from internally labeled CD3+ Jurkat cells corresponding to previously identified CD3 subunits, suggesting that the cross-reactive determinant in keratinocytes is also not CD3. The cross-reactivity is not species specific, in that mAb 446 stained a mouse keratinocyte line, nor is it absolutely keratinocyte specific, in that mAb 446 stained one of the two nonkeratinocyte human epithelial cell lines tested. This study raises the possibility that perturbation of unique CD3 epitopes may have unique effects on T cell surface events and subsequent activation and function.     

Induction of T cell-dependent B cell differentiation by anti-CD3 monoclonal antibodies

Three monoclonal antibodies (mAb) recognizing the CD3 (T3) surface complex each induced B cell differentiation (as measured by PFC generation) in cultures containing T + non-T cells. Irradiation of the T cells before culture usually augmented the PFC response. An IgG2a mAb (454) induced PFC in all donors tested, whereas two IgG1 mAb (147 and 446) induced PFC in only 80% of the donors tested. This heterogeneity in PFC response to IgG1 anti-CD3 mAb strictly paralleled the heterogeneity in proliferative response to IgG1 anti-CD3 mAb and was governed by cells within the non-T population. In IgG1 anti-CD3 high responders (HR), all anti-CD3 mAb tested induced Tac expression. In IgG1 anti-CD3 low responders (LR), mAb 454 induced Tac expression, but mAb 147 did not. However, when the cultures were supplemented with exogenous interleukin 2, Tac expression and PFC generation in response to mAb 147 was similar to the response to mAb 454 in both HR and LR. The addition of anti-Tac to the cultures partially inhibited anti-CD3-induced PFC generation. These studies indicate that anti-CD3 mAb can lead to B cell differentiation under appropriate experimental conditions and may be valuable in studying polyclonal T cell-dependent B cell differentiation in normal and disease states.     

Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys

RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with ⁸⁹Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake.

Studies with ⁸⁹Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44⁺ or CD44^-) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44⁺, resp), PL45 (CD44⁺, non-resp) and HepG2 (CD44^–, non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose.

At 1, 2, 3, and 6 days after injection, ⁸⁹Zr-RG7356 uptake in MDA-MB-231 (CD44⁺, resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44^–, non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of ⁸⁹Zr-RG7356 was similar in MDA-MB-231 (CD44⁺, resp) and PL45 (CD44⁺, non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. ⁸⁹Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356.

⁸⁹Zr-RG7356 selectively targets CD44⁺ responsive and non-responsive tumors in mice and CD44⁺ tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44⁺ tumors may not be sufficient in itself to drive an anti-tumor response.     

A new monoclonal anti-CD3epsilon antibody reactive on paraffin sections.

We generated a new monoclonal antibody (MAb), F7.2.38, by immunizing mice with CD3varepsilongammadelta/CD3omega complexes purified from human T-cells by OKT3 MAb-Sepharose affinity chromatography. Immunoprecipitation experiments and Western blotting analysis showed that MAb F7.2.38 recognized the CD3varepsilon chain in CD3varepsilon cDNA-transfected FOX B-cells and in various T-cell lines. Using flow cytometry on permeabilized or intact cells, the epitope was found to be located in the cytoplasmic tail of the CD3varepsilon chain. Immunohistochemical staining on paraffin-embedded sections showed that the reactivity of MAb F7.2.38 was comparable to that of the commercially available anti-CD3varepsilon polyclonal antibody. Of the 52 well-characterized T-cell lymphomas, 41 were positive for F7. 2.38 (79%), whereas all 37 B-cell lymphomas and 69 non-lymphoid tumors were unreactive. This new anti-CD3varepsilon antibody would be particularly useful for phenotyping T-cell lymphomas on routinely processed paraffin-embedded tissue sections.     

Interleukin-2 receptor monoclonal antibodies have no effect on anti-CD3 monoclonal antibody-mediated cytotoxicity in CD3+ leukemic large granular lymphocytes

We studied the role of Fc receptors and interleukin-2 (IL-2) receptor subunits in anti-CD3 monoclonal antibody (MAb)-mediated cytotoxicity of CD3+ leukemic large granular lymphocytes (LGL). Peripheral blood mononuclear cells from four patients with CD3+ LGL leukemia were cultured with 1 microgram/ml of anti-CD3 MAb. Anti-CD3 MAb-mediated cytotoxicity was not inhibited when K562 target cells were preincubated with heat-aggregated human IgG, suggesting that binding of the effector cell-bound anti-CD3 MAb to Fc receptors of target was not involved in cytotoxicity. Induction of cytotoxicity was not blocked by the addition of either anti-p55 or anti-p75 IL-2 receptor MAbs. These results show that the induction of cytotoxicity by anti-CD3 MAb is not mediated through IL-2 receptor subunits in CD3+ leukemic LGL.     

