trispecific antibody.ppt

TRISPECIFIC ANTIBODY: A NOVEL APPROACH TO TRISPECIFIC ANTIBODY: A NOVEL APPROACH TO CANCER THERAPEUTICSCANCER THERAPEUTICS

CONTENTCONTENTSS

What is trispecific antibody ?What is trispecific antibody ?

How is it developed ?How is it developed ?

Mechanism of actionMechanism of action??

Immune activation through Immune activation through

trispecific antibodytrispecific antibody

Application in cancerApplication in cancer

Clinical trialsClinical trials

Bhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi 2

What is trispecific antibody?What is trispecific antibody?

Antibody which exhibiting three specificities Antibody which exhibiting three specificities is called is called

trispecific antibodytrispecific antibody

It is a glycoprotein withIt is a glycoprotein with intermediate molecular intermediate molecular weight weight

Trispecific antibody Trispecific antibody contain three regioncontain three region which are specific for three different epitopes.which are specific for three different epitopes.


………….……. What is trispecific antibody ? .……. What is trispecific antibody ?

On the basis of structure – two types On the basis of structure – two types

Trispecific F(ab)2 Trispecific F(ab)2 antibodyantibody

Trispecific scFv antibodyTrispecific scFv antibody


How is it developed?How is it developed?

Trispecific antibody (TR3H) is developed by the Trispecific antibody (TR3H) is developed by the help of rDNA technologyhelp of rDNA technology

E.Coli is used as hostE.Coli is used as host

E.Coli TOP 10 – for the construction & E.Coli TOP 10 – for the construction & propagation of vectorpropagation of vector

E.Coli BL 21 star – for the E.Coli BL 21 star – for the

expression of plasmidexpression of plasmid


Steps involved in development of scTsAbSteps involved in development of scTsAb

a) Construction of TR3H vectora) Construction of TR3H vector

vector contains genes of :vector contains genes of :

- anti TAA scFv- anti TAA scFv

- Fc linker- Fc linker

- Anti CD3- Anti CD3

-HSA linker-HSA linker

-Anti CD28-Anti CD28

-C-myc-C-myc


b) Expression and purification of TR3H scTsAbb) Expression and purification of TR3H scTsAbc) Western blottingc) Western blottingd) Flow Cytometry of TR3H scTsAb to effector & d) Flow Cytometry of TR3H scTsAb to effector &

Target cellTarget cell


Mechanism of action?Mechanism of action? Trispceific Antibody form a link between T-Trispceific Antibody form a link between T-

cell, tumor cell and other effector cell.cell, tumor cell and other effector cell.

In case of scTsAb, anti CD3 scFv interacts with In case of scTsAb, anti CD3 scFv interacts with

TCR-CD3 complex and stimulates T-cells.TCR-CD3 complex and stimulates T-cells.

VH domain of anti CD28 antibody stimulate VH domain of anti CD28 antibody stimulate

B7 molecule expressed on APC, interacts with B7 molecule expressed on APC, interacts with

CD28 on T cell.CD28 on T cell.

Anti OC interacts with tumor associated Anti OC interacts with tumor associated

antigen(TAA).antigen(TAA).


TRI contained anti-tumor arm toTRI 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.


Immune activation through trispecific Immune activation through trispecific antibodyantibody

Anti OC anti CD3 and anti CD8 compose scTsAb Anti OC anti CD3 and anti CD8 compose scTsAb is constructed with the intent of recruiting Tcell is constructed with the intent of recruiting Tcell to the ovarian tumor cells where they are to the ovarian tumor cells where they are activated to destroy the tumor cells.activated to destroy the tumor cells.

scTsAb activates the two signals in T cell scTsAb activates the two signals in T cell simultaneously- First signal is induced by anti simultaneously- First signal is induced by anti CD3 domain and second signal is induced by CD3 domain and second signal is induced by anti CD28.anti CD28.

Signal induced by anti CD3 and anti CD28 Signal induced by anti CD3 and anti CD28 activate the three transcription factors NF activate the three transcription factors NF kappa B, NF-AT, AP1.kappa B, NF-AT, AP1.


