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Construction of a recombinant bispecific antibody (diabody) of minimal size for retargeting T-cells to human carcinomas
T cells in cancer
25 June 1997 - Poster presentations
1P.5.10.08 1 Apoptosis and growlng profile induced by different mitogenic signals in PBMC from prostate cancer B. Martinez-Mattin ‘, M.PBrez-Bias2, A. Ptieto ’I F. Esquivel ’, M.P. Hemdndez' , F.Cani6n3, J. Carballido3. M. Alv&ez-Mon ‘. ‘Depl. of Medicine, S&co/ of Medicine, University of A/ca/a, *Dept. of Immunology; School of Medicine, University Complurense of Madrid, 3Lbpt, of Medicine, School of Medicine, lInkersi& of the Andes, Santiago de Chile, 4Service of Urology, Clinica “Puerta de Hieuo”, Madrid, Spain The etiology of the prostatic cancer (Cap), one of the most common cancer in men, is unknown, although it is thought to be multifactorial. Histological observation of the prostate evidences areas of lymphocytic infiltration. It is known that cells of the immune system provide differentiation factors influencing in the differentiation and growing of cancer cells. In previous studies we have found that peripheral blood mononuclear cells (PBMC) from CaP patients show a defective proliferative response to surface (PHA) and cytoplasmatic (PMA) mitogens. Thus we have studied the viability, apoptosis and the change in the isoforms of CD45 in the PBMC from 12 CaP patients and 12 healthy men controls by flow cytometry with an internal standard that allows us to find the absolute number of lymphocytes and their subsets that are in the cultures. The diminished proiiierative response found with many of the mitogans used in culture with lymphocytes from CaP patienes was related to a lower number of lymphocytes in the culture. This effect can not be so much adscribed to an increase in the apoptotic rate but to a lower proliferative response in certain lymphocyte subsets depending on the mitogen used. Hence we found fewer CD8 lymphocytes in cultures from CaP patients than in those from controls stimulated with PHA. Moreover fewer CD4 and CD8 lymphocytes and lower generation of CD45RO were obsetved in cultures from CaP than in those from controls when PMA with anti-CD3 or lonomycin was used. These results indicate the PBMC from CaP patients show a different response to surface and citoplasmatic mitogens and this response is always lower than those found in the controls.
1P.5.10.09 ) Characterization of lymphocytes isolated from cervical (pre-) neoplastic lesions associated with human papillomavirus (HPV) N. Jacobs, W. Al-Saled, S.L. Giannini, P. Hubert, L.-M. Dupuis, J. Boniver, P. Delvenne. University of LIEGE, CHU, Department of Pathology, 64000 Liege, Belgium Our aim has been to identify the factors, such as cytokines that may play a role in the transition from the state of immunosufveillance to a state of immunotolerante, during the progression of cervical lesions and the development of cancer. Previous studies have shown that in cervical pre-neoplastic lesions the production of the type 2 cytokines IL4 (detected by immunohistochemistly) increased with the progression of the tumor. In contrast, the production a type 1 cytokine It2 decreased. The cells producing these cytokines were only detected in the strorna undetlying the lesions. We have developed a technique to isolate and cultivate lymphocytes from the epithelium and the stroma of small lesional biopsies (3-4 mm) to determine the cytokines produced by the lymphocytes infiltrating the lesion. The cytokine expression and production by the lymphocytes derived from normal and lesional biopsies was analysed by RT-PCR and ELISA. Our preliminary experiments demonstrated that lymphocytes derived from the stmma of normal and lesional biopsies produced both IFNy (type 1) and IL4 (type 2). In addition, analyse of the cell surface phenotype has shown that within the lymphocytes culture from the lesional biopsies the percentage of CD8+ T cells is diminished compared to the normal biopsies. We are in the progress of studying a larger number of biopsies and other cytokines to extend these results.
