抗CD3／抗CD20微型双功能抗体裸鼠体内分布研究

目的建立裸鼠皮下人B-淋巴细胞移植瘤模型,研究抗CD3/抗CD20微型双功能抗体在裸鼠体内的分布。方法采用亲和层析分离纯化本室构建的抗CD3/抗CD20微型双功能抗体可溶性表达产物,并用SDS-PAGE、Western     

双功能抗体介导结肠癌细胞杀伤效应的研究

目的：研究抗CD3/抗TNP双功能抗体与TNP化抗癌胚抗原（CEA）McAb结合。在裸鼠体内对结肠癌细胞的杀伤作用。方法：将抗癌胚抗原单抗TNP化。TNP化后的抗CEA单抗（TNP-CEA），抗CD3/抗TNP双功能抗体，人淋巴细胞及结肠癌细胞CL187接种裸鼠体内。计算肿瘤生长体积，观察成瘤率。结果：双功能抗体能够介导PBLs对CL187细胞的杀伤作用。结肠癌细胞CL187的成瘤能力受到抑制，成瘤率为0/4。结论：此双功能抗体在免疫治疗中具有潜在的应用价值。     

双功能抗体抗CD3/抗CD19介导T细胞对靶细胞的杀伤作用

背景与目的:双功能抗体有2个抗原结合臂,一方面具有肿瘤相关抗原,另一方面具有和免疫效应细胞表面分子标记结合的能力,并能有效地使效应细胞靶向杀火肿瘤细胞的作用.本实验探讨抗CD3/抗CD19双功能抗体介导T细胞杀伤靶细胞的作用.方法:利用非放射性荧光染料Calcein-AM进行抗体介导的体外特异性杀伤活性榆测,Ficoll-Hypaque法分离外周血淋巴细胞(peripheral blood lymphocyte,PBL),流式细胞术从中分选T淋巴细胞及CD4+T淋巴细胞和CD8+T淋巴细胞作为效应细胞.首先以Raji细胞为靶细胞,按不同效靶比,不同抗体浓度分组,以PBS、抗CD3scFv+、抗CD3scFv抗体及非相关抗体抗CD3/抗PGP为对照,另设CD19-K562细胞为非相关靶细胞组,比较双功能抗体介导特异性体外杀伤活性.取上述杀伤实验效靶比25:1,抗体浓度500 ng/mL各组,ELISA法比较激活的T细胞分泌IL-2的量,Real-time PcR法检测T细胞因子IL-3、IFN-γ、TNF-α激活后的表达变化.结果:体外介导T细胞杀伤靶细胞Raji的效果比对照组明显增强(P＜0.05),激活T细胞分泌IL-2、IL-3、IFN-γ及TNF-α的表达均明显高于对照组.结论:双功能抗体抗CD3/抗CD19在体外介导T细胞对靶细胞具有较强的杀伤作用.     

肝癌细胞凋亡-抗HBx/抗CD3双功能抗体介导细胞毒性T细胞对靶细胞的杀伤

研究发现，针对效应细胞毒性T淋巴细胞[CTL]表面受体[TCB／CD3复合物]与肿瘤相关抗原的双功能抗体能有效地介导CTL对靶肿瘤细胞的杀伤。为此，我们通过细胞融合法经流式细胞仪[FACS]无菌分选建立了抗HBx／抗CD3二次杂交瘤，应用该二次杂交瘤所分泌的双特性单克隆抗体[BsAb]介导CTL在LTNM4裸鼠人肝癌模型进行了实验性治疗研究。     

