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.     

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.     

Cure of Burkitt's lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 x CD19 tandem diabody, and CD28 costimulation

To increase the valency, stability, and therapeutic potential of bispecific antibodies, we have constructed a tetravalent tandem diabody (Tandab) that is specific to both human CD3 (T-cell antigen) and CD19 (B-cell marker; S. M. Kipriyanov et al., J. Mol. Biol., 293: 41-56, 1999). It was generated by the functional dimerization of a single chain molecule that contained four antibody variable domains (V(H) and V(L)) in an orientation that prevented intramolecular pairing. Compared with a previously constructed heterodimeric CD3 x CD19 diabody, the Tandab exhibited a higher apparent affinity to both CD3+ and CD19+ cells and longer blood retention when injected into mice. Biodistribution studies in mice bearing Burkitt's lymphoma xenografts demonstrated specific accumulation of the radioiodinated Tandab in a tumor site with tumor-to-blood ratios of 1.5, 8.1, and 13.3 at 3, 18, and 24 h, respectively. Treatment of severe combined immunodeficiency mice bearing established Burkitt's lymphoma (5 mm in diameter) with human peripheral blood lymphocytes, Tandab, and anti-CD28 MAbs resulted in the complete elimination of tumors in all of the animals within 10 days. In contrast, mice receiving human peripheral blood lymphocytes in combination with either the diabody alone or the diabody plus anti-CD28 MAbs showed only partial tumor regression. These data demonstrate that the CD3 x CD19 Tandab may be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas.     

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.     

Effect of tetravalent bispecific CD19xCD3 recombinant antibody construct and CD28 costimulation on lysis of malignant B cells from patients with chronic lymphocytic leukemia by autologous T cells

To develop an effective antitumor immunotherapy for B-lineage non-Hodgkin's lymphoma, we constructed a tetravalent tandem diabody (tanDb) specific for both human CD19 (B-cell marker) and CD3 (T-cell antigen). Here, we report the effective killing of malignant primary B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) by autologous T cells induced by tanDb at very low E:T ratios. Mononuclear cells from patients with B-CLL were cultured with bispecific antibody fragments in either the presence or absence of monospecific anti-CD28 antibody. Use of tetravalent tanDbs caused almost quantitative elimination of malignant B cells from the blood samples of 19 patients and some cytotoxic activity in 3 of 23 analyzed cases. In contrast, the structurally similar but bivalent diabody and single-chain diabody demonstrated nearly no antitumor activity in an autologous system. tanDb-induced activation and proliferation of T cells occurred only in the presence of CD19+ target cells. Expression of the B7-1 (CD80) and B7-2 (CD86) molecules on the surface of leukemia cells made unnecessary the additional CD28-costimulation of T cells. When only a few tanDb molecules were present, the effect of CD28 costimulation on T-cell activation was more pronounced. Depending on the patient sample, we observed a 10- to 1,000-fold decrease of the half-maximal concentrations of tanDb for cell lysis. Upon CD28 crosslinking by agonistic MAb, specific tumor cell lysis was found at tanDb concentrations as low as 0.5 pM. These data demonstrate that the tetravalent CD19xCD3 tanDb might be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas.     

Carcinoembryonic antigen (CEA)-specific T-cell activation in colon carcinoma induced by anti-CD3 x anti-CEA bispecific diabodies and B7 x anti-CEA bispecific fusion proteins.

Two bispecific recombinant molecules, an anti-CD3 x anti-carcinoembryogenic antigen (CEA) diabody and a B7 x anti-CEA fusion protein, were tested for their capacity to specifically activate T cells in the presence of CEA-expressing colon carcinoma cells. T-cell activation by the anti-CD3 x anti-CEA diabody required close contact to CEA-positive cells and resulted in diabody-mediated cytotoxicity against the target cells. Additionally, CD28-mediated costimulation in combination with anti-CD3 x anti-CEA diabodies induced activation of autologous T cells in CEA-positive primary colon carcinoma specimens, as determined by flow cytometry. The high specificity of the bispecific diabody approach could be further enhanced by the use of B7 x anti-CEA fusion proteins because the costimulatory CD28-signaling to the T cells strictly depended on the expression of CEA on the target cells. We demonstrate that displaying engagement sites for the T-cell antigens CD3 and CD28 on the surface of colon carcinoma cells is a suitable way to activate and retarget T cells in a highly tumor-specific manner. For clinical purposes, B7 x anti-tumor-associated antigen (TAA) fusion proteins, which are equally effective but more specific compared with anti-CD28 monoclonal anti-bodies, thus may improve the tumor specificity of anti-CD3 x anti-TAA bispecific antibodies. Furthermore, B7-negative tumors can be converted into B7-positive tumors by B7 x anti-TAA fusion proteins without the need for B7 gene transfer to the malignant cells.     

