Therapeutic Antibodies In Cancer Treatments Biology Essay

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The generation of mouse-derived hybridomas by Kohler and Milstein in 1975 and the development of monoclonal antibodies (mAbs) as a highly specific cancer therapy tackled the formidable challenge of differentiating tumour cells from healthy cells and showed a path towards the development of selective therapeutic modality for cancer. To date, more than 400 anticancer mAbs, comprising approximately 25% of all biotechnology products, are under investigation. Half of them are anticancer monoclonal antibodies and a few have been approved for clinical use 1,4(Schematic figure of an antibody is shown in figure 1).antibody picture.jpg

Monoclonal antibodies can be divided into four main categories: murine, chimeric, humanised and human (with the exception of murine mAbs, the other types have a human Fc portion). In general, mAbs can employ various direct and indirect functions for the destruction of tumour cells. They can mediate antibody-dependent cellular cytotoxicity (ADCC) by recruiting the effector cells of the immune system through the engagement of the Fc portion with FcγRI (CD64), FcγRII (CD32), the B isoform of FcγRIII (CD16) on neutrophils and the A isoform of FcγRIII (CD16) on NK cells1,2,3. ADCC can induce tumour destruction and increase antigen presentation and subsequently activate tumour associated T-cell responses. Moreover, complement-dependent cytotoxicity (CDC) can be induced by activating the complement system, which in turn enhances ADCC by the release of chemotactic factors (C5a and C3a). Human IgG1, IgG3 and murine IgG2a are the potent isoforms in the induction of ADCC and CDC. Unmodified mAbs can also mediate tumour cell-killing by inhibiting angiogenesis, inducing apoptosis and blocking relevant receptors particularly activated growth factor receptors 1,2.

Figure 1: Schematic figure of an antibody molecule. It is composed of two identical heavy chains and two identical light chains. Variable domains of heavy(VH) and light chains(VL) form peptide-binding groove. CDRs are the most variable regions located in antigen-binding clefts. The arms of antibody are called Fab( fragment,antigen binding) including one constant and one variable domain from each heavy and light chain. The base of antibodies, Fc (fragment, crystallizable), mediates physiological abilities such as opsonization, cell lysis and degranulation.

Furthermore, to increase the specificity of aggressive treatments (radiotherapy and chemotherapy) and improve the efficiency of immunotherapy, mAbs can be conjugated to radioactive isotopes, toxins, cytotoxic drugs and cytokines to directly target tumour cells. Although murine IgG2a has a weak ability to induce CDC and ADCC, their immunogenicity and short circulating half-life have limited murine mAbs' application in cancer therapy and the immune responses prevent repeated administration from being very successful. Generally speaking, they can only be employed for targeting radioactive elements or cytotoxic agents to tumour cells 1,59. Regardless of the mode of action, human IgG1 is a preferred antibody isoform due to its multiple potential functions for mediating tumour cell death and relatively longer half-life 1(Figure 2).

Figure 2: Direct and indirect effector functions of anti-cancer monoclonal antibodies A. unmodified monoclonal antibodies act through ADCC (antibody-dependent cellular cytotoxicity) and CDC (cytokine dependent cytotoxity). B. monoclonal antibodies can mediate tumour cell death through covalent linkage to cytotoxic agents such as radionuclide, cytokines, immunotoxins and immunoconjugates.

1.1.1. Unmodified monoclonal antibodies:

Chimeric, humanised and human IgG1 mAbs which use ADCC and CDC as effector functions are of commercial and clinical interest. Rituximab (Rituxan), a chimeric IgG1 mAb against CD20, was the first mAb to be approved in the clinical setting as an effective treatment for diffuse large B cell lymphoma, leukaemia, transplant rejection and some autoimmune disorders. It is believed to induce ADCC, CDC and apoptosis by altering intracellular calcium levels1,2,3,5. Alemtuzumab (Campath), a humanised IgG1 mAb against CD52 uses ADCC and CDC as its main effector mechanisms while trastuzumab (Herceptin) mainly down-regulates human epidermal growth factor 2 (HER2) and uses ADCC and CDC as an alternative mechanism. Studies have showen that it can improve survival in advanced breast cancer and is a viable agent in patients with late-stage, metastatic endometrial carcinomas overexpressing HER2/neu 6,7. Altering the function of costimulatory molecules such as CD40 and CD137 is another mechanism by which mAbs can regulate tumour growth (summarized in table 1).