P.5.10.10
kiiopathic CD4+ T iymphocytopania with prickle cell carcinoma of the tongue: A case report
P.E. Manconi, P.F. Biddau, M.L. Di Martino, E. Muggianu, M.G. Cau, A. Ibba, G.S. Del Giacco. Dipatimento Scienze Mediche and Diparfimento Biologia Erd Ew/utiw, Universira di Cag/iari, Cagbatf, k/y
Introduction: We report on one patient with idiopathic CD4+ T cell depletion in the absence of HIV infection, which developed a pickle cell carcinoma of the tongue. PfWnt and Methods:A 28 years old whii woman from Cagliad (Sardinia, Italy) presented to us with mild lymphocytcpenia (total lymphocytes = lOlO/fil, CD4+ = 249/& CD8+ = 425/& CD4/CD8 ratio = 58). Medical history revealed toxoplasmosis in the first months of life and a severe scoliosis. At the beginning of 1998 the patient was accepted in a Medical ward for colyoretinitis by toxoplasma. In September a prickle cell carcinoma of the tongue was detected by surgical biopsy. The patient was then treated with radiotherapy.
451
Before radiotherapy, a defii of CD4+ lymphocytes was noted. Clinical examination revealed: “doll”facies, hyperchmmic speckle, pterygium, feeble voice, ulcer-necrotic lesion of the base of the tongue. One year after the first evaluation, lymphocyte subsets were quantified by lmmunocount (Ortho, Raritan, NJ): total lymphocytes were 4OLI&l), CD4+ (79 cells/flI), CD8+ (181 cells@) and CD4/CD8 ratio (0.4). CD 19+ B cells 83/pl, and CD18+ CD3- NK cells = 125/wl. A feeble depletion of lgG4 and lgG2, without any abnormality of other Ig classes was found in the serum. Conclusion:Pickle cell carcinoma is usually presented only by older people, particularly by heavy smokers. The presence of such a cancer in a non-smocking young girl affected with a severe depletion of CD4 is in agreement with a protective role of these lymphocytes against cancer.
P.5.10.11
Constructlon of a recombinant bispeciflc antlbody (diabodv) of minimal size for retametlna Tcells to human Gcinomas
Wijnand Helfrich, Bart Jan Kroesen, lngrid Molema, Lidia Westers, Loe de Leij. University Hospital Groningen, Oepf. Clinical lmmunolow. Oostersingel59, 9713 EZ Groningen, The Netherlands Bispecific antibodies (BsAb) have strong potential for tumor therapy by retargeting cytotoxic effector cells against human carcinomas. Bsab are traditionally prepared by hybrid hybridoma technology or via chemical coupling of Fab’fmgmerits. Clinical evaluation is however hampered by the diicuity to prepare them in sufficient quantity and purity. In addition, the size and the immunogenicity of the resultant molecules is expected to reduce their efficacy even further. Diabodies are very small recombinant dimeric antibody scFv fragments that can be engineered to harbour two diierent antigen-binding sites. Bispecific diabodies directed against cell-surface antigens are capable of bringing together two cells, such as in cell-targeted therapy. In diabodies, the two antigen-binding sites are at opposite ends of the molecule and separated by approximately 0.85 nm. The average molecular weight of a diabody is only appr. 52 kDa (IgG appr. 150 kDa). In this project a high affinity muline anti-EGP-2 (MOC31) scFv and humanized anti-CD3 (Uchtl v9) scFv were first recast into a diabody format (designated Dia5v9) containing a 5 aa residue linker (G4S) between VH’s and VCs and then petiplasmically secreted from E. coli. The Dia5vS diabody binds to the extracellular domain of the tumor associated antigen EGP-2 (also known as C017-IA antigen) with an affinity close to that of the parental BsF(ab’)B. The specific binding of Dia5v9 to CD3 was demonstrate using a mutine cytototic T cell line stablely transfected with human CD3 epsilon chain. The efficacy of Dia5vg in mediating tumor cell lysis by IL-2 activated human T-cells appeared to be identical to the BsF(ab’) Bis-1 (4 hr 51Cr release assay). This recombinant bispecific antibody may proof to be potent in mtargeting IL-2 activated T-enriched peripheral blood lymphocytes to lyse various carcinoma cells expressing EGP-2 in viw.
