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Therapeutic antibodies sometimes elicit anti-drug antibodies (ADAs) that can affect efficacy and safety. Engineered antibodies that contain artificial amino acid sequences are potentially highly immunogenic, but this is currently difficult to predict. Therefore, it is important to efficiently assess immunogenicity during the development of complex antibody-based formats. Here, we present an in vitro peripheral blood mononuclear cell-based assay that can be used to assess immunogenicity potential within 3 days. This method involves examining the frequency and function of interleukin (IL)-2-secreting CD4 + T cells induced by therapeutic antibodies. IL-2-secreting CD4 + T cells seem to be functionally relevant to the immunogenic potential due to their proliferative activity and the expression of several cytokines. The rates of the donors responding to low and high immunogenic proteins, mAb1, and keyhole limpet hemocyanin were 1.3% and 93.5%, respectively. Seven antibodies with known rates of immunogenicity (etanercept, emicizumab, abciximab, romosozumab, blosozumab, humanized anti-human A33 antibody, and bococizumab) induced responses in 1.9%, 3.8%, 6.4%, 10.0%, 29.2%, 43.8%, and 89.5% of donors, respectively. These data are comparable with ADA incidences in clinical settings. Our results show that this assay can contribute to the swift assessment and mechanistic understanding of the immunogenicity of therapeutic antibodies.

The radiosensitivity of immune responses to allogeneic tumor cells was compared. Delayed hypersensitivity as demonstrated by macrophage migration inhibition and antibody production as detected by immune adherence test were extremely radiosensitive. The radiosensitive nature of macrophage migration inhibition was in marked contrast to he radioresistance of the delayed footpad reaction. The generation of cytotoxicity was relatively radioresistant.

Direct cytotoxicity raised at an early interval after immunization with chicken erythrocytes (CRBC) has been ascribed to O-positive, immunoglobulin (Ig)-bearing lymphocytes. Cytotoxicity of such effector cells was not affected by trypsinization. Cytotoxity was reduced by the treatment of effector cells with anti-IgG serum and complement or by the addition of anti-IgG serum to culture medium for cytotoxicity test. When the effector cells were treated with trypsin before cytotoxicity test, such procedures with anti-IgG serum did not affect their cytotoxicity. Ig molecules on cytotoxic T lympohcytes may not be an essential element for antigen recognition. This explanation may be supported by the fact that cytotoxicity against CRBC could be raised in 600 rad irradiation, thymus-cell transferred mice in the absence of antibody production.

Following priming with alloantigen in vivo, a secondary cytotoxic activity of AKR mice (H-2k) was generated by in vitro stimulation with the same alloantigen. Such secondary effectors could lyse not only targets of a sensitizing haplotype but also third-party targets unrelated to sensitizing or responding cells. Such cross-reactive cytotoxicity of AKR secondary effectors were demonstrated not only by stimulation with C57BL/6 (H-2b) cells but also with C3H/He (H-2k) cells. It was observed that such cross-reactive secondary cytotoxicity was mediated by T lymphocytes, and required protein synthesis in secondary culture. These results suggested the possibility that the antigen receptors on such secondary cytotoxic effector cells have different recognitive specificity from that on primary cytotoxic effector cells.

After administration of indomethacin, an inhibitor of prostaglandin synthesis, the number of splenic T cells increased in normal mice but not in adult-thymectomized or athymic nude mice. The enlarged T-cell population consisted mainly of Lyt-1+2+ cells. This thymus-dependent increase in T-cell population augmented in vivo antibody response to sheep erythrocytes, a T-dependent antigen. The increased T-cell population also included suppressor cells that were eliminated by treatment with anti-Lyt-2 antibody plus complement. These results suggest that increased T cells in the spleen were recruited from the thymus by an indomethacin-mediated mechanism and participated in immune responses as regulator cells.

Immune responses were examined after immunization with chicken erythrocytes (CRBC) in mice. Cytotoxicity of spleen cells was assessed by the release of 51Cr from labelled target cells. (1) At early stages (day 4-7) after primary intraperitoneal immunization, direct cytotoxicity of spleen cells was raised efficiently in C57BL/6 and AKR mice, but not in C3H/He, SL and DDD mice. Delayed hypersensitivity and antibody production were raised to almost the same extent in all the strains at such periods. (2) Effector cells in direct cytotoxicity were theta-positive and IgG-positive, and glass-nonadherent and Nylon wool column-adherent. Effector cells in antibody-dependent cell-mediated cytotoxicity in the presence of antibody to CRBC were eliminated by treatment with anti-IgG serum, but not by treatment with anti-theta serum. (3) Cytotoxicity and antibody production were raised efficiently after intraperitoneal or intravenous immunization, but not after footpad immunization. On the other hand, delayed hypersensitivity developed most efficiently after footpad immunization.

Specific cytotoxicity against histocompatibility antigens of a parental strain was induced in the spleens of sublethally irradiated F1 mice after intravenous transfer of lymphoid cells from another parental strain. Such cytotoxicity reached the highest level at 10 days after the transfer of 3 to 5 X 10(7) spleen cells and was ascribed to be T lymphocytes of donor parental origin. Secondary response of the generation of cytotoxicity was compared among in vivo rechallenge, in vitro MLC and transfer to irradiated F1 mice. Cytotoxicity was not so augmented by in vivo rechallenge. It may be attributable to early elimination of grafts. In in vitro MLC and transfer to irradiated F1 mice, the generation of cytotoxicity appeared earlier with higher magnitudes as compared to primary response. The transfer system to irradiated F1 recipients appears to be more suitable for analyses of the generation of cytotoxic T lymphocytes from memory cells than in vivo rechallenge.

Effects of whole body irradiation with 600 rad on delayed hypersensitivity, direct cytotoxicity and antibody production were examined in mice immunized with chicken erythrocytes (CRBC) or sheep erythrocytes (SRBC) in saline. Delayed footpad reactions to CRBC were not affected by the exposure to irradiation 3 h before or after antigenic stimulation. On the other hand, direct cytotoxic activities in spleen cells and antibody titres to CRBC were reduced by such exposures. Additionally, delayed footpad reactions to SRBC were not affected by the exposure to irradiation. Generation of effector cells responsible for delayed footpad reactions proved to be resistant to irradiation.

Effects of thymectomy of various times after birth on cytotoxicity, delayed footpad reaction and antibody production were examined in 6 week old mice immunized intraperitoneally with chicken erythrocytes. Cytotoxicity and antibody production were abolished by thymectomy within 3 days after birth. Mice thymectomized at 5 days of age or later showed appreciable degrees of cytotoxicity and antibody production. On the other hand, a delayed footpad reaction could be elicited even in mice thymetocmized within 24 h after birth, although the degree of the reaction was reduced by such a treatment. Therefore, T cells responsible for delayed hypersensitivity were less thymus-dependent and required the presence of the thymus for a shorter period in their development that those responsible for cytotoxicity or antibody production.

The effects of thymectomy at various times after birth, 1 and 7 days (Tx-1, Tx-7), on delayed footpad reactions, cell-mediated cytotoxicity (CMC), production of migration inhibitory factor (MIF), and graft-versus-host reactions (GVHR) against allogeneic antigens were determined by experiments with 8-week-old mice. Delayed footpad reactions were detected in Tx-1 mice, but CMC, production of MIF, and the ability to raise GVHR were not detected in such mice. CMC was detected in Tx-7 mice. However, the ability to raise GVHR and to produce MIF was abolished by thymectomy at 7 days after birth. These results suggest that the effector mechanism to mount a GVHR might be mediated by delayed-type hypersensitivity of tuberculin type rather than CMC to host antigens.