Table 1: Examples of unmodified monoclonal antibodies and their mechanisms of action

Unmodified monoclonal antibodies

Mechanisms of action

Rituximab (Rituxan)

Chimeric IgG1 targeting CD20

Induce ADCC, CDC and apoptosis by altering intracellular calcium levels 1,4

Alemtuzumab (Campath)

Humanised IgG1 targeting CD52

Induce ADCC and CDC as main effector mechanisms 1,4

Trastuzumab (Herceptin)

Humanised IgG1 targeting HER2/neu

Down regulates human epidermal growth factor 2 (HER2) and uses ADCC and CDC as an alternative mechanism 1, 3

Pertuzumab (2C4)

Inhibit EGF receptor heterodimerisation and activation 1,2,3

Bevacizumab (Avastin)

Humanised IgG1 targetting VEGF

A potent mAb against metastatic colon cancer, breast cancer and non-small cell lung cancer blocks VEGF and promotes tumour death by inhibiting neoangiogenesis 1,3,4

Edrecolomab

Targets Ep-CAM and induces ADCC 1,5

Ipilimumab (MDX-010)

Tremelimumab (CP-675)

Maintain an active immune response by blocking the inhibitory activity of CTLA-4 1,2,3

RAV12

Targets RAAG12 and induces cell swelling followed by necrosis (oncosis) 2

Cetuximab (Erbitux, chimeric IgG1 targetting EGFR), Nimotuzumab (TheraCIM), Panitumumab (Vectibix) and Matuzumab (EMD72000)

Inhibit epidermal growth factor (EGF) receptor activation by blocking the interaction between the receptor and its ligand 1,2

1.1.2. Modified monoclonal antibodies

Monoclonal antibodies can be used to selectively deliver cytotoxic agents. Radioisotopes (90Y and 131I), immunotoxins which consist of proteins (Pseudomonas exotoxin, Staphylococcus enterotoxin, neocarzinostatin, ricin and gelonin) and small molecules (vinblastine, methotrexate, doxorubicin, calicheamicin and maytansine) can be conjugated to the monoclonal antibodies and directly mediate tumour destruction. 90Y ibritumomab tiuxetan, 131I tositumomab and 131I ch-TNT are approved mAbs for non-Hodgkin's lymphoma and lung cancer. Gemtuzumab ozogamicin is an immunotoxin used to treat acute myelogenous leukemia1,3, 4.

Antibody-cytokine fusion therapy has been evolved to precisely activate the anti-tumour immune responses. Although antibody-IL-2 fusion proteins are the most investigated drugs in this category, other cytokines such as GM-CSF, IL-12, TNF-α, IFN-γ and LT-α have also been scrutinized, albeit with limited success3,4.

1.2. Tumour-Associated Antigens

Anti-cancer monoclonal antibodies can be targeted against tumour cell-surface proteins, the antigens associated with tumour stroma, vasculature and ligands. These targets must be of a specific nature in order to be suitable for mAbs development. They should be expressed on the surface of tumour compartments and should not be internalized rapidly if ADCC and CDC are to be performed. Conversely, internalization is crucial for the cytotoxic activity of immunotoxins. Other characteristics are specific expression in target tissue, having a role in disease pathogenesis (to minimise escape variants) and active expression throughout disease stages and metastatic lesions. They should not be shed or secreted into the circulation to lead the mAbs to the tumour site. Although various glycoproteins, glycolipids and carbohydrates were targeted, some were used with greater frequency (summarized in table 2) 1,2.

Table 2: Top targets for development of anti cancer monoclonal antibodies

Top targets for mAb development

Targeted cancers and examples

Overexpression of EGF receptor family

EGFR (c-erbB-1), HER-2/neu (c-erbB-2), HER3 (c-erbB-3), HER4 (c-erbB-4).

In non-small cell lung cancers, breast cancer, colorectal cancer, head and neck cancers and prostate cancer made them a suitable target for the development of mAbs. More than 21 mAbs have targeted this family.

Epithelial cell adhesion molecule (Ep-CAM)

In colorectal, pancreatic and non-small cell lung cancer

Carcinoembryonic antigen (CEA)

Heavily expressed in adenocarcinomas and GI tract neoplasm while only luminal side of the intestines which is inaccessible to antibodies normally expresses it

TRAILR 1, 2 (tumour necrosis factor-related apoptosis-inducing ligand receptor 1,2)

Induce apoptosis in tumour cells.