抗CD_3/抗CD_(20)微型双功能抗体裸鼠体内分布研究

目的：建立裸鼠皮下人Ｂ－淋巴细胞移植瘤模型,研究抗ＣＤ３／ＣＤ２０。微型双功能抗体在裸鼠体内的分布。方法：采用亲和层析分离纯化本室构建的抗 ＣＤ３／抗 ＣＤ２０微型双功能抗体可溶性表达产物,并用ＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎ ｂｌｏｔ、分子排阻层析和 ＦＡＣＳ鉴定纯化产物;采用５周龄左右的雌性 ＢＡＬＢ／ｃ－ｎｕ,经４Ｇｙ照射、皮下接种 Ｒａｊｉ细胞建立裸鼠皮下人Ｂ－淋巴细胞移植瘤模型,并于眼底静脉丛注射１２５Ｉ标记的抗ＣＤ３／抗ＣＤ２０微型双功能抗体,测定各组织中１２５Ｉ的分布。结果：雌性ＢＡＬＢ／ｃ－ｎｕ经照射、皮下接种１×１０～７～３×１０～７Ｒａｊｉ细胞／只,６～１４天均开始出现移植瘤,移植瘤细胞膜表面标记与 Ｒａｊｉ相同,且在２４小时时肿瘤部位１２５ Ｉ的分布明显低于心、肝、肾、脾,其比值分别为０．０８、０．１９、０．０６和０．５１,而 ７２小时时肿瘤部位１２５Ｉ的分布则高于心、肝、肾、脾,其比值分别为 ２．３２、９．４６、８．２４和９．５０。结论：抗ＣＤ３／抗ＣＤ２０微型双功能抗体能在裸鼠皮下人Ｂ－淋巴细胞移植瘤部位富集,是一个有望用于Ｂ细胞恶性肿瘤临床治疗的双特异性抗体。     

抗CD3/抗CD20偶联物的制备及其介导激活T细胞杀伤活性研究

目的：研究抗CD3/抗CD20抗体偶联物介导的激活T细胞杀伤活性。方法：通过SPDP偶联，经分子筛层析法纯化得到抗CD3/抗CD20抗体偶联物，并用FACS和玫瑰花环试验鉴定纯化产物；采用^51Cr释放试验测定该抗体偶联物介导的体外靶向杀伤活性。结果：纯化的抗CD3/抗CD20抗体偶联物具有与jurkat(CD3^ )和Daudi细胞（CD20^ )的结合活性，且能同时将Jurkat和Daudi细胞结合形成玫瑰花环；在体外能介导激活的T细胞杀伤Daudi细胞。结论：抗CD3/抗CD20抗体偶联物体外能介导激活的T细胞杀伤表达CD20抗原的肿瘤细胞，为B细胞恶性肿瘤临床治疗提供实验依据。     

表达嵌合T细胞受体的T淋巴细胞介导的肿瘤过继性免疫治疗作用研究

目的:构建嵌合T细胞受体(chimeric T cell receptor,chTCR),它包括识别肿瘤相关抗原erbB2的单链抗体、共刺激分子CD28和CD3的信号转导链ζ,将其依次克隆入载体pcDNA3中,电转染入人外周血T淋巴细胞,使嵌合T细胞受体在T细胞表面表达,并验证其在体外和体内的抗肿瘤作用,为肿瘤的过继性免疫治疗提供新思路。方法:抗erbB2的单链抗体(由本所保存),人CD28分子的部分胞外段、跨膜区和胞内段,CD3ζ链的胞内段(分别由RT-PCR的方法获得)依次克隆入载体pcDNA3中,经酶切鉴定正确后,电转染进入人外周血T淋巴细胞,利用流式细胞仪检测T细胞结合Her2蛋白的功能;细胞杀伤试剂金检测T细胞的抗原特异性杀伤功能;并在裸鼠体内验证其对人的乳腺癌细胞株BT-474移植瘤的杀伤效应。结果:成功构建了抗erbB2嵌合T细胞受体并表达在人的T淋巴细胞表面,体外证明能结合Her2肿瘤抗原,并有抗原特异性的杀伤功能,在BALB／c裸鼠体内对表达erbB2的肿瘤细胞BT～474有显著的抑制肿瘤生长的作用。结论:表达嵌合T细胞受体的T淋巴细胞在体外和体内有抗原特异性的杀伤肿瘤的作用,是一种新的肿瘤过继性的免疫治疗策略。     

