TRISPECIFIC ANTIBODY: A NOVEL APPROACH TO CANCER THERAPEUTICS

CONTENTS

What is trispecific antibody ?

How is it developed ?
Mechanism of action?

Immune activation through trispecific

antibody Application in cancer Clinical trials

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

What is trispecific antibody?

Antibody which exhibiting three specificities is called trispecific antibody It is a glycoprotein with intermediate molecular weight

Trispecific antibody contain three region which are specific for three different epitopes.
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Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

.. What is trispecific antibody ?

On the basis of structure two types

Trispecific scFv antibody

Trispecific F(ab)2 antibody

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

How is it developed?

Trispecific antibody (TR3H) is developed by the help of rDNA technology E.Coli is used as host E.Coli TOP 10 for the construction & propagation of vector E.Coli BL 21 star for the expression of plasmid

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

Steps involved in development of scTsAb a) Construction of TR3H vector vector contains genes of : - anti TAA scFv - Fc linker - Anti CD3 -HSA linker -Anti CD28 -C-myc
Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi 6

b) Expression and purification of TR3H scTsAb c) Western blotting d) Flow Cytometry of TR3H scTsAb to effector & Target cell

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

Mechanism of action?

Trispceific Antibody form a link between T-cell, tumor cell and other effector cell. In case of scTsAb, anti CD3 scFv interacts with TCR-CD3 complex and stimulates T-cells.

VH domain of anti CD28 antibody stimulate B7

molecule expressed on APC, interacts with CD28 on T cell.

Anti OC interacts with tumor associated antigen(TAA).

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

TRI contained anti-tumor arm to

(A) In the native condition, with the co stimulation of CD28, the TCR expressed on CTL cell recognized the antigen peptide-MHC on tumor cell and elicited a signal cascade to activate T cell to attack tumor cell MHC I molecule, B7 co-stimulatory signal and antigen presentation ability of patients. (B) Trispecific antibody contained anti-tumor arm to bind to tumor cells, CD3 and CD28 arms to 'pull' and active T cells to attack tumor cells.
Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi 9

Immune activation through trispecific antibody

Anti OC anti CD3 and anti CD8 compose scTsAb is constructed with the intent of recruiting Tcell to the ovarian tumor cells where they are activated to destroy the tumor cells. scTsAb activates the two signals in T cell simultaneously- First signal is induced by anti CD3 domain and second signal is induced by anti CD28. Signal induced by anti CD3 and anti CD28 activate the three transcription factors NF kappa B, NF-AT, AP1.

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

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Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

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Transcription factor enhance the synthesis of IL-2 and its receptor IL-2 activate the proliferation and differentiation of T cell T cell differentiated in to memory and effector T cell population The effector T cell secrete cytokines, activate B cell and cytotoxic killing activity

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

T cell + scTsAb + tumor cell

Activation of transcription factor (NFkB,NF-AT & AP1)

Synthesis of IL-2 & IL-2 receptor

Proliferation and differentiation of T cell

Memory cell & Effector cell

Secretion of cytokines & activation of B cell and CTL

etio
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Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

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Application in cancer

The scTsAb is the platform for the Tcells based immunotherapy against cancer specifically with failure on MHC antigen presentation or absence of costimulatory signal Trispecific antibody induces tumor cell lysis by redirecting T cell mediated cytotoxicity The scTsAb set up a bridge between cancer cell and T cell and create a micro environment for the T cell activation and T cell killing Trispecific antibody mediated immune activation lead to the killing of tumor cell by CTL

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

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Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

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Clinical trials

TR3H -A scTsAb which induce the lysis of ovary tumor cell -It contains anti OC antiCD3 antiCD28 scTsAb having anti SK-OV 3 cell antigen anti CD28 antiCD3 CEA-acTsAb having anti CEAantiCD3 antiCD28 involve in the killing of CEA positive tumor cell

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

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REFERENCES
1. GilBoa E. How tumors escape immune destruction and what we can do about it. Cancer Immunol Immunother, 1999, 48(7): 382385 2. Zhu Z, Lewis GD, Carter P. Engineering high affinity humanized anti-p185HER2/anti-CD3 bispecific F(ab') 2 for efficient lysis of P185her2 3. Kroesen BJ, ter Haar A, Spakman H, Willemse P, Sleijfer DT, de Vries EG, Mulder NH et al. Local antitumor treatment in carcinoma patients with bispecific-monoclonal-antibody-redirected T cells. Cancer Immunol Immunother, 1993, 37(6): 400407

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

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4.Bortoletto N, Scotet E, Myamoto Y, DOro U, Lanzavecchia A (2002) Optimizing anti-CD3 affinity for effective T cell targeting against tumor cells. Eur. J. Immunol. 32: 31023107.
5. SONG Li-Ping, CHENG Ju-Long, WANG XiangBin, ZHANG Zhong, FANG Min, ZHOU ZhiYong, HUANG Hua-Liang*(Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China )

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi

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