1P.5.10.12 1 CD95 ligand expression in primary and metastatlc melanoma: A longitudinal study J.C. Becker, C. Siedel, E.-B. Brbcker. Department of Dermatology, University of WUtzLug, Josef-Schneider-S&. 2,97080 WOtzLn~rg, Germany CD95 and its ligand (CD95L) are cell-surface molecules which interact in the regulation of immune responses. CD95 is expressed on resting T cells and its expression is significantly enhanced within hours of engagement of their antigen receptors. However, initially CD95 on these cells is not functional in mediating apoptosis in response to cmsslinking; this function is acquired several days later. h viw crosslinking of this molecule is mediated by CD95L either as transmembrane or soluble secreted molecule. CD95L expression was first described on activated T cells. Thus, a T cell might have only a few days to perform its effector function before being targeted for destruction by suicide or fratricide. Recently, CD95-CD95L interactions have been accounted for the immune privileged state of eye and testis. Furthermore, the expression of CD95L on different tumors including melanoma have been repotted, suggesting a novel immune escape mechanism. Since CD95L is absent in normal melanocytes, it is supposed to be unregulated dutfng tumotigenesis. Thus, the present study was designed to determine CD95L expression in primary melanomas and subsequent metastases evolving using immunohistology. In primary tumors CD95L expression was present in 2/20 tumors investigated. This expression was vety weak and restricted to a few parts of the tumor. In addition, only thick primaly tumors seem to express CD95L and in both cases metastatic disease developed. However, the limited number of patients studied, does not permit a correlation of CD95L expression on melanoma with the severity of disease. In metastatic melanoma CD95L is present in 8/10 tumors. Metastases of primary tumors displaying CD95L expression maintained on melanoma cells. Apoptosis was observed among tumor infiltrating leukocytes, as detected by in situ terminal deoxytransferasecatalyzed DNA nick end labeling (TUNEL). Thus, as proposed for testes or stromal cells of the anterior chamber of the eye, CD95L-expressing
25 June 1997 - Poster presentations
1P.5.10.08 1 Apoptosis and growlng profile induced by different mitogenic signals in PBMC from prostate cancer B. Martinez-Mattin ‘, M.PBrez-Bias2, A. Ptieto ’I F. Esquivel ’, M.P. Hemdndez' , F.Cani6n3, J. Carballido3. M. Alv&ez-Mon ‘. ‘Depl. of Medicine, S&co/ of Medicine, University of A/ca/a, *Dept. of Immunology; School of Medicine, University Complurense of Madrid, 3Lbpt, of Medicine, School of Medicine, lInkersi& of the Andes, Santiago de Chile, 4Service of Urology, Clinica “Puerta de Hieuo”, Madrid, Spain The etiology of the prostatic cancer (Cap), one of the most common cancer in men, is unknown, although it is thought to be multifactorial. Histological observation of the prostate evidences areas of lymphocytic infiltration. It is known that cells of the immune system provide differentiation factors influencing in the differentiation and growing of cancer cells. In previous studies we have found that peripheral blood mononuclear cells (PBMC) from CaP patients show a defective proliferative response to surface (PHA) and cytoplasmatic (PMA) mitogens. Thus we have studied the viability, apoptosis and the change in the isoforms of CD45 in the PBMC from 12 CaP patients and 12 healthy men controls by flow cytometry with an internal standard that allows us to find the absolute number of lymphocytes and their subsets that are in the cultures. The diminished proiiierative response found with many of the mitogans used in culture with lymphocytes from CaP patienes was related to a lower number of lymphocytes in the culture. This effect can not be so much adscribed to an increase in the apoptotic rate but to a lower proliferative response in certain lymphocyte subsets depending on the mitogen used. Hence we found fewer CD8 lymphocytes in cultures from CaP patients than in those from controls stimulated with PHA. Moreover fewer CD4 and CD8 lymphocytes and lower generation of CD45RO were obsetved in cultures from CaP than in those from controls when PMA with anti-CD3 or lonomycin was used. These results indicate the PBMC from CaP patients show a different response to surface and citoplasmatic mitogens and this response is always lower than those found in the controls.