抗EGFR-抗CD3双功能抗体介导CIK细胞、LAK细胞或PBLS细胞对胃癌荷瘤裸鼠治疗效果的比较

 目的　比较抗EGFR-抗CD3双功能抗体在体内条件下介导CIK细胞、LAK细胞及人类PBLS细胞三类不同的免疫效应细胞对胃癌细胞的杀伤能力,为临床应用该抗体治疗胃癌的细胞选择提供实验指导。方法　采用化学耦联法合成的抗EGFR-抗CD3双功能抗体分别与CIK细胞、LAK细胞及人类PBLS细胞联合经尾静脉注入SGC7901胃癌细胞移植瘤小鼠体内,同时以等量0.9 % NaCl溶液尾静脉注射建立对照,治疗后检测在体情况下4组对胃癌细胞的杀伤能力,进行组间比较。结果　抗EGFR-抗CD3双功能抗体介导CIK细胞治疗组小鼠肿瘤抑制率为（64.9±7.7）%,显著高于抗EGFR-抗CD3双功能抗体介导LAK细胞及人类PBLS细胞组的（43.5±8.2）%和（39.7±6.5）%（均P＜0.05）,治疗结束时平均肿瘤质量为（473.9±37.7）mg,显著低于其他两组的（764.6±88.3）mg和（829.1±104.4）mg（均P＜0.05）。结论　初步的裸鼠模型治疗实验显示由抗EGFR-抗CD3双功能抗体介导的CIK细胞在体内条件下对胃癌治疗作用优于其他常用的免疫效应细胞。     

抗B-CLL-IgM Id×抗CD3双功能抗体对LAK细胞杀伤活性的影响

抗肿瘤双特异性抗体(bispecific antibody,BsAb)由于具有同时与效应细胞及靶瘤细胞两种不同抗原特异性结合的双重特异性,从而提高了效应细胞对靶瘤细胞的特异性杀伤活性,因此,其在肿瘤靶向生物治疗中的应用日益受到重视.本研究通过体外实验,观察了抗人慢性B淋巴细胞白血病LgM独特型×抗CD3双特异性抗体(抗B-CLL-LgM Id×抗CD3 BsAb)对LAK细胞杀伤靶瘤细胞活性的影响,现报道如下.     

抗EGFR／抗CD3双功能抗体对胃癌荷瘤小鼠的免疫治疗研究

目的 通过抗EGFR／抗CD3双功能抗体（EGFR/CD3 BsAb）治疗SGC7901胃癌细胞移植瘤小鼠模型,初步验证该抗体在体内对胃癌细胞的杀伤能力。方法 采取化学偶联法合成的EGFR/CD3 BsAb联合人外周血淋巴细胞（PBLS）经尾静脉给药注入荷SGC7901胃癌细胞移植瘤小鼠体内。实验裸鼠随机分为抗EGFR单抗联合PBLS组（抗EGFR组）、抗CD3单抗联合PBLS组（抗CD3组）、EGFR／CD3 BsAb联合PBLS组（EGFR/CD3 BsAb组）和空白对照组（生理盐水组）。治疗后检测并比较加药各组对胃癌细胞的杀伤能力。结果 EGFR／CD3 BsAb成功制备,分子量为43kD。EGFR／CD3 BsAb组裸鼠的肿瘤抑制率为(57.2±8.6）％,显著高于抗EGFR组的（38.5±6.1）％和抗CD3组的（6.9±7.6）％（P＜0.05）;治疗结束时EGFR／CD3 BsAb组的瘤重为（517.1±45.4）mg,显著低于抗EGFR组的（737.4±54.3）mg和抗CD3组的（1097.9±167.7）mg（P＜0.05）。各组移植瘤组织EGFR免疫组化染色均为强阳性。EGFR／CD3 BsAb组裸鼠肿瘤组织CD3免疫组化染色可见部分细胞呈阳性。透射电镜观察显示,EGFR／CD3 BsAb组和抗EGFR组可见肿瘤坏死。结论 EGFR／CD3 BsAb在体内条件下可能对胃癌有治疗作用。     

Efficient inhibition of multidrug-resistant human tumors with a recombinant bispecific anti-P-glycoprotein x anti-CD3 diabody.