Hematologic neoplasms

More accessible and therapeutic doses are generally less .CD20, CD22 (lymphoma), CD52 (chronic lymphocytic and promyelocytic leukemia), CD40 (lymphoma and multiple myeloma), CD80 (lymphoma) and B cell idiotypes (B-cell lymphoma)

Fibroblast activation protein (FAP)

A stroma target antigen and is expressed by phenotypically different tumour fibroblasts

Tenascin

An extracellular matrix protein presented in stroma of glioma, breast, lung, squamous cell carcinoma, non-Hodgkin lymphoma and targeted by 81C6 mAbs.

Vasculature antigens

Fibronectin extra-domain B (ED-B), prostate specific membrane antigen (PSMA), VEGF receptor 2 and annexin A1 are main tumour vasculature antigens

Targets that inhibit angiogenesis

Vascular endothelial growth factor (VEGF), angiopoietin-1, tie-1, tie-2 and vitaxin

Circulating ligands

TNF-α and IL-6 are under investigation.

The current generation of anti-cancer monoclonal antibodies are limited to the intact antigens presented on the surface of tumour cells. Although many successful mAbs have been developed, the number of cancer patients resistant to current medications is increasing (70%). There are several explanations for this. In the first instance, free TAAs may shed from the tumour and engage the antibody-binding sites which lead to a drop in the number of active antibodies and subsequently their tumour cell-killing ability. Moreover, most targeted antigens are complex molecules, whereas only a single epitope is being recognised by antibodies which confine their efficacy. On the other hand, many potential targets associated with tumour genesis e.g. p53 are intracellular and are thus not accessible to antibody targeting by conventional methods. Therefore, it has been suggested that cell-surface antigens are not ideal therapeutic targets for the generation of mAbs.

The immune system is configured to allow the sentry of intracellular milieu through the peptides displayed on the MHC Class I molecules. These cytosolic oligopeptides (derived from malignant transformation and intracellular pathogens or improperly presented and heavily expressed antigens) are generated by proteasomal and non-proteasomal pathways, displayed on MHC Class I complex and recognised by CD8+ T cell lymphocytes. Since B-cell lymphocytes recognise the tertiary structure of proteins without any MHC restriction, development of antibodies-recognizing peptides presented by MHC molecules on the cell surface is not straightforward. One possibility is to target peptide-MHC complexes with TCRs, but their low affinity and minimal stability has limited their application8. In contrast, antibodies have higher affinities and are easier to handle. Thus, several approaches were performed to target this family of antigens as the next generation of antigens for development of anti-cancer monoclonal antibodies to direct immune responses toward intracellular antigens that are not themselves secreted or displayed on the cell surface. Telomerase catalytic subunit (hTERT), melanoma differentiation antigen gp100, epithelial cell associated mucin (MUC1/CanAg), MAGE 10 , and NY-ESO-1 9 are examples of intracellular antigens presented by human MHC molecules HLA-A1 or HLA-A2 2,4,12.

1.3. MHC Class I - structure, antigen processing and presentation

The MHC Class I molecule - known as Human Leukocyte Antigen (HLA) in humans and "H-2" in mice - is formed of an α chain which is polymorphic and encoded within the MHC region of chromosome 6 and non-polymorphic β2 microglobulin chain encoded in chromosome 15 in humans. The α chain spans the membrane and comprises three domains, α1, α2 and α3. The tertiary folded structure of the α1 and α2 domains forms a highly polymorphic peptide-binding groove on the surface of the MHC Class I molecules, which accommodates the presented peptide fragment of 8 to 10 amino acids with similar anchor residues. The α3 domain interacts with β2 microglobulin and facilitates the stability of the molecules but neither of them is involved in the formation of the peptide-binding cleft (Fig 3). CD8 molecules are cell-surface markers of cytotoxic T cells and recognise the MHC Class I complexes. They interact mainly with an invariable part of the α3 domain and the base of the α2 domain73.

MHC Class I presents a wide variety of peptides generated from incomplete, mutated or unfolded self proteins or foreign antigens on the cell surface to be recognised by CD8+ cytotoxic T cell lymphocytes and if necessary initiate an immune response. This allows the surveillance of the entire intracellular milieu by the adaptive immune system (Figure 4). The bound peptides are the integral part of the MHC molecules to avoid peptide exchange at the surface of the cells. They are mainly generated as N-extended peptides by the proteasomal processing of intracellular antigens and undergo further sequential trimming in the cytosol and ER. Dendritic cells, to a limited extent, display the extracellular proteins on the MHC type I by cross-presentation 7,32,33.