1P.5.10.09 ) Characterization of lymphocytes isolated from cervical (pre-) neoplastic lesions associated with human papillomavirus (HPV) N. Jacobs, W. Al-Saled, S.L. Giannini, P. Hubert, L.-M. Dupuis, J. Boniver, P. Delvenne. University of LIEGE, CHU, Department of Pathology, 64000 Liege, Belgium Our aim has been to identify the factors, such as cytokines that may play a role in the transition from the state of immunosufveillance to a state of immunotolerante, during the progression of cervical lesions and the development of cancer. Previous studies have shown that in cervical pre-neoplastic lesions the production of the type 2 cytokines IL4 (detected by immunohistochemistly) increased with the progression of the tumor. In contrast, the production a type 1 cytokine It2 decreased. The cells producing these cytokines were only detected in the strorna undetlying the lesions. We have developed a technique to isolate and cultivate lymphocytes from the epithelium and the stroma of small lesional biopsies (3-4 mm) to determine the cytokines produced by the lymphocytes infiltrating the lesion. The cytokine expression and production by the lymphocytes derived from normal and lesional biopsies was analysed by RT-PCR and ELISA. Our preliminary experiments demonstrated that lymphocytes derived from the stmma of normal and lesional biopsies produced both IFNy (type 1) and IL4 (type 2). In addition, analyse of the cell surface phenotype has shown that within the lymphocytes culture from the lesional biopsies the percentage of CD8+ T cells is diminished compared to the normal biopsies. We are in the progress of studying a larger number of biopsies and other cytokines to extend these results.
P.5.10.10
kiiopathic CD4+ T iymphocytopania with prickle cell carcinoma of the tongue: A case report
P.E. Manconi, P.F. Biddau, M.L. Di Martino, E. Muggianu, M.G. Cau, A. Ibba, G.S. Del Giacco. Dipatimento Scienze Mediche and Diparfimento Biologia Erd Ew/utiw, Universira di Cag/iari, Cagbatf, k/y
Introduction: We report on one patient with idiopathic CD4+ T cell depletion in the absence of HIV infection, which developed a pickle cell carcinoma of the tongue. PfWnt and Methods:A 28 years old whii woman from Cagliad (Sardinia, Italy) presented to us with mild lymphocytcpenia (total lymphocytes = lOlO/fil, CD4+ = 249/& CD8+ = 425/& CD4/CD8 ratio = 58). Medical history revealed toxoplasmosis in the first months of life and a severe scoliosis. At the beginning of 1998 the patient was accepted in a Medical ward for colyoretinitis by toxoplasma. In September a prickle cell carcinoma of the tongue was detected by surgical biopsy. The patient was then treated with radiotherapy.
451
Before radiotherapy, a defii of CD4+ lymphocytes was noted. Clinical examination revealed: “doll”facies, hyperchmmic speckle, pterygium, feeble voice, ulcer-necrotic lesion of the base of the tongue. One year after the first evaluation, lymphocyte subsets were quantified by lmmunocount (Ortho, Raritan, NJ): total lymphocytes were 4OLI&l), CD4+ (79 cells/flI), CD8+ (181 cells@) and CD4/CD8 ratio (0.4). CD 19+ B cells 83/pl, and CD18+ CD3- NK cells = 125/wl. A feeble depletion of lgG4 and lgG2, without any abnormality of other Ig classes was found in the serum. Conclusion:Pickle cell carcinoma is usually presented only by older people, particularly by heavy smokers. The presence of such a cancer in a non-smocking young girl affected with a severe depletion of CD4 is in agreement with a protective role of these lymphocytes against cancer.
