Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Microbial Production of Pharmaceuticals
Antibody production in E. coli

Hybridomas, like most other Animal Cell Cultures, grow relatively slowly, do not reach high densities, and require complex and expensive media. The Monoclonal Antibodies obtained this way are very costly, which limits their widespread clinical use. To address this issue, attempts have been made to develop a kind of "bioreactor" based on genetically modified Bacteria, plants, and animals. For the effective delivery and function of certain immunotherapeutic agents, a single antigen-binding region of an antibody (Fab or Fv fragment) is often sufficient, meaning that the presence of the antibody's Fc fragment is not required.

Figure 10.12 illustrates the method for producing functional antibodies using E. coli (Fig. 10.12).

1. Using mRNA isolated from antibody-producing Cells (B lymphocytes) of mice or humans, cDNA is synthesized.

2. Separate PCR Amplification of cDNAs encoding the H and L chains is performed.

3. Amplified cDNAs are digested with specific Restriction Endonucleases and then inserted into a bacteriophage λ-based vector. The cDNAs of the H and L chains contain different, chain-specific endonuclease sites, which facilitates the specific insertion of each nucleotide sequence into its respective vector. At this stage, multiple different segments of the H and L chains are cloned (Fig. 10.13, A and B).

4. The cDNA of one H chain and one L chain are inserted into a common "combinatorial" vector, so that both chains are synthesized in the bacteriophage, forming a "fully functional" Fv fragment (Fig. 10.13, C).

The synthesis of H and L chains occurs during the lytic cycle of bacteriophage λ, allowing for the screening of the combinatorial bacteriophage clone library to determine their antigen-binding activity.

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Fig. 10.12. Generation of a combinatorial cDNA library of antibody V_L and V_H regions using E. coli.

At the stage of joining the H and L chain cDNAs in a single vector, a wide spectrum of different antibody genes is generated. Some of these encode unique binding sites that would be impossible to obtain using conventional hybridoma technology. The mammalian antibody pool comprises 10⁶–10⁸ different antibodies. A phage library contains approximately the same number of clones; thus, a single combinatorial library can be expected to produce as many different antibodies (Fv molecules) as any mammal. Furthermore, once an initial combinatorial library is established, L and H chains can be shuffled to obtain Fv fragments that recognize novel epitopes. Even greater diversity can be achieved using random mutagenesis. Since millions of phage plaques can be screened in a relatively short time, identifying Fv fragments with the desired Specificity takes only 7 to 14 days. In comparison, screening a few hundred hybridoma cell lines typically takes months.

Bacteriophage λ-Based Vectors are not well suited for producing large quantities of protein molecules. To solve this problem, a vector was designed in which the H and L chain DNAs are inserted into a site flanked by plasmid DNA. This plasmid, containing the H and L chain DNAs, can be excised from the vector and used to transform E. coli (Fig. 10.12). As part of the plasmid, the Fv fragment DNA will replicate multiple times in E. coli cells, yielding a large amount of product that can be used for both diagnostic and therapeutic purposes. When creating combinatorial libraries, filamentous Bacteriophages M13 or fd can be used instead of phage λ (Fig. 10.14). In these cases, the corresponding antibody fragment is synthesized as part of a fusion protein displayed On the surface of the phage particle. Screening of the combinatorial antibody fragment library can be performed using an enzyme-linked immunosorbent assay (ELISA). The method is as follows: aliquots from the library are added to the wells of a microtiter plate coated with the target antigen. The wells are washed to remove unbound phage particles. A conjugate consisting of an antibody that binds to the phage coat protein and an enzyme is added to each well. The wells are washed to remove unbound conjugate, and a chromogenic substrate is added to each. The substrate is cleaved by the phage-bound enzyme, coloring the wells containing phage particles that carry antibodies to the target antigen. The Selection process and subsequent purification of bacteriophages synthesizing an antibody fragment specific to the desired antigen is much simpler in this case than when counting bacteriophage λ plaques. Once the phage synthesizing the desired antibody fragment is isolated, the DNA encoding this fragment can be extracted and subcloned into an expression vector. Different antibody variants with increased affinity for the target antigen can be generated by replacing DNA fragments of the V_L and V_H regions or through random mutagenesis.

