Cristina Caldas

Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen.

Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.

Cellular autoreactivity against heat shock protein 60 in renal transplant patients: peripheral and graft-infiltrating responses

Autoreactivity to heat shock protein 60 (Hsp60) has been implicated in the pathogenesis and regulation of chronic inflammation, especially in autoimmune diseases. In transplantation, there is a lack of information regarding the cytokine profile and specificity of cells that recognize self‐Hsp60 as well as the kinetics of autoreactivity following transplantation. We studied the cellular reactivity of peripheral and graft‐infiltrating lymphocytes against Hsp60 in renal transplant patients. Cytokine production induced by this protein in peripheral blood mononuclear cells indicated a predominance of interleukin (IL)‐10 during the late post‐transplantation period, mainly in response to intermediate and C‐terminal peptides. Patients with chronic rejection presented reactivity to Hsp60 with a higher IL‐10/interferon (IFN)‐γ ratio compared to long‐term clinically stable patients. Graft‐infiltrating T cell lines, cocultured with antigen‐presenting cells, preferentially produced IL‐10 after Hsp60 stimulation. These results suggest that, besides its proinflammatory activity, autoreactivity to Hsp60 in transplantation may also have a regulatory role.

CD4+CD25+Foxp3+ Indirect Alloreactive T cells from Renal Transplant Patients Suppress Both the Direct and Indirect Pathways of Allorecognition

Alloreactive T cells recognize donor antigens by two routes: direct and indirect pathways of allorecognition. Although the direct pathway is reported to be dominant in allograft rejection, indirect allorecognition also plays an important role. Indirect alloreactivity is also observed in renal transplant patients irrespective of rejection. Previously we showed a predominance of interleukin (IL)‐10 induced by indirect allorecognition of donor human leucocyte antigen (HLA)‐DR peptides, suggesting the existence of indirect alloreactive T cells displaying regulatory activity. In the present work, our objective was to characterize these regulatory T cells. We detected indirect alloproliferation of peripheral blood mononuclear cells (PBMC) from renal transplant patients, induced by donor HLA‐DR peptides, dependent on IL‐4 or IL‐10, suggesting regulatory activity as part of the alloreactive T‐cell repertoire. PBMC‐derived indirect alloreactive T‐cell lines were established and produced both inflammatory and regulatory cytokines. We showed that two of these T‐cell lines which were able to inhibit both direct and indirect alloproliferation of another T‐cell line from the same patient presented a CD4+CD25+Foxp3+ T‐cell population. These data support the idea that indirect alloreactive T cells may also have regulatory activity and may contribute to the maintenance of the human renal allograft.

Angiostatin-like molecules are generated by snake venom metalloproteinases

Angiostatin is a plasminogen-derived anti-angiogenic factor composed of its first four kringle structures. This molecule is generated by proteolytic cleavage of plasminogen by some proteolytic enzymes in vitro. Since venoms of viper snakes are a rich source of both serine- and metalloproteinase, we hypothesized that angiostatin-like polypeptides could be generated during the envenomation after snake bites and play a pathophysiological role in the local tissue damage and regeneration. Our results showed that crude venoms from several species of Bothrops snakes were able to generate angiostatin-like polypeptides and purified metalloproteinases but not serine proteinases from Bothrops jararaca and Bothrops moojeni venoms were responsible for their generation in vitro. The putative plasminogen cleavage sites by the crude venoms and purified proteinases were determined by N-terminal amino acid sequencing of the angiostatin-like molecules. Angiostatin-like peptides derived from human plasminogen digestion by jararhagin, a metalloproteinase isolated from B. jararaca venom, inhibited endothelial cell proliferation in vitro. These results indicate that angiostatin-like molecules can be generated upon snakebite envenomations and may account for the poor and incomplete regenerative response observed in the damaged tissue.