Overexpressing of P-glycoprotein (Pgp) has been shown to be responsible for cancer resistance to multiple chemotherapeutic agents. Immunotherapy with biological agents, such as bispecific antibodies (BsAbs), may represent a promising approach to overcome the emergence of drug resistance. Here we constructed a recombinant BsAb, a diabody, with specificities to both CD3 on human T-lymphocyte and Pgp on cancer cells. The diabody was produced in Escherichia coli in a soluble functional form and purified by an affinity chromatography with a yield of >4 mg/l culture medium in shaker flask. The diabody binds to both CD3 on T-lymphocytes and Pgp on multidrug-resistant (MDR) tumor cells with affinities that are comparable to its respective parental single chain Fv molecules. In the presence of activated human peripheral blood lymphocytes (PBLs), the diabody mediates effectively the lysis of the Pgp-overexpressing human leukemia K562/A02 and epidermoid carcinoma KBv(200) cells, but is much less potent in mediating the lysis of the parent K562 and KB cells. Further, the diabody localized selectively within the K562/A02 xenografts in mice. When combined with activated PBL, the diabody significantly inhibited the growth of K562/A02 and KBv(200), but had no effect on K562 and KB xenografts. In contrast, treatment with doxorubicin, a standard chemotherapeutic agent, only inhibited the growth of K562 and KB, but had no effect on K562/A02 and KBv(200) xenografts. Taken together, our results suggest that the anti-Pgp x anti-CD3 diabody may have a great potential in the treatment of various MDR cancers.     

Disulfide-stabilized diabody antiCD19/antiCD3 exceeds its parental antibody in tumor-targeting activity

Background

A diabody is a bispecific antibody that is capable of recruiting a polyclonal T cell to antibody target-expressing tumor cells. However, the two chains of diabodies tend to dissociate because they are integrated non-covalently. Therefore, it is necessary to remodel the diabody to increase its stability in order to enhance the antitumor activity.

Methods

We constructed an antiCD3×antiCD19 diabody with one binding site for the T cell antigen receptor (TCRCD3) and the other for the B cell-specific antigen (CD19) by recombinant gene engineering technology. Cysteine residues were introduced into the V domains of the anti-CD3 segment. The stability and cytotoxicity of the two diabodies were compared in vitro and vivo.

Results

The disulfide-stabilized (ds) diabodies produced by Escherichia coli were secreted with high yields in a fully active form without a decrease in affinity. Compared with the parental diabody, the disulfide-stabilized (ds) diabody proved more stable in vitro and in vivo without reducing binding affinity. Both were able to effectively eliminate human lymphoma Raji cells by redirecting T lymphocytes in vitro and in vivo, but the ds diabody was more effective in inhibiting the growth of xenografts transplanted in BALB/C nude mice.

Conclusion

The antiCD3×antiCD19 ds diabody is more suitable for a controlled polyclonal T cell therapy of human CD19-positive B cell malignancies than its parental diabody.     

Efficient inhibition of human B-cell lymphoma xenografts with an anti-CD20 x anti-CD3 bispecific diabody.

Bispecific antibodies have been exploited both as cancer immunodiagnostics and as cancer therapeutics, and have shown promise in several clinical trials in cancer imaging and therapy. A number of bispecific antibodies against B-cell markers have been shown to be effective in vitro in mediating tumor cell lysis and in vivo in inhibiting tumor growth in animal models. We have constructed a bispecific diabody from the variable genes encoding two hybridoma-derived monoclonal antibodies directed against human CD20 on B cells and CD3 on T cells. The anti-CD20 x anti-CD3 diabody was expressed in a single Escherichia coli host and purified by a one-step affinity chromatography. The bispecific diabody bound as efficiently to both CD20- and CD3-positive cells as the respective parental antibodies, and was capable of cross-linking CD20-positive tumor cells and human T lymphocytes as shown by cellular rosetting. The diabody effectively lysed human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). Further, when combined with human PBL and interleukin-2, the diabody significantly prolonged the survival of nude mice inoculated with human B-lymphoma cells. Taken together, our results suggest that an anti-CD20 x anti-CD3 diabody may have significant clinical application in the treatment of human CD20-positive B-cell malignancies.     