Heterodimeric bispecific antibody-derivatives against CD19 and CD16 induce effective antibody-dependent cellular cytotoxicity against B-lymphoid tumor cells

Bispecific scFv antibody-derivatives (bsscFvs) recruiting natural killer (NK) cells for the lysis of malignant cells have therapeutic potential. However, a bsscFv specific for the B-lymphoid tumor antigen CD19 and the trigger molecule CD16 on NK cells had similar affinities for both antigens (42 and 58nM, respectively) and was not optimal for cytotoxicity. Therefore, a bispecific tribody (bsTb) was constructed with two binding sites for CD19 and one for CD16. This bsTb contained a CD19-specific Fab fragment carrying a CD16-specific scFv fused to its light chain and a CD19-specific scFv fused to its heavy chain. The bsTb was compared with a bispecific bibody (bsBb) lacking the CD19-specific scFv. The bsTb had 3-fold greater avidity for CD19 than the bsBb (8 and 24nM, respectively), while both had equal affinity for CD16 (56nM). Both molecules mediated antibody-dependent cellular cytotoxicity (ADCC) of leukemia-derived SEM cells and primary cells from leukemia patients. The bsTb showed half-maximum effective concentrations (EC(50)) of 55pM and promoted equal lysis as the bsBb and the bsscFv at 6- and 12-fold lower concentrations, respectively. Among these three molecules the bsTb showed the most promising in vitro properties which are anticipated to be displayed also in vivo.     

CD3X anti-nitrophenyl bispecific diabodies: universal immunotherapeutic tools for retargeting T cells to tumors.

We developed a universal recombinant bispecific molecule (BiMol) that is capable of redirecting cytotoxic T cells to tumor cells via tagged anti-tumor ligands such as antibody fragments or cytokines. A recombinant bispecific diabody with binding specificities for the CD3 molecule on T cells as well as for the hapten nitrophenyl (NIP) was produced. This bispecific molecule is capable of redirecting cytotoxic T cells to kill a series of malignant cells, including B cell lymphoma, Hodgkin's lymphoma, and colon carcinoma via NIP-conjugated ligands to tumor-associated antigens. Cytotoxic activity of the diabody was found to be comparable to tetradoma-derived bispecific antibodies with similar specificities. Our findings demonstrate that universal CD3xanti-NIP diabodies could be used for T cell based cellular immunotherapy in a variety of human malignancies. Additionally, these bispecific molecules allow fast and economic testing of tumor-associated antigens on malignant cells for their potential use as immunotherapeutic target structures if corresponding hapten-conjugated antibodies or ligands are available.     

靶向4-1BBL 和CD3 双信号的协同抗肿瘤作用机制研究*

目的:探讨人4-1BBL 胞外区/抗CD20融合蛋白增强抗CD3/抗CD20diabody介导的靶向杀伤作用及其机制。方法:通过表达纯化人4-1BBL 胞外区/抗CD20融合蛋白及抗CD3/抗CD20diabody,利用台盼蓝计数观察联合应用人4-1BBL 胞外区/抗CD20融合蛋白及抗CD3/抗CD20diabody对淋巴细胞增殖的影响;采用ELISA 检测白介素-2（IL- 2）水平。RT-PCR 检测穿孔素和颗粒酶mRNA 的表达。Calcein 检测其联合应用抗CD3/抗CD20双功能抗体及PBL 对靶细胞Raji 细胞的杀伤作用。结果:人4-1BBL 胞外区/抗CD20融合蛋白增强抗CD3/抗CD20diabody对靶细胞Raji 细胞的杀伤作用,其机制可能是促进淋巴细胞增殖,减少细胞死亡,促进IL- 2 分泌及上调穿孔素和颗粒酶mRNA 表达。结论:人4-1BBL 胞外区/抗CD20融合蛋白与抗CD3/抗CD20diabody联合应用,分别靶向4-1BBL 和CD3 双信号发挥协同抗肿瘤作用,为肿瘤免疫治疗提供了新的思路。      