Evolution of anti-CD20 monoclonal antibody therapeutics in oncology

Approval of an anti-CD20 chimeric monoclonal antibody, rituximab, has revolutionized cancer treatment and also validated CD20 targeting for providing benefit and improvement of overall response rate in B cell malignancies. Although many patients have benefited from the treatment of rituximab, there are still significant numbers of patients who are refractory or develop resistance to the treatment. Here we discuss pre-clinically well-defined potential mechanisms of action for rituximab and review the ways next generation anti-CD20 monoclonal antibodies can potentially exploit them to further enhance the treatment of B cell malignancies. Although the relative importance of each of these mechanism remains to be established in the clinic, well-designed clinical trials will help to define the efficacy and understanding of which effector activity of modified next generation anti-CD20 mAb will be important in the treatment of B-cell malignancies.Key words: CD20, NHL, CLL, monoclonal antibody, next generation anti-CD20 antibodies, ADCC, CDC, ADCP, PCD, rituximab     

Adaptive immune defects and delayed rejection of allogeneic tumor cells in beige mice

The effect of the beige (bg) mutation on adaptive allogeneic tumor rejection was examined by monitoring tumor cell survival in vivo using [131I]iododeoxyuridine-prelabeled cells. Accelerated elimination of allogeneic tumor cells normally begins 8 days after ip injection and is due to active immune responses. Two independent mutations to beige on two different inbred backgrounds (C57BL/6J bgJ and DBA/2JCo bg8J) were tested, and bg/bg mice showed a 1-day delay in immune elimination of allogeneic cells. This delayed rejection was not due to a defect in clearing label from dead cells, nor to an inability to effect antibody-induced killing in vivo. Both humoral and cell-mediated responses against the allogeneic tumor cells were significantly lower in bg/bg than in +/bg mice.     

Regulation of cytolytic activity in CD3- and CD3+ killer cell clones by monoclonal antibodies (anti-CD16, anti-CD2, anti-CD3) depends on subclass specificity of target cell IgG-FcR

Anti-CD3 MAb can inhibit MHC-restricted cytolytic activity of CD3+ mature cytotoxic T cells. In particular effector-target cell combinations, however, anti-CD3 MAb enhance or induce cytolysis by cross-linking CD3+ effector and IgG-FcR+ target cells. Virtually all natural killer (NK) cells or NK cell-derived clones are CD3-4-8- but do express CD2 and CD16 (IgG-FcR) antigens. We have studied how these cell surface molecules are involved in the regulation of cytolytic activities. The addition of anti-CD2 MAb to effector and target cells was found to induce conjugate formation of the IgG-FcR+ target cells with the effector cell and nonspecific cytolysis of, for instance, the P815 mouse mastocytoma cells. Enhancement or induction of conjugate formation and cytolysis of IgG-FcR+, P815, U937, and Daudi cells was also accomplished by using anti-CD16 MAb (e.g., Leu-11c (B73.1) or CLB Fc-gran 1 (VD2) MAb). Some human and mouse tumor cell lines (K562, P815, and U937) appear to express distinct types of IgG-FcR, showing different affinities for distinct subclasses of MAb (e.g., IgG1, IgG2a), but another line (Daudi) expresses only one type of IgG-FcR preferentially binding IgG1 MAb. Here we demonstrate that IgG-FcR on the effector cells can act as activation sites because anti-CD3 as well as anti-CD16 MAb of IgG1 and IgG2a subclasses can induce lytic activity of target cells bearing the relevant IgG-FcR. These data demonstrate that induction of conjugate formation and cytolysis by MAb occur when the target cells bear IgG-FcR with "specificity" for those MAb. Thus, besides via CD3, cytolytic activity by mature T and NK cells also can be induced via the CD2 and CD16 antigens on these cells.     

Effects of in vivo administration of anti-CD3 monoclonal antibody on T cell function in mice. II. In vivo activation of T cells

Anti-CD3 mAb are known to be both immunosuppressive and mitogenic to T cells in vitro. However, only immunosuppression has been observed after in vivo administration of these mAb. The present study demonstrates that T cell activation does occur after in vivo administration of anti-CD3 mAb to mice, evidenced by increased IL-2R expression on T cells, CSF secretion, and extra-medullary hematopoiesis in the spleen. These effects required multivalent cross-linking of the mAb, since F(ab')2 fragments failed to induce them. However, the F(ab')2 fragments did induce modulation of CD3/TCR from the surface of T cells, demonstrating that TCR modulation is not sufficient to induce activation. In addition, interaction of the TCR with either intact or F(ab')2 fragments of the mAb led to increased expression of CD8 in vivo, suggesting that the F(ab')2 fragments of anti-CD3 mAb might be capable of inducing a T cell to undergo some, but not all, of the changes involved in reaching a fully activated state. Further study of the activating effects of anti-CD3 mAb might increase the understanding of the mechanisms of in vivo T cell activation and might also be exploited clinically to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myelodysplastic disorders.