Fig. 10.13. Engineered DNA regions from a combinatorial Fv fragment cDNA library cloned into bacteriophage λ. A and B. DNA fragments of the L (A) and H (B) chains were separately inserted into bacteriophage λ-based vectors. C. Each EcoRI library was digested, and DNA fragments from the H-chain library were ligated with DNA fragments from the L-chain library, resulting in a combinatorial library containing all possible combinations of L and H chain fragments, with expression of the joined fragment in a single vector. pLac — E. coli lac promoter, RBS — ribosome binding site.

Having developed Methods for obtaining Fv fragments, researchers sought to determine whether a single protein chain consisting only of V_L and V_H domains could form a functional antigen-binding molecule. Computer modeling of the three-dimensional Structure OF THE proposed single-chain antibody showed that to adopt the conformation required for antigen binding, the V_L and V_H domains must be separated by a linker peptide. With this in mind, the V_L and V_H DNAs, synthesized from the cDNA template of a cloned monoclonal antibody, were joined to a chemically synthesized DNA linker, creating a V_L-DNA—linker—V_H-DNA construct. The corresponding single-chain protein was synthesized in E. coli, purified, and found to have an antigen affinity and specificity similar to those of the intact monoclonal antibody. Thus, functional single-chain antibodies can be easily produced using E. coli.

Single-chain antibodies could find widespread clinical application in cases where Fc effector Functions are not required, and the small molecular size (the molecular mass of a single-chain antibody is approximately 27 kDa, compared to 150 kDa for immunoglobulin G) offers distinct advantages. Furthermore, a sequence encoding a specific protein can be fused to the single-chain antibody, yielding a bifunctional molecule that can bind to a specific target while exhibiting a particular activity.

Fig. 10.14. Generation of a combinatorial cDNA library of antibody Fv fragments using the filamentous bacteriophage M13. The cDNAs of the V_L and V_H regions were amplified by PCR and then ligated using DNA encoding a short linker peptide. The resulting DNA fragments, comprising the single-chain antibody cDNA combinatorial library, were inserted into the M13 phage genome, fused to phage Gene 3, which encodes a phage surface protein. In M13, three protein molecules are synthesized from gene 3; therefore, each recombinant M13 phage containing the single-chain antibody cDNA combinatorial library will display three molecules of the fusion protein consisting of the gene 3 product and the single-chain antibody.

Another experiment was also conducted: instead of joining the V_L and V_H chains with a short peptide, the framework region Amino Acids were modified to form a disulfide bridge between them. The efficacy of this disulfide-stabilized Fv molecule conjugated to a Cancer-cell-destroying toxin was compared with that of a single-chain Fv molecule conjugated to the same toxin (Fig. 10.15). It was found that the disulfide-stabilized and single-chain Fv immunotoxins possessed identical activity and specificity, but the former was several times more stable. This suggests that in certain situations, disulfide-stabilized Fv molecules may be preferable to single-chain Fv molecules.

Fig. 10.15. Schematic representation of a single-chain Fv immunotoxin (A) and a disulfide-stabilized Fv immunotoxin (B).

Anti-HIV Drugs

Scientists have not yet succeeded in developing a sufficiently effective vaccine against the HUMAN IMMUNODEFICIENCY VIRUS (HIV), which causes Acquired Immunodeficiency Syndrome (AIDS). In parallel with vaccine development, research is ongoing to find other agents capable of slowing down the disease progression.

HIV infects a specific type of lymphocyte, namely helper T cells (T_H cells). Normally, during The Development of an Immune Response, T_H cells bind degradation products of specific Antigens and release factors that stimulate other immune system cells to participate in the immune response. T_H cells play a key role in this process, but in HIV infection, they cease to function. Once the virus enters a T_H cell, it is shielded from the host's immune system and begins its destructive effect on the T_H cells.

✵ Viral Replication within the infected cell leads to its lysis.

✵ The infected cell acts as a factory producing the HIV glycoprotein (gp120), which causes the destruction of T_H cells and other T lymphocytes.