MHC I.jpgAg binding groove.jpg

Figure 3: the schematic picture of MHC class I and its antigen binding groove

Figure 4: processing and presentation of intracellular antigens on MHC class I complex. Cytoplasmic proteins are degraded by proteasome followed by further trimming in endoplasmic reticulum. peptide fragment of 8 to 10 amino acids are loaded on MHC class I 73.

1.4. MHC tetramer production and its applications

Recombinant MHC multimers, coupled with flow cytometry, have been developed to identify antigen-specific T cells. Multimers differ in their valency, applied expression system and peptide-loading strategy. Tetramers, by far the most popular reagents, are conventionally generated by refolding soluble MHC α chain with β2m. Soluble MHC monomers are then biotinylated using Biotin Ligase and converted into tetravalent by adding the correct ratio of streptavidin or avidin. Tetramers are conjugated to fluorochromes (PE and APCs) to be visualized and enumerated by FACS analysis 19, 13.

The expression system used for the generation of MHC classes I and II are generally different due to the difference in their refolding processes 20. MHC Class I is produced in bacterial cells such as Esherichia coli and refolded conveniently in vitro, while the refolding process of MHC type II is cumbersome and therefore eukaryotic cells such as baculovirus infected insect cells 21or Drosophila cell transfectants are used 22.

There are several techniques for loading the antigenic peptide in different stages. In the case of MHC Class II molecules, peptides can be genetically linked to one of the MHC chains, while peptides are included during in vitro production or after the generation of the MHC Class I monomer or even multimers20.

HLA multimer technology has helped us to study the frequency, phenotype and function of antigen-specific T-cells13. Tetramers are able to identify antigen-specific T-cells targeting viruses, tumours, and transplantation antigens with delicate sensitivity. Several types of viral infections have been studied particularly frequently such as EBV, CMV, LCMV, HCV, VSV, influenza, parvovirus B19 and HIV. Among bacterial infections Listeria monocytogenes has dominated. Analysis of anti-tumour responses and the evaluation of post-immunotherapy responses in patients suffering from cancer as well as estimation of the heterogeneity of memory repertoire are other areas of research13.

Although tetramers were first designed as diagnostic tools, their ability to manipulate T-cell response has made them a promising therapeutic strategy. Potential clinical applications are for the selection and expansion of desired T-cells (Adoptive T cell transfer) and the removal of unwanted T-cells following hematopoietic stem cell transplantation (reduction of GVHD)(Figure 5). MHC tetramers are able to identify and tolerise autoreactive T-cells in autoimmune disorders, for example autoreactive CD4+ T-cells specific for GAD65 555-567(minitope) in type I diabetes 26. Another novel strategy was pioneered by Dimopoulos and colleagues. They joined MHC tetramer technology and intracellular cytokine staining (ICS) to identify the antigen presentation specificity of tumour cells for the development of cancer vaccine targets with no need for T-cell cloning, T-cell culture and no bias was seen by IFN γ production of unknown cells18.

tetramer applications.jpg

Figure 5: Therapeutic applications of MHC multimers. a. In tumour and viral infections desired Tcells are selected and expanded (Adoptive T cell transfer). b,c. unwanted T cells are depleted following hematopoietic stem cell transplantation (reduction of GVHD) and autoimmune disorders13.

1.5. T-cell receptor mimic (TCRm) antibodies and their therapeutic potential

Therapeutic vaccines for the treatment of cancer and particular types of viruses are developed to induce T-cell mediated immune responses. To measure the potency of such vaccines, several culture-based qualitative and semi-quantitative assays such as Limiting dilution assay (LDA), ELISPOT and ICS have been developed but the underestimation of the immune responses was the main limitation. Since the concentration of MHC bound to the specific antigen on the surface of vaccine treated APCs directly correlates with the intensity of the cytotoxic responses, monoclonal antibodies with the unique specificity for antigen-specific MHC-restricted T-cells (TCR mimic antibodies) were employed as a reliable tool to detect the presentation and intensity of CTL responses directed against tumours and viral infections17. They can elucidate structural and functional MHC-Peptide-TCR interactions in detail, quantify the number of MHCs bound to the specific peptide on the surface of the antigen-presenting cells and localize APCs within the normal and diseased tissues. Their ability to inhibit MHC-Peptide-TCR interactions, opens up a possible role in the regulation of autoimmune disorders in vivo 8. Furthermore, recombinant TCR mimic antibodies can be isolated from a phage display library by expression in E.coli cells 8or generating in hybridomas30. Several studies have combined genetic immunization with hybridoma technology to obtain high affinity monoclonal antibodies.