Construction and characterization of a bispecific diabody for retargeting T cells to human carcinomas

We describe the construction of a recombinant bispecific antibody fragment in the diabody format with specificity for both the well-established human pancarcinoma associated target antigen EGP2 (epithelial glycoprotein 2, also known as the CO17-1A antigen or KSA) and the CD3ϵ chain of human TCR/CD3 complex. The murine anti-EGP2 (MOC31) single chain variable fragment (scFv) and the humanized anti-CD3 (Ucht1v9) scFv were cast into a diabody format (designated Dia5v9) using a short 5 amino acid Gly-Ser linker between immunoglobulin heavy-chain and light-chain variable domains. Purification of the poly-histidine tagged Dia5v9 was achieved from extracts of protease deficient Escerichia coli by IMAC chromatography. The Dia5v9 diabody showed strong binding to both EGP2 and CD3 in transfected cells. The in vitro efficacy of Dia5v9 in mediating tumor cell lysis by interleukin-2 activated human T cells appeared to be similar to that of the hybrid-hybridoma–derived BsF(ab′)₂ Bis1 (anti-EGP2/anti-CD3) in a standard 4-hr ⁵¹Cr-release assay. This small and partially humanized recombinant bispecific antibody fragment may be valuable for T-cell–based immunotherapeutical treatment protocols, retargeting activated peripheral blood T lymphocytes to lyse various human carcinomas in vivo. Int. J. Cancer 76:232–239, 1998.© 1998 Wiley-Liss, Inc.     

Bispecific CD3 × CD19 diabody for T cell-mediated lysis of malignant human B cells

For the treatment of minimal residual disease in patients with leukemias and malignant lymphomas, we constructed a heterodimeric diabody specific for human CD19 on B cells and CD3ϵ chain of the T cell receptor complex. The bispecific diabody was expressed in Escherichia coli using a vector containing a dicistronic operon for co-secretion of V_H3-V_L19 and V_H19-V_L3 single-chain Fv fragments (scFv). It was purified in one step by immobilized metal affinity chromatography (IMAC) from the periplasmic extract and culture medium. Flow cytometry experiments revealed specific interactions of the diabody with both CD3 and CD19 positive cells, to which it bound with affinities close to those of the parental scFvs. It was less stable than anti-CD3 scFv but more stable than anti-CD19 scFv when incubated in human serum at 37°C. In cytotoxicity tests, the diabody proved to be a potent agent for retargeting peripheral blood lymphocytes to lyse tumor cells expressing the CD19 antigen. The efficiency of cell lysis compared favorably with that obtained with a bispecific antibody (BsAb) of the same dual specificity that was prepared by the quadroma technique. Int. J. Cancer 77:763–772, 1998. © 1998 Wiley-Liss, Inc.     

Monocyte chemoattractant protein-1 gene modification of multidrug-resistant human lung cancer enhances antimetastatic effect of therapy with anti-P-glycoprotein antibody in SCID mice

Distant metastases and multidrug resistance are critical problems in the therapy of human small cell lung cancer (SCLC). In this study, we investigated whether transduction of the monocyte chemoattractant protein-1 (MCP-1) gene into multidrug-resistant (MDR) human lung cancer cells affected the formation of metastases or their inhibition by the anti-P-glycoprotein (P-gp) monoclonal antibody (MAb) MRK16. MDR human SCLC (H69/VP) cells were transduced with the human MCP-1 gene inserted into the expression vector BCMGSNeo. MCP-1 gene transduction had no effect on drug sensitivity, the expression of surface antigens or the in vitro proliferation of H69/VP cells. Using the metastatic model of NK cell-depleted SCID mice, H69/VP cells transduced with the MCP-1 gene were inoculated intravenously (i.v.) and formed metastatic colonies in the liver, kidneys and lymph nodes, similar to those formed by parent or mock-transduced cells. However, systemic treatment of the mice with MRK16 reduced the metastases of H69/VP cells in the liver, kidneys and lymph nodes, and was significantly more effective in inhibiting the metastases of MCP-1 producing H69/VP than those of mock-transduced cells. MCP-1 gene transduction significantly prolonged the survival of tumor-bearing mice treated with MRK16. Our findings suggest that local production of MCP-1 in the tumor site increases the anti-P-gp antibody-dependent cell-mediated cytotoxicity, and the MCP-1 gene-induced modification of MDR human SCLC cells thereby enhances the antimetastatic effect of therapy with anti-P-gp antibody. Thus, the accumulation of effector cells in the tumor site is a very important factor in the therapy using the anti-P-gp antibody.     

Anti-CD19 antibodies inhibit the function of the P-gp pump in multidrug-resistant B lymphoma cells.

