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

Li, Wei; Fan, Daiming; Yang, Moon-Sik; Shi, Ruizan; Yan, Yan; Jiang, Linlin; Yan, Chunhong; Li, Shuangjing; Wang, Min; Wang, Jianxiang; Xiong, Dongsheng

doi:10.1007/s13402-012-0101-9

Cited by 10 publications

(13 citation statements)

References 23 publications

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“…Cells were maintained in RPMI 1640 (Gibco, Grand Island, NY, USA) containing 10 % fetal bovine serum (FBS) and grown at 37 °C with 5 % CO 2 . Bi-specific anti-CD3 × anti-CD19 diabody and its disulfide-stabilized format (ds-diabody) constructed previously are stored in our lab [ 31 ].…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we constructed a novel bi-specific anti-CD3 × anti-CD19 diabody and its disulfide-stabilized format (ds-diabody) [ 31 ]. The cytotoxicity of these two diabodies showed no dramatic differences in vitro.…”

Section: Introductionmentioning

confidence: 99%

“…The cytotoxicity of these two diabodies showed no dramatic differences in vitro. However, in vivo, the ds-diabody was more efficient than its parental diabody in inducing tumor cell lysis because of its increased stability [ 31 ]. Osada et al .…”

Section: Introductionmentioning

confidence: 99%

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Redirection of CD4+ and CD8+ T lymphocytes via an anti-CD3 × anti-CD19 bi-specific antibody combined with cytosine arabinoside and the efficient lysis of patient-derived B-ALL cells

Fan

Yang

et al. 2015

J Hematol Oncol

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BackgroundB-acute lymphoblastic leukemia (B-ALL) is derived from B cell progenitors. Recently, the development of appropriate combinations of chemotherapy and immunotherapy represents a promising approach for eliminating cancer. We previously constructed an anti-CD3 × anti-CD19 bi-specific antibody in a diabody configuration and its disulfide-stabilized format (ds-diabody). The combination of the diabody or ds-diabody and Ara-C was highly effective in enhancing the cytotoxicity of T cells against the CD19+ human leukemia cell-line, Nalm-6, both in vitro and in vivo. This study verified whether B-ALL patient-derived cells were sensitive to the diabody or ds-diabody and low-dosage Ara-C combination.MethodsThis study aimed to detect the B7 family members B7.1 (CD80) and B7.2 (CD86) that were expressed in B-ALL patient-derived cells pre-treated by Ara-C (0.25 μM) and to determine the targeted killing ability of T cell subtypes induced by the diabody or ds-diabody combination with Ara-C both in vitro and in vivo. We also determined the levels of the cytokines that were released by activated CD4+ or CD8+ T cells during therapy.ResultLow-dose Ara-C enhanced CD80 and CD86 expression in nearly 50 % of specimens of B-ALL patient-derived cells. A combination of diabody or ds-diabody and Ara-C enhanced T cell against B-ALL cells in vitro and in vivo. Both CD8+ and CD4+ T cells were potently activated. Expression of CD25 and CD69 was augmented equally by CD4+ or CD8+ T cells. However, CD8+ T cells made the major contribution by redirecting target cell lysis in a granzyme B and perforin-dependent mechanism. CD4+ T cells played an important immunomodulatory role by secreting IL2. Consequently, IL3, IL6, TNFα, and IFNγ were also released by CD4+ or CD8+ T cells following diabody-mediated T cell activation.ConclusionT cell therapy induced by diabody or ds-diabody combined with low dose of Ara-C was effective against cancer cell-lines and in clinical trials. In vivo, the ds-diabody was more efficient than its parent diabody due to its enhanced stability.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Redirection of CD4+ and CD8+ T lymphocytes via an anti-CD3 × anti-CD19 bi-specific antibody combined with cytosine arabinoside and the efficient lysis of patient-derived B-ALL cells