Although the application of TCR mimic antibodies in the treatment of tumours was first confined to delivering toxins and drugs to the tumour site, Wittman and colleagues in their cutting-edge research have showed that TCRm antibodies could potentially activate components of the innate immune system and kill specific tumour cell lines. They have developed a murine IgG2a TCR mimic antibody-targeted intracellular GVL peptide from human chorionic gonadotropin β (hCGβ) bound to HLA-A2 and demonstrated that it can mediate tumour cell lysis by both CDC and ADCC in a human breast cell line carcinoma in vitro. They have also revealed the in vivo prophylactic ability of the 3.2 G1 TCRm antibody, as the implantation and growth of MDA-MB-231tumour cells were inhibited in nude mice28.

Recently, Verma and colleagues have provided proof of the therapeutic potential of this antibody, as it can impede the growth of MDA-MB-231 and MCF-7 tumours in orthotopic models of the breast cancer or even eliminate them at the highest dose without attacking the normal tissue of the breast27.

1.6. Difficulties in generating T-cell receptor mimic antibodies

Although they have huge implications in diagnosis and treatment of tumours, viral infections, autoimmunity and transplantation, the production of monoclonal antibodies with precise specificity for T-cell receptors is technically demanding. As explained earlier, one efficient method for generating TCR mimic antibodies is using hybridoma technology which involves the immunization of HLA/A*0201 transgenic mice with the specific tumour or viral peptide fragments contained in human MHC Class I tetramer (Figure 6). It was demonstrated that immunization with human MHC molecules results in the development of unwanted antibodies against structural regions which are not involved in TCR recognition. Since the human MHC α3 domain interacts with low affinity with the murine CD8 molecule, the vast majority of undesired antibodies are targeted to this region35,36,37. This complicates the isolation of TCR mimic antibodies. Moreover, assessing the efficacy of vaccines and evaluating different vaccination regimens in HLA-A2 transgenic mice needs high HLA-A2 restricted cytotoxic T-cells and high staining efficacy which could not be obtained with the poor interaction of the human MHC Class I α3 domain of tetramers with murine CD8 molecules35,36,37.

TCR generation.jpg

3

1

4

2

5

Figure 6: generation of TCR mimic monoclonal antibodies. 1:mouse is immunized with tetramers and mouse spleen produces plasma cells that secrete Ab against the tetramers. 2:myeloma cells unable to produce Abs are selected. 3: plasma cells from mouse spleen is fused with myeloma cells to produce hybridomas. 4: cells are transformed to H.A.T medium.5: hybridomas that produce antibodies specific to tetramers are selected and grown in bulk (Encyclopaedia, Britannica, 1999).

1.7. Chimeric tetramers - potential in CTL evaluation and promoting TCRm generation

The generation of chimeric human-murine MHC Class I tetramers in which the human α3 domain is substituted with the murine counterpart can increase the affinity of MHC-I CD8 interactions without interfering with the peptide-binding groove. These recombinant tetramers are able to increase the staining efficiency which the leads to a more accurate evaluation of CTL responses in transgenic mice.

To date, a few successful attempts have been made to produce chimeric tetramers. Engineering of tetramers containing a non-specified H-2D murine α3 domain by Ren and colleagues was the proof of concept. Furthermore, Choi and colleagues produced a human-murine chimeric HLA-A2 tetramer containing the murine MHC allele H-2Kb α3 domain coupled with human β2 microglobulin (A2Kb). The recombinant A2Kb tetramers were used for the evaluation of CTL responses in HHD mice (H-2Dd-/-/β2m-/-) vaccinated with "DNA-prime recombinant vaccinia virus (rVV) boost strategy". They showed the higher staining capability for cytotoxic T cells compared to unmodified A2 tetramers34.

HHD molecules - a human HLA-A*0201 α1/α2 linked to the mouse H-2Db α3 transmembrane and cytoplasmic domains - is a successful chimeric monochain devised by Pascolo and colleagues at the University of Edinburgh. A human β2 microglobulin is covalently bound to its heavy chain by a 15 amino acid linker. Another chimeric monochain with the same heavy chain coupled with the murine β2 microglobulin (MHD) was engineered as well. Their efficiency was evaluated in H-2Dd-/-/β2m-/- double knockout mice (HHD) and compared with the fully human HHH monochain. The expression of HHD and HHH molecules on the cell surface was far better than was seen with the MHD constructs. The superior interaction of mice CD8 with the mice α3 part leads to better recognition of cytotoxic responses by HHD chimeric monochains. These data showed therefore that the development of tetramers with more murine character may not function as well as HHD for the detection of cytotoxic responses 38.