Overcoming the challenges associated with CD3+ T-cell redirection in cancer

The development of bispecific antibodies that redirect the cytotoxic activity of CD3+ T cells to tumours is a promising immunotherapeutic strategy for the treatment of haematological malignancies and solid cancers. Since the landmark FDA approval at the end of 2014 of the anti-CD3 × anti-CD19 bispecific antibody blinatumomab (Blincyto^®) for the treatment of relapsed/refractory B-cell acute lymphoblastic leukaemia, ~100 clinical trials investigating the safety and efficacy of CD3+ bispecific T-cell redirectors for cancer have been initiated. However, despite early success, numerous challenges pertaining to CD3+ T-cell redirection in the context of cancer exist, including the recruitment of counterproductive CD3+ T-cell subsets, the release of systemic cytokines, the expansion of immune checkpoint molecules, the presence of an immunosuppressive tumour microenvironment, tumour antigen loss/escape, on-target off-tumour toxicity and suboptimal potency. The aim of the present review is to discuss novel approaches to overcome the key challenges associated with CD3+ bispecific T-cell redirection in order to achieve an optimal balance of anti-tumour activity and safety.Subject terms: Oncology, Translational research     

Cytotoxic and antitumor activity of a recombinant immunotoxin composed of disulfide-stabilized anti-TAC Fv fragment and truncated pseudomonas exotoxin

Disulfide-stabilized Fv (dsFv)-immunotoxins are recombinant immunotoxins in which the inherently unstable Fv moiety, composed of the V_H-V_L heterodimer, is stabilized by a disulfide bond engineered between structurally conserved framework positions of V_H and V_L. Anti-Tac(dsFv)-PE38KDEL is composed of such a dsFv, directed to the α subunit of the IL2 receptor (IL2R), and containing a truncated form of Pseudomonas exotoxin (PE38KDEL). We have found this new type of immunotoxin to be indistinguishable in its in vitro activity and specificity from its single-chain immunotoxin counterpart, anti-Tac(Fv)-PE38KDEL. We have now examined the therapeutically relevant factors, including stability, pharmacokinetics, and antitumor activity of this new disulfide-stabilized Fv-immunotoxin. We found that anti-Tac(dsFv)-PE38KDEL was specifically cytotoxic to human activated T-lymphocytes in addition to IL2R bearing cell lines. Anti-Tac(dsFv)-PE38KDEL was considerably more stable at 37° C in human serum and in buffered saline than the single-chain immunotoxin, anti-Tac(Fv)-PE38KDEL. The half-life in blood was similar for both immunotoxins (approx. 20 min). The therapeutic potential of the disu Hide-stabilized immunotoxin was evaluated using an animal model of immunodeficient mice bearing subcutaneous tumor xenografts of human IL2R-bearing cells. Anti-Tac(dsFv)-PE38KDEL caused complete regression of tumors with no toxic effects in mice. Because dsFv-immunotoxins are more stable and can be produced with significantly improved yields compared to scFv-immunotoxins, dsFv-immunotoxin may be more useful for therapeutic applications. © 1994 Wiley-Liss, Inc.     

Anti-CD3-based bispecific antibody designed for therapy of human B-cell malignancy can induce T-cell activation by antigen-dependent and antigen-independent mechanisms

Anti-CD3 × anti-B-cell antigen bispecific monoclonal antibodies (bsAbs) can redirect T-cell-mediated lysis toward malignant B cells. Clinical trials with CD3-based bsAbs have shown toxicity in patients which is likely related to non-specific T-cell activation and targeting. Our current studies were designed to explore the mechanisms responsible for the observed in vivo toxicity by evaluating the immunologic effects of 2 different bsAb preparations in vitro. 1D10 was used as the tumor specific arm of the bsAbs. This antibody reacts with a variant of HLA-DR found on a majority of pre-B- and B-cell malignancies, and normal B cells in some individuals. Anti-CD3 served as the T-cell specific arm. A 1D10 × anti-CD3 bispecific IgG (bsIgG) produced using the hybrid-hybridoma method was compared to a 1D10 × anti-CD3 bispecific F(ab′)₂ [bsF(ab′)₂] produced using the leucine zipper technique. In cytotoxicity assays, both bsIgG and bsF(ab′)₂ induced lysis by pre-activated T cells of 1D10 (+) malignant B cells. bsIgG at high concentrations also induced lysis of 1D10 (−) tumor cells, while bsF(ab′)₂ did not. Proliferation of T cells induced by bsIgG and bsF(ab′)₂ was also evaluated. Both forms of bsAbs induced T-cell proliferation in the presence of antigen (+) Raji cells, while only bsIgG did so in the presence of antigen (−) malignant B cells. bsF(ab′)₂ induced T-cell activation in the absence of any tumor cells when testing was performed on samples where the 1D10 target antigen was present on normal peripheral blood B cells. We conclude that non-specific T-cell activation from bsAbs can occur in an antigen-independent manner due to the Fc/Fc receptor (FcR) interaction, or in an antigen-dependent manner when antigen is expressed on normal or tumor cells. Both mechanisms may have been responsible for the toxicity observed in prior clinical studies. Int. J. Cancer77:251–256, 1998. © 1998 Wiley-Liss, Inc.     