Treatment with Encapsulated Hsp60 Peptide (p277) Prolongs Skin Graft Survival in a Murine Model of Minor Antigen Disparity

The increased expression of heat shock protein (Hsp)60 in different kinds of graft tissues has been associated with a proinflammatory role and rejection. However, there are very few reports in which treatment with Hsp60 delays skin allograft rejection. The aim of this work was to evaluate the capacity of encapsulated human Hsp60‐derived peptide p277 to delay graft rejection in two murine models of skin transplantation with minor antigen disparities. Briefly, BALB/c mice and C57BL/6 were intranasally pre‐treated with five doses of Hsp60 p277 peptide encapsulated in polylactide‐co‐glycolide acid microspheres (PLGM), and received skin grafts from DBA2 mice and 129/B6 (F1) mice respectively. The treatment with the peptide increased skin graft survival more than 20 days in both the mouse strains, mainly in C57BL/6 recipients (P < 0.05). Also, p277‐treated BALB/c and C57BL/6 mice showed IL‐10 and IFN‐γ production, induced by p277 peptide. For the first time, a mucosal schedule using the Hsp60 C‐terminal peptide p277 encapsulated in PLGM showed some survival prolongation of skin grafts bearing minor antigen disparities. Our results suggest a potential role for Hsp60‐based therapy and the mucosal route as a useful tool to control the inflammatory response to allografts.

Diversity of physiological cell reactivity to heat shock protein 60 in different mouse strains

Abstract Heat shock proteins (Hsp) are families of highly conserved molecules and immunodominant antigens in some infections and in autoimmune diseases. Some reports suggest that different regions of the Hsp60 molecule induce distinct immune responses. However, there are no reports comparing physiological T-cell reactivity to Hsp60 in mice. In this study, we have analyzed T-cell proliferation and cytokine production induced by Hsp60, under physiological conditions, in three mouse strains bearing distinct major histocompatibility complex (MHC) backgrounds. Proliferative response predominantly was found in C57BL/6 mice, mostly induced by N-terminal and intermediate Hsp60 peptides (P < 0.0001). Interferon-γ (IFNγ) production was broadly induced by different regions of Hsp60 in all three mouse strains, although response was focused in different peptide groups in each strain. We did not observe an exclusive Th1 or Th2 cytokine profile induced by any particular region of Hsp60. However, we identified a strain hierarchy in IL-10 production induced by Hsp60 peptides from different regions, mostly detected in C3H/HePas, and in BALB/c, but not in C57BL/6 mice. In contrast, IL-4 production only was induced by the intermediate and C-terminal region peptides in both C3H/HePas and BALB/c mice. Our data give original information on physiological cellular reactivity to Hsp60. We also have identified peptides with the capacity to induce the production of anti-inflammatory cytokines, bringing perspectives for their use in immunotherapy of chronic inflammatory diseases and allograft rejection.

Contrasting roles of donor and recipient TGFB1 and IFNG gene polymorphic variants in chronic kidney transplant rejection.

OBJECTIVE To assess the long-term impact (minimum of 3 years follow-up) of polymorphisms in cytokine genes in donor:recipient pairs on the results of the transplant. METHODS We compared genetic cytokine polymorphisms and the primary factors of risk for the development of chronic rejection in paired groups of renal transplant patients with and without chronic allograft nephropathy [CAN]. RESULTS Multivariate analysis indicated that the presence of the high-production TT genotype (codon 10) of the transforming growth factor beta-1 (TGFB1) was protective in receptors (p=0.017), contrasting with the increased risk when present in donor samples (p=0.049). On the other hand, in the case of the gamma interferon studied, the greater frequency of the high production allele was protective in the analysis of the donor group (p=0.013), increasing the risk of chronic nephropathy of the allograft when present in the recipients (p=0.036). CONCLUSION Our results highlight the importance of TGFB1 genotyping in donors, and indicate that polymorphisms in the gene of this cytokine in donor cells might contribute to the development of chronic allograft nephropathy.

Design Approach Regarding Humanization and Functionality of an Anti-CD18 Monoclonal Antibody

In this work the closest human germline sequence was used as the framework on to which to graft murine CDRs. The proposed fully humanized version displays the same staining pattern to different cell subpopulations like CD4, CD8 and memory CD45RO lymphocytes, when compared with the original anti-CD18 MAb, reinforcing the germline approach as a successful strategy to humanize antibodies with maintenance of affinity and selectivity.