Transcription factor enhance the synthesis of IL-2 and it’s 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

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 etioBhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi 12

Application in cancerApplication in cancer The scTsAb is the platform for the Tcells The scTsAb is the platform for the Tcells

based immunotherapy against cancer based immunotherapy against cancer specifically with failure on MHC antigen specifically with failure on MHC antigen presentation or absence of costimulatory presentation or absence of costimulatory signalsignal

Trispecific antibody induces tumor cell lysis Trispecific antibody induces tumor cell lysis by redirecting T cell mediated cytotoxicityby redirecting T cell mediated cytotoxicity

The scTsAb set up a bridge between cancer The scTsAb set up a bridge between cancer cell and T cell and create a micro cell and T cell and create a micro environment for the T cell activation and T environment for the T cell activation and T cell killingcell killing

Trispecific antibody mediated immune Trispecific antibody mediated immune activation lead to the killing of tumor cell by activation lead to the killing of tumor cell by CTLCTLBhanu Pratap, Department of Biochemistry, Jamia Hamdard, New Delhi 14

Clinical trialsClinical trials

TR3HTR3H

-A scTsAb which induce the lysis of ovary -A scTsAb which induce the lysis of ovary tumor celltumor cell

-It contains anti OC × antiCD3 × antiCD28-It contains anti OC × antiCD3 × antiCD28 scTsAb having anti SK-OV 3 cell antigen × scTsAb having anti SK-OV 3 cell antigen ×

anti CD28 × antiCD3anti CD28 × antiCD3 CEA-acTsAb having anti CEA×antiCD3 CEA-acTsAb having anti CEA×antiCD3

×antiCD28 – involve in the killing of CEA ×antiCD28 – involve in the killing of CEA positive tumor cellpositive tumor cell


1. GilBoa E. How tumors escape immune destruction 1. GilBoa E. How tumors escape immune destruction and what we can do about it. Cancer Immunol and what we can do about it. Cancer Immunol Immunother, 1999, 48(7): 382Immunother, 1999, 48(7): 382 －－ 385385

2. Zhu Z, Lewis GD, Carter P. Engineering high 2. Zhu Z, Lewis GD, Carter P. Engineering high affinity humanized anti-p185HER2/anti-CD3 affinity humanized anti-p185HER2/anti-CD3 bispecific F(ab') 2 for efficient lysis of P185her2bispecific F(ab') 2 for efficient lysis of P185her2

3. Kroesen BJ, ter Haar A, Spakman H, Willemse P, 3. Kroesen BJ, ter Haar A, Spakman H, Willemse P, Sleijfer DT, de Vries EG, Mulder NH et al. Local Sleijfer DT, de Vries EG, Mulder NH et al. Local antitumor treatment in carcinoma patients withantitumor treatment in carcinoma patients with

bispecific-monoclonal-antibody-redirected T cells. bispecific-monoclonal-antibody-redirected T cells. Cancer Immunol Immunother, 1993, 37(6): 400Cancer Immunol Immunother, 1993, 37(6): 400 －－407407

REFERENCESREFERENCES


4.Bortoletto N, Scotet E, Myamoto Y, D’Oro U, 4.Bortoletto N, Scotet E, Myamoto Y, D’Oro U, Lanzavecchia A Lanzavecchia A (2002) Optimizing anti-CD3 (2002) Optimizing anti-CD3 affinity for effective T cell targeting against affinity for effective T cell targeting against tumor cells. Eur. J. Immunol. 32: 3102–3107.tumor cells. Eur. J. Immunol. 32: 3102–3107.

5. SONG Li-Ping, CHENG Ju-Long, WANG Xiang-5. SONG Li-Ping, CHENG Ju-Long, WANG Xiang-Bin, ZHANG Zhong, FANG Min, ZHOU Zhi-Bin, ZHANG Zhong, FANG Min, ZHOU Zhi-Yong, HUANG Hua-Liang*(Institute of Genetics Yong, HUANG Hua-Liang*(Institute of Genetics and Developmental Biology, the Chinese and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China Academy of Sciences, Beijing 100101, China ))


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trispecific antibody.ppt

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