In the case of TCR mimic monoclonal antibodies, chimeric tetramers were able to direct the generation of antibodies towards the peptide-binding regions of the MHC molecule by making the structural parts of the molecule less immunogenic. This ensured that unwanted antibodies were spared and made the purification of practical antibodies straightforward.

Recombinant HHD is a valuable target in the development of monoclonal antibodies as it is derived from the most common human HLA (HLA-A2) and its functionality has been confirmed for other applications. Moreover, generation of further chimeric tetramers with the same human heavy chain and different murine compartment for use in common murine expression systems in development of monoclonal antibodies, Balb/c mice, can improve the efficiency of TCR mimic antibody generation ( for more details see the next chapter).

1.8. Chimeric tetramers for use in Balb/c mice

Balb/c mice, an albino inbred widespread lab strain, are commonly used to generate monoclonal antibodies as they develop plasmocytomas when injected with mineral oil. Neethling and colleagues generated TCR mimic antibodies by immunization of this strain with peptide-HLA-A*0201 complex and Quil-A adjuvant17. The same procedure was utilized by Weidanz via "eIF4G (720)-HLA-Alow asterisk0201 complex"30. Moreover, Timusk developed TCRm antibodies by genetic immunization of the same strain39.

Successful employment of Balb/c mice by Wittman for the development of TCR mimic antibodies after immunization with fully human tetramers "HLA-A2-hCG beta47-55 peptide (designated GVL/A2) complex" showed the practicability of this strain27,28. Although HHD chimeric monomer seems to be a perfect construct for the generation of TCR mimic monoclonal antibodies, the HHD strain of mice have not been used for this intention. As a result, TCRm antibody development could be hampered as it needs optimization of the hybridoma-fusion techniques in this strain. On the other hand, the HHD construct might not be compatible with Balb/c mice for the generation of monoclonal TCR mimic antibodies. Consequently, a combination of these two systems - HHD constructs and Balb/c strain of mice- and the production of chimeric tetramers compatible with both strains could increase the chance of generating TCR mimic monoclonal antibodies.

1.9. Antibody repertoire screening

To identify antibodies generated against the peptide-binding groove, HLA-A*0201 control tetramers were refolded using influenza A matrix protein-derived peptide ("Flu Peptide", amino acid sequence GILGFVFTL). Flu peptide is a widespread standard peptide for the generation of control tetramers. Since the structural regions are similar between control tetramers and those developed using tumour-associated antigens, antibodies that can bind to control tetramers are considered nonspecific and removed from the repertoire 50(Figure 7).

Figure 7: Immunising mice with human MHC Class I tetramers provokes an immune response against the entire tetramer - many non-useful antibodies are generated. Producing chimeric murine/human tetramers may minimise production of unwanted antibodies.

1.10. Aims of the dissertation

As discussed in detail earlier, the aim of this project is therefore to generate C57BL/6 and Balb/c compatible chimeric MHC Class I tetramers by substituting the structural parts of MHC Class I (α3 and β2 microglobulin) with their mouse counterparts (Figure 8).

To achieve this, the chimeric constructs' and mouse β2m sequences were amplified by PCR and cloned into the TOPO vector followed by sub-cloning into the pET9C plasmid vector. The expression vector was first transformed into DH-5α to produce high levels of plasmid DNA followed by transformation into BL21 (DE3) for protein expression. Monomers were refolded in the presence of antigenic peptide and β2 microglobulin, biotinylated, tetramerized and analysed by FACS. These steps are summarized in figure 9.

These tetramers could then be used to generate therapeutic TCR mimic monoclonal antibodies in C57BL/6 and Balb/c mice against various tumour antigens, potentially with improved efficiency due to the decreased immunogenicity of the tetramers.

Figure 8: A: Schematic figures of human MHC class I and mice α3 domains ;HHD (H-2Db) and BALB/C (H-2Dd) mice

B: Schematic figures of engineered constructs; HHD (HLA-A2.1α1 α2 H-2Db), MM (HLA-A2.1α1 α2 ,H-2Dd), LS (HLA-A2.1α1 α2 ,H-2Dd). As illustrated , the human part of the LS construct has 3 amino acids more than the human domain of the MM construct

protocol.jpg

Figure 9: Steps of chimeric tetramer production

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