全人源食管癌单链抗体scFv 基因文库的构建

 目的 制备全人源化食管癌单链抗体scFv基因文库。方法 取食管癌病人癌肿周围淋巴结作为B细胞的来源，提取总RNA，用RT-PCR的方法获得抗体可变区基因cDNA文库。首先分别网格筛选确定扩增VH和VL基因片段的引物对，以cDNA为模板扩增VH和VL基因片段，再以它们为模板分别扩增VH-linker与VL-linker，用SOE-PCR技术将它们拼接成SCFv，再引入酶切住点Sfi I和Not I，胶回收PCR产物获得SCFv。将SCFv基因克隆入噬菌粒载体pCANT心5E后电转入Ecoli TG1。PCR法鉴定抗体基因插入率，1．5％琼脂糖凝胶电泳鉴定阳性克隆酶切产物。结果 食管癌周围淋巴结的提取总RNA琼脂糖电泳结果中可见清晰的28S、18S条带；VH基因的大小约为450bp。VL基因为350bp，组装后的scFv基因约为850bp。PCR连接产物的转化效率为2×10^7efu／μg，SCFv的阳性插入率为91．7％（22／24）。结论 食管癌相关的人源单链抗体基因文库的构建为进一步筛选单链抗体库奠定了基础。     

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.     

Development of a novel small antibody that retains specificity for tumor targeting

Background

For the targeted therapy of solid tumor mediated by monoclonal antibody (mAb), there have different models of rebuilding small antibodies originated from native ones. Almost all natural antibody molecules have the similar structure and conformation, but those rebuilt small antibodies cannot completely keep the original traits of parental antibodies, especially the reduced specificity, which gravely influences the efficacy of small antibodies.

Methods

In this study, authors developed a novel mimetic in the form of V_HFR1_C-10-V_HCDR1-V_HFR2-V_LCDR3-V_LFR4_N-10for a parental mAb induced with human breast cancer, and the mimetic moiety was conjugated to the C-terminal of toxicin colicin Ia. The novel fusion peptide, named protomimecin (PMN), was administered to MCF-7 breast cancer cells to demonstrate its killing competency in vitro and in vivo.

Results

Compared with original antibody-colicin Ia (Fab-Ia) and single-chain antibody-colicin Ia (Sc-Ia) fusion proteins, PMN retained the targeting specificity of parental antibody and could specifically kill MCF-7 cells in vitro. By injecting intraperitoneally into BALB/c athymic mice bearing MCF-7 tumors, with reduced affinity, PMN significantly suppressed the growth of tumors compared with control mice treated by toxicin protein, Fab-Ia protein, Sc-Ia protein or by PBS (p < 0.05).

Conclusion

This novel mimetic antibody retained original specificity of parental antibody, and could effectively guide killer moiety to suppress the growth of breast cancer by targeted cell death.     

Genetic alteration of a bispecific ligand-directed toxin targeting human CD19 and CD22 receptors resulting in improved efficacy against systemic B cell malignancy

A bispecific ligand-directed toxin (BLT) called DT2219ARL consisting of two scFv ligands recognizing CD19 and CD22 and catalytic DT₃₉₀ was genetically enhanced for superior in vivo anti-leukemia activity. Genetic alterations included reverse orienting VH-VL domains and adding aggregation reducing/stabilizing linkers. In vivo, these improvements resulted in previously unseen long-term tumor-free survivors measured in a bioluminescent xenograft imaging model in which the progression of human Raji Burkitt's lymphoma could be tracked in real time and in a Daudi model as well. Studies showed DT2219ARL was potent (IC₅₀s 0.06–0.2 nM range) and selectively blockable. Imaging studies indicated the highly invasive nature of this B cell malignancy model and showed it likely induced pre-terminal hind limb paralysis because of metastasis to spinal regions prevented by DT2219ARL. DT2219ARL represents a new class of bispecific biological that can be continually improved by genetic mutation.     