After chemotherapy, tumor cells with multidrug resistance (MDR) often emerge. MDR is attributable to the expression of membrane transport proteins that inhibit the cellular influx and increase the efflux of many chemotherapeutic drugs. One such protein is P-glycoprotein (P-gp), which functions as an ATP-dependent active transporter. Recently, an anti-P-gp monoclonal antibody (MAb) that inhibits P-gp has been described. Previous studies from our laboratory using the anti-CD19 B-cell lymphoma-reactive MAb, HD37, have suggested that HD37 may also influence MDR. To test this directly, we used Namalwa/MDR1 cells to study the effect of HD37 on the efflux of rhodamine 123 from these cells. We found that HD37 and three other anti-CD19 MAbs inhibited the efflux of rhodamine 123 from Namalwa/MDR1 cells with approximately 50% of the efficiency of the well-known chemosensitizer, verapamil. In contrast, MAbs against seven other molecules expressed on these cells were ineffective. The inhibitory activity of HD37 did not require an Fc portion; F(ab')2 fragments were effective, but Fab' fragments were not, suggesting that higher avidity binding and/or cross-linking of CD19 are necessary. We could find no evidence that HD37 recognizes a cross-reactive epitope on P-gp, modulates P-gp from the cell surface, or enhances the ATPase activity of membranes from treated cells.     

Enhanced in vitro lysis of human ovarian carcinomas with activated peripheral blood lymphocytes and bifunctional immune heteroaggregates

Heteroaggregated monoclonal antibodies (HA) comprised of an anti-CD3 and an anti-tumor associated antigen (TAA) antibody were evaluated for their ability to lyse fresh human ovarian cancer cells. HAs were able to significantly enhance the in vitro lysis of fresh ovarian tumor cells by in vitro activated peripheral blood lymphocytes (PBLs). HAs did not increase tumor lysis using fresh PBLs or PBLs cultured overnight in vitro. HA efficacy required both anti-CD3 and anti-TAA antibodies to be present in the same molecule, implying that physical bridging between the effector and target cell by the HA may be a requirement for their activity in cell lysis. The presence of anti-CD3 antibody in the PBL cultures enhanced cell growth and did not block the efficacy of the anti-CD3 containing HAs. The frequent expression of multiple TAAs in human ovarian cancers and the ability to recruit cytotoxic T-cell activity with HAs in vitro give promise to the future of this form of immunotherapy in the clinical setting.     

Combined therapy with anti-P-glycoprotein antibody and macrophage colony-stimulating factor gene transduction for multiorgan metastases of multidrug-resistant human small cell lung cancer in NK cell-depleted SCID mice.

Our aim was to determine the antimetastatic potential of anti-P-glycoprotein (P-gp) antibodies (Abs) against multidrug-resistant (MDR) human small cell lung cancer (SCLC) cells expressing P-gp. Human SCLC cells H69 (P-gp negative) and its etoposide-resistant variant H69/YP (P-gp positive) were used. H69 and H69/VP cells injected i.v. metastasized to the liver, kidneys and systemic lymph nodes of NK cell-depleted severe combined immunodeficient (SCID) mice. H69/VP cells, but not H69 cells, were resistant to treatments with vindesine. Treatment with mouse-human chimeric anti-P-gp Ab (MH162) and its mouse counterpart (MRK-16) reduced metastasis of H69/VP cells in various organs and prolonged the survival of tumor-bearing mice, although they were less effective if injected at late times (after 28 days). Treatment with another mouse anti-Pgp Ab, MRK-17, was effective only against liver metastasis. MH162 and MRK-16 efficiently induced Ab-dependent cellular cytotoxicity (ADCC) by peritoneal macrophages against H69/VP cells in vitro, but MRK-17 was less effective, in accordance with their in vivo antimetastatic potential. Gene transfection of macrophage colony-stimulating factor (M-CSF) into H69/VP cells to augment macrophage-mediated ADCC resulted in inhibition of metastasis to the liver and lymph nodes, but not kidneys. Combined treatment with a low dose of MRK-16 completely cured metastasis of M-CSF transfectant, but not of the mock transfectant. Our findings suggest that while anti-P-gp Abs had antimetastatic potential against SCLC cells expressing P-gp, combined treatment with M-CSF gene transduction to augment the therapeutic efficacy of anti-P-gp Abs may be beneficial for eradicating metastatic MDR SCLC in humans.