✵ The infected cell fuses with other T_H cells, forming a syncytium that is unable to perform the functions characteristic of individual T_H cells.

The primary consequence of HIV infection is the inability of the body's immune system to protect it against common bacterial and viral infections, which ultimately lead to the patient's death despite Treatment with Antibiotics and other agents.

In The First stage of HIV infection, an interaction occurs between the viral envelope glycoprotein with a molecular mass of 120 kDa (gp120) and the receptor on The surface of T_H cells—CD4 (Fig. 10.16, A). In vitro, infection of T_H cells is blocked by antibodies to CD4; the process is also slowed down by an excess of free CD4 protein. However, neither of these approaches leads to the destruction of the virus. One approach that provides both protection of T_H cells and inactivation of the virus involves creating a chimeric protein consisting of a CD4 molecule fragment and an immunoglobulin Fc fragment. The properties of this protein, called CD4-immunoadhesin, are determined by its constituent parts: the CD4 component binds gp120 and blocks HIV, while the immunoglobulin component slows down the degradation of the molecule in plasma and mediates its binding to cells bearing the antibody receptor. Upon binding of CD4-immunoadhesin to a free viral particle or an infected cell, antibody-dependent cellular cytotoxicity is triggered, which ensures the destruction of the virus or the infected cell.

Fig. 10.16. HIV INFECTION AND ITS therapy. A. Binding of HIV to a T_H cell is mediated by the contact of the viral protein gp120 with the T_H cell surface protein CD4. B. On the surface of an HIV-infected cell is the gp120 protein, to which a free chimeric CD4–toxin complex can bind. Once inside the infected cell, the toxin portion of the chimeric molecule kills it.

Another approach to controlling the progression of HIV infection involves creating a system to label HIV-infected cells for their specific destruction. For example, by fusing two DNA fragments, one encoding the CD4 receptor and the other encoding the intracellular Pseudomonas toxin (exotoxin A), we obtain a gene encoding a chimeric protein with combined properties (Fig. 10.17). Pseudomonas exotoxin A is a 66 kDa protein consisting of three domains: domain I is responsible for cell binding, domain II for translocation into The Cell, and domain III for transferring ADP-ribose to eukaryotic elongation factor 2 (EF-2), which leads to its inactivation. Instead of domain I, the chimeric CD4–Pseudomonas exotoxin A protein contains most of the CD4 sequence (Fig. 10.17), thereby possessing both the cytotoxic activity of Pseudomonas exotoxin and the gp120-binding activity of CD4. Since the gp120 glycoprotein is present on the surface of all HIV-infected cells, the CD4 domain of the chimeric protein binds exclusively to these cells. Upon binding to the infected cell, the chimeric protein enters it with the assistance of domain II of Pseudomonas exotoxin A. The exotoxin portion of the chimeric protein then inactivates the elongation factor EF-2, which is involved in Protein Synthesis. This halts further protein synthesis, ultimately leading to cell death. Thus, the CD4 domain 'marks' the HIV-infected cells, while the exotoxin acts as a 'hired killer'.

When synthesized in E. coli, the chimeric protein forms insoluble cytoplasmic inclusion bodies. They are dissolved in guanidine hydrochloride and isolated using rapid dilution and anion-exchange Chromatography. The protein obtained in this manner was successfully tested in control cell cultures. However, an immune response against the Pseudomonas component of the chimeric protein may occur in The Human Body, and it might need to be administered alongside an immunosuppressant, such as cyclosporine. It should be kept in mind that the above-described method of combating HIV infection is in the early Stages of development, although it may prove highly effective in the future. Such immunotherapeutics are highly potent, allowing them to be used in low doses and minimizing side effects on The Immune System. In addition, they may prove useful for treating various neoplasms and, in some cases, replacing Chemotherapy. Other cytotoxic Proteins, such as diphtheria toxin or the plant toxin ricin, can also be targeted to infected cells. Nevertheless, even under optimal circumstances, it will be several years before the THERAPEUTIC USE OF recombinant exotoxins becomes routine.

Fig. 10.17. Genetically engineered chimeric CD4–Pseudomonas exotoxin A complex. The bacteriophage T7 promoter of E. coli was used.