P.5.10.11
Constructlon of a recombinant bispeciflc antlbody (diabodv) of minimal size for retametlna Tcells to human Gcinomas
Wijnand Helfrich, Bart Jan Kroesen, lngrid Molema, Lidia Westers, Loe de Leij. University Hospital Groningen, Oepf. Clinical lmmunolow. Oostersingel59, 9713 EZ Groningen, The Netherlands Bispecific antibodies (BsAb) have strong potential for tumor therapy by retargeting cytotoxic effector cells against human carcinomas. Bsab are traditionally prepared by hybrid hybridoma technology or via chemical coupling of Fab’fmgmerits. Clinical evaluation is however hampered by the diicuity to prepare them in sufficient quantity and purity. In addition, the size and the immunogenicity of the resultant molecules is expected to reduce their efficacy even further. Diabodies are very small recombinant dimeric antibody scFv fragments that can be engineered to harbour two diierent antigen-binding sites. Bispecific diabodies directed against cell-surface antigens are capable of bringing together two cells, such as in cell-targeted therapy. In diabodies, the two antigen-binding sites are at opposite ends of the molecule and separated by approximately 0.85 nm. The average molecular weight of a diabody is only appr. 52 kDa (IgG appr. 150 kDa). In this project a high affinity muline anti-EGP-2 (MOC31) scFv and humanized anti-CD3 (Uchtl v9) scFv were first recast into a diabody format (designated Dia5v9) containing a 5 aa residue linker (G4S) between VH’s and VCs and then petiplasmically secreted from E. coli. The Dia5vS diabody binds to the extracellular domain of the tumor associated antigen EGP-2 (also known as C017-IA antigen) with an affinity close to that of the parental BsF(ab’)B. The specific binding of Dia5v9 to CD3 was demonstrate using a mutine cytototic T cell line stablely transfected with human CD3 epsilon chain. The efficacy of Dia5vg in mediating tumor cell lysis by IL-2 activated human T-cells appeared to be identical to the BsF(ab’) Bis-1 (4 hr 51Cr release assay). This recombinant bispecific antibody may proof to be potent in mtargeting IL-2 activated T-enriched peripheral blood lymphocytes to lyse various carcinoma cells expressing EGP-2 in viw.
1P.5.10.12 1 CD95 ligand expression in primary and metastatlc melanoma: A longitudinal study J.C. Becker, C. Siedel, E.-B. Brbcker. Department of Dermatology, University of WUtzLug, Josef-Schneider-S&. 2,97080 WOtzLn~rg, Germany CD95 and its ligand (CD95L) are cell-surface molecules which interact in the regulation of immune responses. CD95 is expressed on resting T cells and its expression is significantly enhanced within hours of engagement of their antigen receptors. However, initially CD95 on these cells is not functional in mediating apoptosis in response to cmsslinking; this function is acquired several days later. h viw crosslinking of this molecule is mediated by CD95L either as transmembrane or soluble secreted molecule. CD95L expression was first described on activated T cells. Thus, a T cell might have only a few days to perform its effector function before being targeted for destruction by suicide or fratricide. Recently, CD95-CD95L interactions have been accounted for the immune privileged state of eye and testis. Furthermore, the expression of CD95L on different tumors including melanoma have been repotted, suggesting a novel immune escape mechanism. Since CD95L is absent in normal melanocytes, it is supposed to be unregulated dutfng tumotigenesis. Thus, the present study was designed to determine CD95L expression in primary melanomas and subsequent metastases evolving using immunohistology. In primary tumors CD95L expression was present in 2/20 tumors investigated. This expression was vety weak and restricted to a few parts of the tumor. In addition, only thick primaly tumors seem to express CD95L and in both cases metastatic disease developed. However, the limited number of patients studied, does not permit a correlation of CD95L expression on melanoma with the severity of disease. In metastatic melanoma CD95L is present in 8/10 tumors. Metastases of primary tumors displaying CD95L expression maintained on melanoma cells. Apoptosis was observed among tumor infiltrating leukocytes, as detected by in situ terminal deoxytransferasecatalyzed DNA nick end labeling (TUNEL). Thus, as proposed for testes or stromal cells of the anterior chamber of the eye, CD95L-expressing