微型双功能抗体介导人T细胞杀伤耐药实体瘤细胞

目的 探讨抗P-糖蛋白（P-gP）／抗CD3微型双功能抗体介导人T细胞杀伤耐药实体瘤细胞的作用。方法 采用抗E-tag亲和层析柱分离纯化抗P-gp／抗CD3微型双功能抗体,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定;采用^51Cr释放实验,测定抗P-gP／抗CD3微型双功能抗体介导的人T细胞体外靶向杀伤活性;采用多药耐药（MDR）细胞系KB／MDR、敏感KB细胞裸鼠移植瘤模型,测定该微型双功能抗体介导的体内靶向杀伤活性。结果 纯化的抗P-gp／抗CD3微型双功能抗体,在相对分子质量28000和26000处各有1条蛋白带。在抗P-gp／抗CD3微型双功能抗体存在的情况下,激活的T淋巴细胞能够裂解KB／MDR细胞,且随着效靶比的增高而增高。抗P-gp／抗CD3微型双功能抗体联合人T淋巴细胞能有效抑制KB／MDR细胞裸鼠移植瘤的生长,而对敏感KB细胞移植瘤的生长无抑制作用。结论 抗P-gP／抗CD3微型双功能抗体在体内外均能介导人T淋巴细胞杀伤表达P-gP抗原的KB／MDR细胞,是有望用于耐药实体瘤临床治疗的双特异性抗体。     

A bispecific recombinant immunotoxin, DT2219, targeting human CD19 and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma.

A novel bispecific single-chain fusion protein, DT2219, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT(390)) fused to two repeating sFv subunits recognizing CD19 and CD22 and expressed in Escherichia coli. Problems with yield, purity, and aggregation in the refolding step were solved by incorporating a segment of human muscle aldolase and by using a sodium N-lauroyl-sarcosine detergent-based refolding procedure. Problems with reduced efficacy were addressed by combining the anti-CD19 and anti-CD22 on the same single-chain molecule. DT2219 had greater anticancer activity than monomeric or bivalent immunotoxins made with anti-CD19 and anti-CD22 sFv alone and it showed a higher level of binding to patient leukemia cells and to CD19(+)CD22(+) Daudi or Raji cells than did anti-CD19 and anti-CD22 parental monoclonal antibodies. The resulting DT2219, mutated to enhance its avidity, was cytotoxic to Daudi cells in vitro (IC(50) = 0.3 nmol/L). In vivo, DT2219 was effective in a flank tumor therapy model in which it significantly inhibited tumor growth (P < 0.05) and in a systemic model in which it significantly prolonged survival of severe combined immunodeficient mice with established Daudi (P < 0.008) compared with controls. DT2219 has broader reactivity in recognizing B-cell malignancies, has more killing power, and requires less toxin than using individual immunotoxin, which warrants further investigation as a new drug for treating B leukemia/lymphoma.     

Structural design of disialoganglioside GD2 and CD3‐bispecific antibodies to redirect T cells for tumor therapy

Antibody‐based immunotherapy has proven efficacy for patients with high‐risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single‐chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T‐cell‐mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti‐GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH–VL orientation, the (GGGGS)₃ linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2‐positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure‐based method to assist in designing BsAb for T‐cell‐mediated therapy.     

Extremely potent,rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody

A recent study reported on an anti-CD19/anti-CD3 single-chain bispecific antibody (bscCD19xCD3) exhibiting high activity against human B lymphoma cell lines (L?ffler et al., Blood 2000;95:2098-103). In the present study, we have explored in detail the in vitro efficacy, T-cell donor variability, binding characteristics, specificity, kinetics and interleukin-2 (IL-2) dependence of bscCD19xCD3. We found that a majority of human donor T cells tested (n = 86) gave half-maximal B-lymphoma cell lysis (ED(50)) within a range of 10-50 pg/ml bscCD19xCD3, corresponding to sub-picomolar concentrations of the bispecific antibody. Under identical experimental conditions, the anti-CD20 monoclonal antibody rituximab had an at least 100,000-fold lower in vitro efficacy. The extreme potency of bscCD19xCD3 was in sharp contrast to the relatively low affinity of the anti-CD3 and anti-CD19 single-chain Fv portions in K(D) ranges of 10(-7) and 10(-9) M, respectively. Cell lysis by bscCD19xCD3 was predominantly mediated by the population of CD8/CD45RO-positive T cells. Both immortalized CD4- and CD8-positive human T-cell clones were highly active effector cells as well. Cell lysis by bscCD19xCD3 was rapid and specific. The respective parental monoclonal antibodies inhibited cell lysis and CD19-negative cells were not harmed by T cells in the presence of high amounts of bscCD19xCD3. The potent T-cell stimulus IL-2 could not markedly augment the activity of bscCD19xCD3-stimulated T cells. In conclusion, bscCD19xCD3 could redirect unstimulated cytotoxic T cells against CD19-positive cells in an unexpectedly potent, rapid and specific fashion.