Fan

Yang

et al. 2015

J Hematol Oncol

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“…Since the heavy chain (V H ) and the light chain (V L ) domains of antiCD3Fv are associated non-covalently, they dissociated relatively easily. To enhance the stability of the fusion protein, we employed disulfide covalent bonds to lock the two chains together as we have done before in the antiPgp × antiCD3 diabody and the antiCD19 × antiCD3 diabody, both of which showed a higher antitumor activity in animal models [ 31 , 32 ]. As expected, the disulfide-stabilized antiCD3Fv-⊿IL3 improved the serum stability by the disulfide cross-linking of the CD3 Fv fragments.…”

Section: Discussionmentioning

confidence: 99%

“…The fragments of antiCD3V L and antiCD3*V L (containing a cysteines at position Ser-100 of CD3V L ) containing the restriction enzyme site MluI and the G4S linker were obtained by polymerase chain reaction (PCR) from the plasmid pLH-T3a-19a and pLH-T3a*-19a [ 32 ], using the primer pairs P1/P2 (Table 1 , all primers were synthesized by Invitrogen Life Technologies, USA), respectively. The antiCD3V H and antiCD3*V H (containing a cysteines at position Gly-44 of CD3V H ) segments containing the restriction enzyme site MluI and the G4S linker were generated similarly by PCR from the plasmid pLH-19a-T3a and pLH-19a-T3a* [ 32 ], respectively, using the primer pairs P5/P6. The fragment of ⊿IL3 (the end of eight amino acids were deleted) was obtained by PCR from cDNA of human peripheral blood mononuclear cells, using the primer pairs P3/P4 (including the restriction enzyme site NheI and the G4S linker) or P3/P7 (both containing the G4S linker).…”

Section: Methodsmentioning

confidence: 99%

AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells

Fan

Zhang

et al. 2015

J Hematol Oncol

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BackgroundLeukemic stem cells (LSCs) are frequently seen as a cause of treatment failure and relapse in patients with acute myeloid leukemia (AML). Thus, successful new therapeutic strategies for the treatment of AML should aim at eradicating LSCs. The identification of targets on the cell surface of LSCs is getting more and more attention. Among these, CD123, also known as the interleukin-3 (IL3)-receptor α chain, has been identified as a potential immunotherapeutic target due to its overexpression on LSCs in AML as well as on AML blasts, rather than normal hematopoietic stem cells.MethodsWe constructed a CD123-targeted fusion protein antiCD3Fv-⊿IL3, with one binding site for T cell antigen receptor (TCRCD3) and the other for CD123, by recombinant gene-engineering technology. Cysteine residues were introduced into the V domains of the antiCD3Fv segment to enhance its stability by locking the two chains of Fv together with disulfide covalent bonds. The stability and cytotoxicity of the two fusion proteins were detected in vitro and in vivo.ResultsBoth fusion proteins were produced and purified from Escherichia coli 16C9 cells with excellent yields in fully active forms. High-binding capability was observed between these two fusion proteins and human IL3R, leading to the specific lysis of CD123-expressing cell lines KG1a; also, mononuclear cells from primary AML patients were inhibited in a colony forming assay in vitro, presumably by redirecting T lymphocytes in vitro. In addition, they displayed an antileukemic activity against KG1a xenografts in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, especially disulfide-stabilized (ds)-antiCD3Fv-⊿IL3 for its improved stability.ConclusionsThese results suggest that both fusion proteins display the antileukemic activity against CD123-expressing cell lines as well as leukemic progenitors in vitro and in vivo, especially ds-antiCD3Fv-⊿IL3. They could be the promising candidates for future immunotherapy of AML.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0109-5) contains supplementary material, which is available to authorized users.

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Building blocks for bispecific and trispecific antibodies

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2019

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

Abstract: 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.

Cited by 10 publications

References 23 publications

Redirection of CD4+ and CD8+ T lymphocytes via an anti-CD3 × anti-CD19 bi-specific antibody combined with cytosine arabinoside and the efficient lysis of patient-derived B-ALL cells

Redirection of CD4+ and CD8+ T lymphocytes via an anti-CD3 × anti-CD19 bi-specific antibody combined with cytosine arabinoside and the efficient lysis of patient-derived B-ALL cells

AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells

Building blocks for bispecific and trispecific antibodies

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